CD2結合蛋白（CD2AP）和其相互作用蛋白的製作方法

2024-04-04 01:05:05 1

本發明涉及cd2相關蛋白(cd2ap)及其相互作用的蛋白，尤其是cd2ap與C型肝炎病毒(hcv)非結構蛋白ns5a之間的相互作用，cd2ap和胰島素受體底物1(irs1)之間的相互作用，以及cbl-b/cbl和irs1之間的相互作用，並進一步涉及下調cd2ap表達的試劑和方法，操縱cd2ap和hcvns5a之間的相互作用而抑制hcv裝配的試劑和方法，操縱cd2ap和irs1之間的相互作用而治療二型糖尿病病人的試劑和方法,和操縱cbl-b/cbl和irs1之間的相互作用而治療二型糖尿病病人的試劑和方法。此外，本發明也涉及到基於cd2ap的血清學和膽汁檢測脂肪肝和二型糖尿病的試劑和方法。
背景技術：
：C型肝炎病毒(hcv)，flavivirus的一員，是一種單股正鏈rna病毒，基因組9.6kb(1)。hcv感染全球約1億8000萬人，導致嚴重的慢性肝病(2)。hcv的正常靶點是肝細胞。進入宿主細胞後，hcv脫掉其基因組rna的外殼，並翻譯出多聚蛋白前體，然後在宿主和病毒蛋白酶的切割下產生三個結構蛋白和七個非結構蛋白，非結構蛋白在病毒rna複製，裝配和釋放中起重要作用(3)。慢性hcv感染通常會導致脂肪肝，主要病症是富含中性脂的囊泡的異常積累。hcv的脂滴主要組分也是中性脂，是病毒顆粒進行裝配的場所。(4,5)。脂滴是細胞中廣泛存在的一種獨特的細胞器，具有一個磷脂單層。脂滴參與許多生物學過程，如能量儲存、脂質代謝、免疫及信號轉導。在hcv感染的細胞中，脂滴的表面布滿hcv的核心蛋白和ns5a，ns5a分布在在外表面(5-7)，核心蛋白和ns5a雙雙附著到脂滴，對傳染性hcv顆粒的組裝和釋放是必不可少的(6,8,9)。ns5a可以分為三個結構域，d1，d2和d3。d1和d2為rna複製所需，d3有助於病毒的組裝和釋放。另外，通常認為,ns5ad1負責靶向脂滴而d3負責結合核心蛋白(10,11)。將含有ns5a的hcv複製複合體轉運到脂滴，取決於核心蛋白/ns5a與細胞骨架之間的相互作用。微管和肌動蛋白絲抑制劑的處理會抑制hcv複製複合體的移動(12)。沉默微管動力蛋白dynein，顯著降低ns5a陽性結構到脂滴的遠程移動性(13,14)。然而，並沒有證據顯示dynein和ns5a存在直接作用。因此，轉運過程中與ns5a相互作用的宿主蛋白了解的仍然不完全肝臟是系統代謝的主要器官，肝臟功能的失衡極大地促胰島素抗性和2型糖尿病(t2dm)的發生(15)。在導致胰島素抗性的分子中，irs-1是一個支架蛋白，在胰島素級聯中起著重要作用。許多體內和體外研究表明，降低irs的細胞水平可能是胰島素抗性的一種機制(15-22)。胰島素受體是一個酪氨酸激酶，其底物irs1的穩定性，主要是通過蛋白酶體降解蛋白水平來調節。研究表明，蛋白酶體介導降解irs-1可能參與了胰島素和胰島素樣生長因子(igf-1)信號通路的下調，導致胰島素抗性(23-27)。irs1的泛素化顯示為胰島素誘導irs1蛋白酶體降解的前提；irs-1的n-端區域包括ph、ptb結構域，被證明是靶向irs-1到泛素蛋白酶體降解途徑所必須的(28)。胰島素抗性通常導致肝纖維化和脂肪變性，在hcv感染狀況下尤其如此(29)。胰島素通過誘導其受體順式磷酸化，驅動營養儲存和組織生長，其受體是一個二聚體跨膜受體酪氨酸激酶(rtk)。這導致胰島素受體底物(irs)1和2的磷酸化，激活一個廣泛的信號網絡包括但不限於phosphatidylinositol-2-kinase/akt/mtor和ras/mek/erk通路。irs-1是一個信號適配器蛋白，在人類中由irs-1基因編碼。這是一個131kda的蛋白質，胺基酸序列有1242個殘基。它包含一個位於n-末端的pleckstrin同源(ph)結構域和位於40個殘基的下遊的ptb結構域，接著是不保守的c-末端尾巴。irs-1在信號傳輸中起著關鍵的作用，信號從胰島素和胰島素樣生長因子-1(igf-1)受體到胞內信號通路，如pi3k/akt和erkmap激酶通路。胰島素受體(ir)對irs-1酪氨酸磷酸化引入了多個可以和帶有sh2蛋白同源結構域的蛋白，如pi3k、grb-2/sos複合物和shp2相互結合的位點。技術實現要素：本發明提供下調個體cd2ap表達的方法。在一些實施例中，其特徵在於，該方法包括：施用一個cd2ap下調配伍到個體，其中cd2ap下調配伍通過sirna/shrna,crispr/cas9,crispr/cpf1,talen或zfns來工作；因此，個體肝組織的cd2ap表達被下調。在一些實施例中，其特徵在於，所述cd2ap下調配伍包括至少一個選自序列號3-20(針對人)或59-76(針對狗)的sirna/shrnai多聚核苷酸序列或至少一個crispr/cas9,crispr/cpf1載體，其中所述crispr/cas9,crispr/cpf1載體包含一個選自序列號21-56(針對人)或77-103(針對狗)的引導多聚核苷酸序列。本發明提供一個下調cd2ap在個體肝組織表達的藥物配伍，其特徵在於，所述配伍包括至少一個選自序列號3-20(針對人)或59-76(針對狗)的sirna/shrnai多聚核苷酸序列或至少一個crispr/cas9,crispr/cpf1載體，其中所述crispr/cas9,crispr/cpf1載體包含一個選自序列號21-56(針對人)或77-103(針對狗)的引導多聚核苷酸序列。本發明提供篩選能夠下降cd2ap和hcv非結構蛋白ns5a相互作用的候選試劑的方法，其特徵在於，所述方法包括：提供表達cd2ap和ns5a的細胞；讓候選試劑作用於表達cd2ap和ns5a的細胞；檢測候選試劑對cd2ap和ns5a相互作用的影響；其中，如果引起cd2ap和ns5a相互作用下降一個預定閾值，選定該候選試劑；其中，所述預定閾值被定義為cd2ap和ns5a相互作用下降至少70％，優選80％。本發明提供降低cd2ap和ns5a相互作用的藥物配伍，其特徵在於，所述藥物配伍包括至少一個多肽，含有5-40個胺基酸，優選10-30個胺基酸，更優選15-25個胺基酸；其中所述多肽衍生於序列號2的3-58胺基酸，序列號2的111-165胺基酸，序列號2的271-327胺基酸，和序列號105的353-466胺基酸。本發明提供篩選能夠下降cd2ap和irs1相互作用的候選試劑的方法，其特徵在於，所述方法包括：提供表達cd2ap和irs1的細胞；讓候選試劑作用於表達cd2ap和irs1的細胞；檢測候選試劑對cd2ap和irs1相互作用的影響；其中，如果引起cd2ap和irs1相互作用下降一個預定閾值，選定該候選試劑；其中，所述預定閾值被定義為cd2ap和irs1相互作用下降至少70％，優選80％。本發明提供降低cd2ap和irs1相互作用的藥物配伍，其特徵在於，所述藥物配伍包括至少一個多肽，含有5-40個胺基酸，優選10-30個胺基酸，更優選15-25個胺基酸；其中所述多肽衍生於序列號2或58的3-58胺基酸，序列號2或58的111-165胺基酸，和序列號2或58的271-327胺基酸。本發明提供篩選能夠下降cbl-b/cbl和irs1相互作用的候選試劑的方法，其特徵在於，所述方法包括：提供表達cbl-b/cbl和irs1的細胞；讓候選試劑作用於表達cbl-b/cbl和irs1的細胞；檢測候選試劑對cbl-b/cbl和irs1相互作用的影響；其中，如果引起cbl-b/cbl和irs1相互作用下降一個預定閾值，選定該候選試劑；其中，所述預定閾值被定義為cbl-b/cbl和irs1相互作用下降至少70％，優選80％。本發明提供下調個體肝組織中cbl-b/cbl表達的方法，其特徵在於，該方法包括：施用一個cbl-b/cbl下調配伍到個體，其中cbl-b/cbl下調配伍通過sirna/shrna,crispr/cas9,crispr/cpf1,talen或zfns來工作；因此，個體肝組織的cbl-b/cbl表達被下調。在一些實施例中，所述cbl-b/cbl下調配伍包括至少一個選自序列號112-124和196-208(針對人)或161-170和246-255(針對狗)的sirna/shrnai多聚核苷酸序列或至少一個crispr/cas9,crispr/cpf1載體，其中所述crispr/cas9,crispr/cpf1載體包含一個選自序列號125-158和209-243(針對人)或171-192和256-280(針對狗)的引導多聚核苷酸序列。本發明提供一個下調cbl-b/cbl在個體表達的藥物配伍，其特徵在於，所述cbl-b/cbl下調配伍包括至少一個選自序列號112-124和196-208(針對人)或161-170和246-255(針對狗)的sirna/shrnai多聚核苷酸序列或至少一個crispr/cas9,crispr/cpf1載體，其中所述crispr/cas9,crispr/cpf1載體包含一個選自序列號125-158和209-243(針對人)或171-192和256-280(針對狗)的引導多聚核苷酸序列。本發明提供治療個體的hcv感染，其特徵在於，所述治療包括：施用一個配伍，其包含至少一個選自序列號3-20的sirna/shrnai多聚核苷酸序列；施用一個配伍，其包含至少一個crispr/cas9,crispr/cpf1載體，其中所述crispr/cas9,crispr/cpf1載體包含一個選自序列號21-56的引導多聚核苷酸序列；或者施用一個配伍，其包含一個降低cd2ap和ns5a相互作用的試劑。本發明提供治療患有糖尿病的個體，其特徵在於，所述治療包括：施用一個配伍，其包含至少一個選自序列號3-20(針對人)或59-76(針對狗)的sirna/shrnai多聚核苷酸序列；施用一個配伍，其包含至少一個crispr/cas9,crispr/cpf1載體，其中所述crispr/cas9,crispr/cpf1載體包含一個選自序列號21-56(針對人)或77-103(針對狗)的引導多聚核苷酸序列；或者，施用一個配伍，其包含一個降低cd2ap和irs1相互作用的試劑。本發明提供治療患有糖尿病的個體，其特徵在於，所述治療包括：施用一個配伍，其包含至少一個選自序列號112-124和195-208(針對人)或161-170和246-255(針對狗)的sirna/shrnai多聚核苷酸序列；施用一個配伍，其包含至少一個crispr/cas9,crispr/cpf1載體，其中所述crispr/cas9,crispr/cpf1載體包含一個選自序列號125-158和209-243(針對人)或171-192和256-280(針對狗)的引導多聚核苷酸序列；或者，施用一個配伍，其包含一個降低cbl-b/cbl和irs1相互作用的試劑。本發明提供檢測個體肝臟異化的診斷方法。在實施例中，所述方法包括，提供來自個體的肝臟樣品，讓肝臟樣品接觸檢測cd2ap表達的檢測試劑；因此，當cd2ap表達被檢測到時，提示該個體肝臟異化。異化包括hcv感染和糖尿病。本發明提供檢測個體肝臟異化的診斷試劑盒。在實施例中，所述試劑盒包括，cd2ap蛋白特異抗體或cd2apmrna特異的多聚核苷酸探針，和一個次級試劑，能夠檢測與cd2ap蛋白結合的抗體或來自cd2apmrna的信號。通過如下結合附圖對優選實施例的詳細描述，本發明的目的和優點是顯而易見的。附圖說明本發明的優選實施方案現在將參考附圖進行說明，其中類似的附圖標記表示相同的元件。圖1顯示bioid構件,ns5a-bira*-ha的結構域示意圖。圖2顯示免疫印跡圖片。huh7細胞轉導了表達ns5abira*-ha的慢病毒載體，在添加或不添加50μm生物素的完全培養基中孵育24h。全細胞裂解物進行10％sds-page分析；分離蛋白質用辣根過氧化物酶標記的鏈黴親和素來檢測。圖3所示sds-page凝膠與考馬斯亮藍染色的圖片。細胞裂解物的製備與圖2相同。細胞裂解物通過鏈黴親和素瓊脂糖珠進行親和純化。純化的蛋白質進行10％sds-page分析和考馬斯亮藍染色。將箭頭指示的生物素處理樣品的特定條帶進行質譜分析。圖4顯示免疫印跡圖片。免疫共沉澱分析293t細胞中cd2ap和ns5a蛋白之間的相互作用。293t細胞轉染了單獨的ha標記cd2ap(prk-ha-cd2ap)或ha標記cd2ap(prk-ha-cd2ap)連同flag標記的ns5a；ns5a來自2a型hcv的jfh1菌株(prk-flag-ns5a)。轉染36小時後，細胞裂解物進行免疫共沉澱(co-ip)，將細胞裂解液與鼠源的flag抗體(標為f)或對照igg1(標為igg)進行co-ip。用兔源的ha抗體和flag抗體進行wb檢測。裂解液中ha-cd2ap的表達量相同。圖5顯示免疫印跡照片。免疫共沉澱分析hcv感染期間cd2ap和ns5a蛋白之間的相互作用。huh7.5.1細胞感染hcvjfh1或未感染為對照。感染後72h，收集細胞進行裂解。細胞裂解液與cd2ap抗體(左圖)或抗ns5a抗體(中間圖)進行co-ip，然後用ns5a抗體和cd2ap抗體進行wb檢測。ns5a的表達證實hcv的感染(右上圖)。圖6顯示免疫印跡照片。hcvjfh1感染huh7.5.1細胞72小時，細胞裂解液與ns5a抗體或對照小鼠igg1進行co-ip，然後用ns5a抗體和cd2ap抗體進行wb檢測ip複合物。只有ns5a抗體可以沉澱cd2ap，igg1對照未拉下cd2ap。圖7顯示免疫螢光染色照片。cd2ap和ns5a在hcv感染的huh7.5.1細胞中共定位。huh7.5.1細胞感染hcvj399em(如圖6所示hcv2a相同株，但ns5a是gfp標記的)(下面板)或未感染(上面板)72小時。然後用兔源的cd2ap抗體和alexfluor555標記的二抗染色cd2ap(紅色)，綠色代表ns5a-egfp的定位。圖8所示全長和截短cd2ap示意圖。cd2ap的n-端含有三個sh3結構域。從n端到c端分別是sh3-1，sh3-2和sh3-3。圖9顯示免疫印跡照片。cd2ap截短體與ns5a的相互作用。將ha標記的ns5a和flag標記的cd2ap截短體共轉293t細胞。轉染後36小時，將細胞裂解液與鼠源的flag(f)抗體或者igg1對照(igg)進行co-ip。ip複合物用兔源的ha和flag抗體進行檢測。ip下來的cd2ap截短體用星號標記。分子量在20-35kda之間的非特異性譜帶未在此顯示。圖10顯示全長和截短ns5a的示意圖。ns5a由三個結構域組成；如圖所示，它們由兩個低複雜度的序列連接(lcs1和lcs2)。圖11顯示免疫印跡照片。ns5a截短體與cd2ap的相互作用。293t細胞轉染了ha標記的cd2ap和其中一個flag標記的ns5a蛋白或截短突變體。轉染後36小時，將細胞裂解液與鼠源的flag(f)抗體或者igg1對照(igg)進行co-ip。ip複合物用兔源的ha和flag抗體進行檢測。發現ns5a的第三結構域與cd2ap特異性相互作用。抗flag抗體純化的ns5a截短體用星號表示。圖12顯示ns5a與全長及截短cd2ap的定位。在huh7.5.1細胞中穩定表達帶有mcherry標籤的全長cd2ap或者sh3結構域缺失的截短cd2ap(cd2ap：331-639)，然後用hcv-j399em感染該細胞。48h後，共聚焦顯微鏡觀察cd2ap(紅色)和ns5a(綠色)的運動並進行實時跟蹤。ns5a與全長cd2ap共定位(左圖)，但不與截短cd2ap共定位(右圖)。圖13是一個曲線圖顯示一個ns5a/cd2ap複合物的共移動。如圖12所示，感染後72h實時圖像跟蹤cd2ap(紅色)和ns5a(綠色)顯示了ns5a和cd2ap的共移動。使用volocity軟體分析圖13方框1中所示的ns5a/cd2ap共定位複合物的速度。圖14所示照片顯示ns5a/cd2ap複合物的共移動依賴於肌動蛋白聚合。穩定表達帶有mcherry標籤的全長cd2ap的huh7.5.1細胞用hcv-j399em感染48小時，然後用1μg/ml細胞鬆弛素b(上圖)或10μm秋水仙素(下圖)處理1小時。或者藥物處理1小時後，更換不加藥物的新鮮培養基培養4小時後(右圖)對活細胞進行共聚焦顯微鏡觀察。細胞鬆弛素b處理後ns5a和cd2ap沒有共定位(左上圖)。然而，秋水仙鹼處理不影響ns5a和cd2ap的共定位(左下圖)。更換培養基後培養四小時，cd2ap和ns5a的共定位重新出現(右上圖面板)，而秋水仙素處理的細胞，在更換培養基後後並無區別(右下圖)。圖15是一個曲線圖顯示秋水仙素處理後方框3所示的ns5a/cd2ap共定位點的速度。在微管聚合被抑制後，cd2ap/ns5a複合物沒有任何共移動。圖16顯示免疫印跡照片。將nc和cd2ap沉默細胞(c4#,c6#)進行脂滴分離，然後用特異性的抗體進行檢測。左側是ns5a在脂滴上的表達水平，右側是細胞裂解液中各種蛋白的表達。adrp和calnexin分別是脂滴和內質網的標記物。用imagej軟體進行ns5a的定量分析。在hcv亞複製子細胞con1中將cd2ap下調後，ns5a在脂滴上的表達水平下降。然而，細胞裂解液中ns5a的表達水平沒有受到cd2ap下調的影響(右側)。圖17是螢光照片，顯示下調cd2ap減少脂滴的量。將對照nc和cd2ap沉默細胞6#用含有bsa或含有0.5mm油酸-bsa複合物的dmem培養16小時。細胞用中性脂染料進行脂滴染色。下調cd2ap顯著降低由oa刺激的脂滴量。通過volocity軟體分析油酸刺激後單個細胞中脂滴的區域面積，證明對照細胞和cd2ap下調細胞間存在顯著性差異該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。(黑色柱狀圖，p＜0.01)。圖18是螢光照片，顯示過表達cd2ap可以功能性拯救脂滴的生物合成。cd2ap下調細胞(6#)轉染cd2ap拯救突變體(6#-res)或空載體(6#-nc)。然後，用單獨bsa或0.5mm的oa-bsa複合物處理細胞16小時。然後細胞進行脂滴染。通過volocity軟體分析油酸刺激後單個細胞中脂滴的區域面積，證明脂滴的量在cd2ap拯救後基本恢復(黑色柱狀圖，p＜0.05)。圖19是免疫螢光照片，顯示在cd2ap下調細胞(6#)中表達hcv核心蛋白沒有拯救脂滴的積累。ha標籤核心蛋白轉染對照(nc)或cd2ap蛋白下調(6#)細胞，染色脂滴。hcv核心蛋白被抗ha抗體染色(綠色)。脂滴如上染色(紅色)。通過volocity軟體分析油酸刺激後單個細胞中脂滴的區域面積，證明在cd2ap下調細胞中表達核心蛋白沒有拯救脂滴脂滴的量(黑色柱狀圖，p＜0.05)。圖20是免疫螢光照片，顯示在cd2ap拯救細胞(6#-res)和對照細胞(6#-nc)中表達hcv核心蛋白，cd2ap拯救細胞顯示ld的積累比對照細胞顯著增多。核心蛋白的表達並沒有回覆cd2ap下調細胞中脂滴的表達量。通過volocity軟體分析油酸刺激後單個細胞中脂滴的區域面積，證明脂滴的量在cd2ap拯救後基本恢復(黑色柱狀圖，p＜0.05)。圖21是一個柱狀圖，顯示感染後72h，與對照細胞相比，下調cd2ap降低hcvmrna水平。cd2ap下調(4#和6#)或對照(nc)huh7.5.1細胞感染hcvjfh172小時。提取細胞內總rna並進行定量rt-pcr檢測hcvmrna。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)。圖22顯示免疫印跡照片。全細胞裂解物免疫印跡cd2ap，hcvns5a，核心蛋白和β肌動蛋白，表明cd2ap的下調後ns5a和核心蛋白的降低。圖23是一個柱狀圖，顯示下調cd2ap顯著降低上清中hcvrna拷貝數。來自cd2ap下調(4#和6#)或對照(nc)細胞上清中hcvrna拷貝數用rt-pcr定量。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)。圖24是一個柱狀圖，顯示下調cd2ap顯著抑制報告基因的螢光素酶活性。cd2ap下調(4#和6#)或對照(nc)細胞感染了含有renila螢光素酶基因的報告病毒j399em+lm。72小時後檢測螢光素酶活性。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)，n.s.(無顯著差別)。圖25是一個柱狀圖，顯示cd2ap拯救細胞部分恢復細胞內hcvmrna。下調了cd2ap的huh7.5.1細胞(6#)轉染了cd2ap拯救突變體(6#-res)或對照質粒(6#-nc)然後在moi為0.1感染jfh1。感染後72h，相對qrt-pcr分析檢測到細胞內hcvrna水平，cd2ap拯救細胞和對照細胞相比顯著增加。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)，n.s.(無顯著差別)。圖26顯示免疫印跡照片。cd2ap拯救細胞部分恢復hcv蛋白。細胞裂解物來自感染hcvjfh1的cd2ap拯救細胞，對cd2ap,核心蛋白,ns5a進行免疫印跡實驗，顯示部分拯救ns5a和核心蛋白。圖27是一個柱狀圖，顯示與對照細胞(nc)相比，cd2ap下調(4#&6#)不影響hcvpp進入。細胞轉染了hcvpp，螢光素酶活性檢測48小時後進行。cd2ap下調後對hcv進入無顯著影響。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)，n.s.(無顯著差別)。圖28是一個柱狀圖，顯示比較對照細胞(nc)，在複製子細胞con1中的cd2ap下調(4#&6#)沒有減少hcv亞基因組的複製。與對照相比，下調cd2ap顯著降低了cd2apmrna(空盒，p＜0.01)，下調cd2ap沒有減少細胞內hcvrna水平(黑盒)。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)，n.s.(無顯著差別)。圖29是一個柱狀圖，顯示與對照細胞(nc)相比，下調cd2ap(4#和6#)不影響依賴於hcv-ires的翻譯。細胞轉染了phcv-ires。48h後，用雙螢光素酶報告基因檢測系統(promega)測定螢光素酶活性。翻譯效率決定於螢火蟲螢光素酶(f-luc)活性與renilla螢光素酶(r-luc)活性的比例。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)，n.s.(無顯著差別)。圖30是一個柱狀圖，顯示下調cd2ap顯著降低細胞內hcv滴度(p＜0.05)。cd2ap下調(4#&6#)或對照(nc)huh7.5.1細胞感染了j399em，moi為1。收集細胞顆粒，定量測定細胞內病毒滴度。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)，n.s.(無顯著差別)。圖31是一個柱狀圖，顯示下調cd2ap還顯著降低上清液hcv滴度(p＜0.01)。培養上清液72小時後收集，定量釋放到胞外的病毒滴度。該實驗重複3次，定量數據誤差顯示的是平均值±s.e.m，雙尾的學生t檢驗用於統計分析。**(p<0.01)，*(p<0.05)，n.s.(無顯著差別)。圖32所示(a)脂滴(ld)和hcvns5a結合的照片，和(b)一個ns5a陽性ld的柱狀圖。對於(a)，穩定的cd2ap下調(4#和6#)和對照細胞(nc)感染了jfh-1，然後進行免疫螢光染色脂滴(紅色)和hcvns5a(綠色)。核用dapi染色(藍色)。下調cd2ap顯著降低脂滴與hcv蛋白ns5a的共定位。對於(b)，ns5a陽性脂滴的定量顯示，cd2ap的下調顯著降低hcv感染的細胞中ns5a在脂滴上的定位。對照(nc)和cd2ap下調(4#&6#)細胞分別共有161，104，和87的細胞計數。統計分析不同組間差異為*(p＜0.05)，**(p＜0.01)。圖33所示(a)脂滴和hcv核心蛋白結合的照片，和(b)一個核心蛋白陽性ld的柱狀圖。對於(a)，穩定的cd2ap下調(4#和6#)和對照細胞(nc)感染了jfh-1，然後後免疫螢光染色脂滴(紅色)和hcv核心蛋白(綠色)。核用dapi染色(藍色)。下調cd2ap顯著降低脂滴與hcv核心蛋白的共定位。對於(b)，核心蛋白陽性脂脂滴的定量顯示，cd2ap的下調顯著降低hcv感染的細胞中核心蛋白在脂滴上的定位。對照(nc)和cd2ap下調(4#&6#)細胞分別共有161，104，和87的細胞計數。統計分析不同組間差異為*(p＜0.05)，**(p＜0.01)。圖34顯示免疫印跡照片。胰島素受體底物1(irs1)的表達在cd2ap下調細胞中顯著上調。cd2ap下調(4#&6#)和對照(nc)細胞感染了jfh-1。感染後72h，全細胞裂解物進行免疫印跡實驗，針對irs1或胰島素受體(ir)及其磷酸化形式。比較對照細胞，顯著增加的irs1和p-irs1在cd2ap下調細胞中檢測到。比較對照細胞，在cd2ap下調細胞中ir和p-ir有小幅增加。圖35顯示免疫印跡照片。降解irs1是蛋白酶依賴的。mg132處理過的huh7.5.1細胞進行免疫印跡實驗，運用特異性抗體檢測處理後不同時間irs1的表達水平(0，0.25，0.5，1，2，4h10μmmg132處理)。圖36顯示免疫印跡照片。cd2ap下調細胞對蛋白酶體抑制劑處理更不敏感。cd2ap下調(4#&6#)和對照(nc)細胞經dmso(-)或(+)μmmg132處理兩小時。全細胞裂解物irs1的特異性抗體進行免疫印跡實驗。圖37顯示免疫印跡照片。irs1的泛素化在cd2ap下調細胞(4#&6#)中比對照(nc)細胞要少。細胞在完全培養基中培養48小時後收穫。用抗irs1抗體純化細胞裂解物。純化的蛋白為了polyubiquitin和irs1進行免疫印跡實驗。圖38顯示免疫印跡照片。irs1、cd2ap、cbl-b/cbl存在於同一蛋白複合物。huh7.5.1細胞裂解物進行免疫共沉澱反應，抗irs1抗體(左面板)。cd2ap和irs1共純化。huh7.5.1細胞裂解物進行免疫共沉澱反應,抗cbl-b抗體(中面板)或抗cbl抗體(右面板)。irs1與cbl-b/cbl共同純化。圖39顯示在huh7.5.1細胞中irs1、cd2ap、cbl-b/cbl的共定位照片。細胞進行染色，抗irs1抗體(紅色)和抗cd2ap抗體(綠色)。細胞質中觀察到cd2ap和irs1共定位(左面板)。細胞進行染色，抗irs1抗體(紅色)和抗cbl-b抗體(綠色)(中面板)或抗cbl抗體(綠色)(右面板)。細胞質中觀察到irs1和cbl-b或cbl共定位。圖40是免疫印跡照片。huh7.5.1細胞中下調cbl-b或cbl的表達，運用sirna，針對cbl-b或cbl；用相應抗體免疫印跡cbl-b或cbl蛋白。處理後的肌動蛋白印跡作為上樣量對照。2#和3#是cbl-b特異的兩個sirna；1#和4#是cbl特異的兩個sirna。nc是陰性對照sirna。數據顯示，在cbl-b(左面板)或cbl(右面板)下調的huh7.5.1細胞中irs1的水平出現明顯的上升。圖41顯示免疫印跡照片。與對照(nc)細胞相比，akt-ampk-hsl軸線在cd2ap下調細胞(4#&6#)中被活化。總的細胞裂解物用針對ampk信號通路的不同抗體進行了免疫印跡。p-akt(s473)上升，但總akt不上升；p-ampk(t172)下降，但是總ampk不減少；p-hsl(s554)下降，但總hsl不下降；erk或p-erk無變化。圖42顯示免疫跡照片。p-akt的表達水平，與對照細胞相比，在cd2ap拯救細胞中得以恢復。來自cd2ap拯救細胞和對照細胞的裂解物，用抗akt(s473)和抗akt抗體進行了免疫印跡。cd2ap下調細胞比對照細胞表達更多的p-akt。當cd2ap下調細胞中cd2ap被拯救時，cd2ap拯救細胞中的p-akt比對照細胞中更少。圖43顯示免疫印跡照片。dorsomophin(ampk抑制劑)的處理，與對照細胞(nc)相比，cd2ap下調細胞(4#&6#)減少了p-ampk和p-hsl水平。細胞在完全培養基中培養48小時，然後用dmso或dorsomorphin(5μm)處理四小時。全細胞裂解物用特異抗體進行了免疫印跡。圖44是一個曲線圖，顯示hcv感染小鼠模型中肝組織hcv滴度的時間變化過程。感染後不同時間點用qpcr定量肝組織hcv滴度。前兩周為hcv急性感染期而感染兩周後是慢性感染期。圖45是一個曲線圖，顯示hcv感染小鼠模型中血清hcv滴度的時間過程。感染後不同時間點用qpcr定量血清hcv滴度。圖46顯示感染hcv小鼠在感染後不同時間後經cd2ap染色的小鼠肝組織切片的照片。hcv感染誘導cd2ap表達。在感染後1個月，2個月和4個月的肝組織切片中出現cd2ap染色，和脂肪肝的出現相對應。圖47顯示hcv感染和非hcv感染患者的肝組織經ccd2ap染色的照片。非hcv感染患者的肝組織沒有顯示cd2ap染色，但是hcv感染病人的肝組織中顯示cd2ap染色。圖48顯示糖尿病患者的肝組織經ccd2ap染色的照片。全部7個病人在他們的肝組織中顯示cd2ap染色。具體實施方式可以通過引用以下的本發明的某些實施例的詳細描述而更容易地理解本發明。在本申請中，為了更充分地描述本發明所涉及領域狀態，當出版物被引用，這些出版物的公開內容的全部經引用而併入本申請。除非另有說明，本發明的實踐將採用分子生物學(包括重組技術)，微生物學，細胞生物學，生物化學，核酸化學和免疫學的現有技術，這些是在本領域的技能之內。這些技術在文獻中已有完全解釋，如分子克隆：實驗室說明書，第三版(sambrook和russel，2001年)(30)；分子生物學當代程序(fmausubel等主編，1987年)(31)；蛋白分析與純化-實驗技術(rosenberg，1996年)(32)；蛋白分析方法：實驗室程序實用指南(copeland，2013年)(33)；免疫學現有程序(coligan等主編，1999年)(34)。本發明發現hcv裝配需要cd2相關蛋白(cd2ap)參與；cd2ap是一個支撐分子，調節肌動蛋白細胞骨架。cd2ap相互作用於hcv非結構蛋白ns5a，通過依賴肌動蛋白的方式轉運ns5a到細胞機器，然後通過微管依賴的方式靶向到脂滴脂滴。ns5a和cd2ap之間的相互作用需要cd2ap的sh3結構域和ns5a的第三結構域。正常肝細胞不表達cd2ap。在cd2ap表達的細胞中，下調cd2ap表達顯著降低hcv裝配和增殖。cd2ap是一個接頭蛋白，具有三個sh3結構域；其單倍劑量不足是人類腎小球疾病易感性的一個決定因素(35)。cd2ap通過e3連接酶下調細胞表面受體酪氨酸激酶活性(36-39)。此外，cd2ap已被證明正刺激pi3k信號，參與脂質代謝的一個信號通路(40,41)。本發明也發現cd2ap相互作用於irs1。在cd2ap表達的肝細胞中，下調cd2ap增加irs1蛋白的水平。糖尿病病人的肝組織中觀察到cd2ap表達。本發明也發現cd2ap，cbl-b/cbl和irs1共定位於同一蛋白質複合物。cbl-b/cbl是e3連接酶。已知cbl-b/cbl相互作用於cd2ap。本發明發現cbl-b/cbl相互作用於irs1，顯示為cbl-b/cbl與irs1共純化和共定位。當cbl-b/cbl水平用sirna下調，irs1水平上調。在一些實施例中，本發明提供在一個主體中下調cd2ap表達的方法。主體是人或狗。在一些實施例中，優選下調主體肝組織的肝細胞中的cd2ap表達。該下調cd2ap表達的方法包括：給主體注射cd2ap下調配伍，從而下調主體肝組織的cd2ap表達。在一些實施例中，cd2ap下調配伍包括sirna/shrnai多核苷酸，特異針對cd2ap(序列號1(人)和57(狗))，編碼由seqidno2(人)和58(狗)分別代表的胺基酸序列。在一些實施例中，該cd2ap特異的sirna/shrnai多核苷酸與選自序列號3-20(針對人)(表1)或序列號59-76(針對狗)(表3)的核苷酸序列相互補。在一些實施例中，cd2ap下調配伍包括crispr/cas9,crispr/cpf1載體，特異針對主體中的cd2ap。cd2ap特異的crispr/cas9,crispr/cpf1載體包括先導多核苷酸，選自序列號21-56(針對人)(表2)或序列號77-103(針對狗)(表4)。此外，類轉錄活化因子核酸酶(talen)和鋅指核酸酶(zfn)技術也能用於下調cd2ap表達。表1.用於下調人cd2ap表達的sirna/shrnai序列seqidno#核苷酸序列seqidno3gctggaaggagaactaaatggseqidno4ggagaactaaatgggagaagaseqidno5ggacttccagctggaggaattseqidno6ggagctgaaagtgggagatatseqidno7gctgaaagtgggagatattatseqidno8gctgaaagtgggagatattatseqidno9gcccaggacgattcagaaactseqidno10gctgggcctacttcacctataseqidno11gccagtaatttactgagatctseqidno12gcttcatctcactgcaaatagseqidno13ggaagtttccagcagatttcaseqidno14agccgagggtctgggcaaaseqidno15agccgagggtctgggcaaaseqidno16tgaagagactggtaggagaseqidno17ctaaatgggagaagaggaaseqidno18aggatgaactggagctgaaseqidno19ggtaacagatgatggtgaaseqidno20ggaaacagatgatgtgaaa表2.用於下調人cd2ap表達的crispr/cas9,crispr/cpf1靶標序列表3.用於下調狗cd2ap表達的sirna/shrnai序列表4.用於下調狗cd2ap表達的crispr/cas9,crispr/cpf1靶標序列seqidno#核苷酸序列seqidno77aaaggcagacactcaaccgccggseqidno78atgtattgaagtgagacacctggseqidno79atgatgtgggactccatcccaggseqidno80agggcgtgacccccaagtcctggseqidno81tgtattgaagtgagacacctgggseqidno82gggcgtgacccccaagtcctgggseqidno83ccatgcaggaagcatgatgtgggseqidno84ggggtcacgccctgagccaaaggseqidno85tccatgcaggaagcatgatgtggseqidno86attgaagtgagacacctgggtggseqidno87gactccatcccaggacttgggggseqidno88gagtgtctgcctttggctcagggseqidno89tgggactccatcccaggacttggseqidno90agacacctgggtggctccggcggseqidno91tgagtgtctgcctttggctcaggseqidno92ggactccatcccaggacttggggseqidno93gtgacccccaagtcctgggatggseqidno94ggcggttgagtgtctgcctttggseqidno95gtgagacacctgggtggctccggseqidno96cccacatcatgcttcctgcatggseqidno97gggactccatcccaggacttgggseqidno98taacgcaactttctattttttggseqidno99ctcacttcaatacatttttaaggseqidno100ccagttaaaaagaaaatctaaggseqidno101ctcaaccgccggagccacccaggseqidno102taaagcaactttctattttttggseqidno103ccttagattttctttttaactgg在一些實施例中，本發明提供一個藥物配伍，用於下調主體中cd2ap表達。主體是人或狗。在一些實施例中，優選下調主體肝組織的肝細胞中的cd2ap表達。該下調cd2ap表達的方法包括：給主體注射cd2ap下調配伍，從而下調主體肝組織的cd2ap表達。在一些實施例中，cd2ap下調配伍包括sirna/shrnai多核苷酸，特異針對cd2ap(序列號1(人)和57(狗))，編碼由seqidno2和58分別代表的胺基酸序列。在一些實施例中，該cd2ap特異的sirna/shrnai多核苷酸與選自序列號3-20(針對人)(表1)或序列號59-76(針對狗)(表3)的核苷酸序列相互補。在一些實施例中，cd2ap下調配伍包括crispr/cas9,crispr/cpf1載體，特異針對主體中的cd2ap。cd2ap特異的crispr/cas9,crispr/cpf1載體包括先導多核苷酸，選自seq是nos21-56(針對人)(表2)或序列號77-103(針對狗)(表4)。在一些實施例中，本發明提供了一種方法篩選候選藥物，能夠降低cd2ap和hcv非結構蛋白ns5a蛋白之間的相互作用。cd2ap的胺基酸序列為序列號2或其變體，其中變體被定義為一種胺基酸序列，與序列號2有至少80％，更優選90％，或甚至更優選95％的同源性。序列號2由序列號1的核酸序列所編碼，其中cd2ap變體的編碼核酸序列，與序列號1具有至少80％，更優選90％，或甚至更優選95％的同源性。ns5a的胺基酸序列為序列號105或其變體，其中變體被定義為一種胺基酸序列，與序列號105有至少80％，更優選90％，或甚至更優選95％的同源性。序列號105是由序列號104的核酸序列所編碼，其中ns5a變體的編碼核酸序列，與序列號104具有至少80％，更優選90％，或甚至更優選95％的同源性。該方法包括提供表達cd2ap和ns5a細胞，用候選藥物處理cd2ap和ns5a表達細胞，然後檢測候選藥物對cd2ap和ns5a相互作用的影響；如果候選藥物減少了cd2ap和ns5a相互作用而達到一個預定的閾值，選定候選藥物。表達cd2ap和ns5a細胞可以是任何合適的原代細胞或細胞系。在一些實施例中，合適的細胞是這樣的一些細胞系，自身表達cd2ap，而ns5a的表達可以由轉染ns5a表達載體；細胞系優選是肝癌細胞系。在一些實施例中，合適的細胞是hcv感染的肝細胞。測定cd2ap和ns5a的相互作用，是任何合適的一個可以測量或確定cd2ap和ns5a之間的相互作用的方法。在一些實施例中，該法是免疫共沉澱，共定位，cd2ap和ns5a聯動的聚焦活細胞成像；如何進行這些實驗是公知的；因此，細節不在此贅述。確定候選藥物是否有效降低cd2ap和ns5a相互作用的預定閾值定義為降低至少70％，更優選80％，cd2ap和ns5a蛋白之間的相互作用。例如，在免疫共沉澱實驗，預定閾值是，候選藥物處理過的細胞中，cd2ap或ns5a的免疫共沉澱量，與沒有經過候選藥物處理的細胞相比，減少至少70％，更優選80％。在一些實施例中，本發明提供了減少cd2ap和ns5a相互作用的藥物配伍。在一些實施例中，該藥物配伍包含一種多肽，具有5-40胺基酸，優選10-30個胺基酸，更優選15-25個胺基酸，其多肽是序列號2的胺基酸3-58，111-165和271-327，和序列號105的胺基酸353-466的衍生物。一個衍生物定義為一個多肽，與相應系列具有至少80％，優選90％，或更優選95％同源性。在一些實施例中，本發明提供了一種方法篩選候選藥物，能夠降低cd2ap和irs1之間的相互作用。cd2ap的胺基酸序列為序列號2或58或其變體，其中變體被定義為一種胺基酸序列，與序列號2或58有至少80％，更優選90％，或甚至更優選95％的同源性。序列號2或58由序列號1或57的核酸序列所編碼，其中cd2ap變體的編碼核酸序列，與序列號1或57具有至少80％，更優選90％，或甚至更優選95％的同源性。irs1的胺基酸序列為序列號107(針對人)或109針對狗或其變體，其中變體被定義為一種胺基酸序列，與序列號107或109有至少80％，更優選90％，或甚至更優選95％的同源性。序列號107或109是分別由序列號107或108的核酸序列所編碼，其中irs1變體的編碼核酸序列，與序列號106或108具有至少80％，更優選90％，或甚至更優選95％的同源性。該方法包括提供表達cd2ap和irs1細胞，用候選藥物處理cd2ap和irs1表達細胞，然後檢測候選藥物對cd2ap和irs1相互作用的影響；如果候選藥物減少了cd2ap和irs1相互作用而達到一個預定的閾值，選定候選藥物。表達cd2ap和irs1細胞可以是任何合適的原代細胞或細胞系。在一些實施例中，合適的細胞是表達cd2ap和irs1的細胞系。測定cd2ap和irs1的相互作用，是任何合適的一個可以測量或確定cd2ap和irs1之間的相互作用的方法。在一些實施例中，該法是免疫共沉澱，共定位；如何進行這些實驗是公知的；因此，細節不在此贅述。確定候選藥物是否有效降低cd2ap和irs1相互作用的預定閾值定義為降低至少70％，更優選80％，cd2ap和irs1蛋白之間的相互作用。例如，在免疫共沉澱實驗，預定閾值是，候選藥物處理過的細胞中，cd2ap或irs1的免疫共沉澱量，與沒有經過候選藥物處理的細胞相比，減少至少70％，更優選80％。在一些實施例中，本發明提供了減少cd2ap和irs1相互作用的藥物配伍。在一些實施例中，該藥物配伍包含一種多肽，具有5-40胺基酸，優選10-30個胺基酸，更優選15-25個胺基酸，其多肽是序列號2或58的胺基酸3-58，111-165和271-327的衍生物。一個衍生物定義為一個多肽，與相應系列具有至少80％，優選90％，或更優選95％同源性。在一些實施例中，本發明提供了一種方法篩選候選藥物，能夠降低cbl-b/cbl和irs1之間的相互作用。cbl-b的胺基酸序列為序列號111或160，或其變體，其中變體被定義為一種胺基酸序列，與序列號111或160有至少80％，更優選90％，或甚至更優選95％的同源性。序列號111和160由序列號110和159的核酸序列所分別編碼，其中cbl-b變體的編碼核酸序列，與序列號110或159具有至少80％，更優選90％，或甚至更優選95％的同源性。cbl的胺基酸序列為序列號194和245或其變體，其中變體被定義為一種胺基酸序列，與序列號194或245有至少80％，更優選90％，或甚至更優選95％的同源性。序列號194和245由序列號193和244的核酸序列所編碼，其中cbl變體的編碼核酸序列，與序列號193或244具有至少80％，更優選90％，或甚至更優選95％的同源性。irs1的胺基酸序列為序列號107或109，或其變體，其中變體被定義為一種胺基酸序列，與序列號107或109有至少80％，更優選90％，或甚至更優選95％的同源性。序列號107或109是由序列號106或108的核酸序列所分別編碼，其中irs1變體的編碼核酸序列，與序列號106或108具有至少80％，更優選90％，或甚至更優選95％的同源性。該方法包括提供表達cbl-b/cbl和irs1細胞，用候選藥物處理cbl-b/cbl和irs1表達細胞，然後檢測候選藥物對cbl-b/cbl和irs1相互作用的影響；如果候選藥物減少了cbl-b/cbl和irs1相互作用而達到一個預定的閾值，選定候選藥物。表達cbl-b/cbl和irs1細胞可以是任何合適的原代細胞或細胞系。在一些實施例中，合適的細胞是表達cbl-b/cbl和irs1的細胞系。測定cbl-b/cbl和irs1的相互作用，是任何合適的一個可以測量或確定cbl-b/cbl和irs1之間的相互作用的方法。在一些實施例中，該法是免疫共沉澱，共定位；如何進行這些實驗是公知的；因此，細節不在此贅述。確定候選藥物是否有效降低cbl-b/cbl和irs1相互作用的預定閾值定義為降低至少70％，更優選80％，cbl-b/cbl和irs1蛋白之間的相互作用。例如，在免疫共沉澱實驗，預定閾值是，候選藥物處理過的細胞中，cbl-b/cbl或irs1的免疫共沉澱量，與沒有經過候選藥物處理的細胞相比，減少至少70％，更優選80％。在一些實施例中，本發明提供在一個主體中下調cbl-b/cbl表達的方法。主體是人或狗。在一些實施例中，優選下調主體肝組織的肝細胞中的cbl-b/cbl表達。該下調cbl-b/cbl表達的方法包括：給主體注射cbl-b/cbl下調配伍，從而下調主體肝組織的cbl-b/cbl表達。在一些實施例中，cbl-b/cbl下調配伍包括sirna/shrnai多核苷酸，特異針對cbl-b/cbl(序列號110或159或序列號193或244)，編碼由seqidno111或160或序列號110或245分別代表的胺基酸序列。在一些實施例中，該cbl-b/cbl特異的sirna/shrnai多核苷酸與選自序列號112-124(表5)或161-170(表7)和序列號195-208(表9)或246-255(表11)的核苷酸序列互補。在一些實施例中，cbl-b/cbl下調配伍包括crispr/cas9,crispr/cpf1載體，特異針對主體中的cbl-b/cbl。cbl-b/cbl特異的crispr/cas9,crispr/cpf1載體包括先導多核苷酸，選自序列號125-158(表6)或171-192(表8)和序列號209-243(表10)或256-280(表12)。此外，類轉錄活化因子核酸酶(talen)和鋅指核酸酶(zfn)技術也能用於下調cbl-b/cbl表達。表5.用於下調人cbl-b表達的sirna/shrnai序列seqidno#nucleiacidsequenceseqidno112gcctgatacatatcagcatseqidno113gcggaattggaatttcttaseqidno114gcatgccgatgctagacttseqidno115gcctgatacatatcagcatseqidno116ggagagaatgtatgaagaacaseqidno117gcggaattggaatttcttagcseqidno118gcacgactacagaaatatagcseqidno119ggaatatcttacagaccatacseqidno120gcaccaaacccggaagctataseqidno121gcctggatctaattcagaaagseqidno122ggaatcacagcgagttcaaatseqidno123ggaacacatggtccatcttcaseqidno124gcatagtctcattgaacattc表6.用於下調人cbl-b表達的crispr/cas9,crispr/cpf1靶標序列表7.用於下調狗cbl-b表達的sirna/shrnai序列seqidno#nucleiacidsequenceseqidno161cccaccatatatacttgatseqidno162cctgatacatatcagcattseqidno163gcgggcaataagactctttseqidno164gcagaaatacagcaccaaaseqidno165gcaccaaacctggaagctaseqidno166gcaatatcttacagaccatseqidno167ccacaccacatgaccatatseqidno168gcctcctcccttaagagatseqidno169ccttcatcccatcctgtttseqidno170cctctgatccagtgccatt表8.用於下調狗cbl-b表達的crispr/cas9,crispr/cpf1靶標序列表9.用於下調人cbl表達的sirna/shrnai序列seqidno#nucleiacidsequenceseqidno195ccagacaatccctcacaatseqidno196ggacacctcatgtgcacatseqidno197ccaggcctctacggcctttseqidno198ccagaaagctttggtcattseqidno199gcctgattgggctcatgaaggseqidno200gggaacattctccagacaatcseqidno201gcttcagggaaggcttctattseqidno202gggaaggcttctatttgtttcseqidno203ggacacctcatgtgcacatccseqidno204gcagaatcccgacctcaaagaseqidno205ggagcaatgtgagggtgaagaseqidno206gcctctacggcctttggatacseqidno207gctgtacgtatgaagcaatgtseqidno208ggtactcctaccaggacatcc表10.用於下調人cbl表達的crispr/cas9,crispr/cpf1靶標序列表11.用於下調狗cbl表達的sirna/shrnai序列seqidno#nucleiacidsequenceseqidno246ccagaagttcattcacaaaseqidno247ggaacatcctccagacgatseqidno248ccagacgatccctcacaatseqidno249gcttcagggaaggcttctaseqidno250gcaggaatcagaaggccaaseqidno251cctttctgccgatgtgaaaseqidno252gctgatgattctctctttaseqidno253gcttctggctcccttcataseqidno254gcatctgccaatgccatttseqidno255gctgcacatatgaagcaat表12.用於下調狗cbl表達的crispr/cas9,crispr/cpf1靶標序列seqidno#nucleiacidsequenceseqidno256cccggagccgccgccgcccccggseqidno257tgccgggcgggtgggggctgaggseqidno258cggcctcatcgggctcatgaaggseqidno259ggagctcttcttcacgttgccggseqidno260caacgtgaagaagagctccggggseqidno261ggggctcgggcggcctcatcgggseqidno262ggcaacgtgaagaagagctccggseqidno263gcaacgtgaagaagagctccgggseqidno264gggggctcgggcggcctcatcggseqidno265gtgaagaagagctccggggccggseqidno266tgaagaagagctccggggccgggseqidno267cgtccttcatgagcccgatgaggseqidno268aagaagagctccggggccgggggseqidno269gaagaagagctccggggccggggseqidno270gatgaggccgcccgagcccccggseqidno271gtggtggtggtgcggctggaaggseqidno272aagagctccggggccgggggcggseqidno273cacctcagcccccacccgcccggseqidno274cggcggcggctccgggggctcggseqidno275agctccggggccgggggcggcggseqidno276gcgggtgggggctgaggtggtggseqidno277tccggggccgggggcggcggcggseqidno278gccgccgccgcccccggccccggseqidno279cgggcgggtgggggctgaggtggseqidno280gccgggggcggcggcggctccgg在一些實施例中，本發明提供cbl-b/cbl下調配伍，用於下調主體中cbl-b/cbl表達。主體是人或狗。在一些實施例中，優選下調主體肝組織的肝細胞中的cbl-b/cbl表達。該下調cbl-b/cbl表達的方法包括：給主體注射cbl-b/cbl下調配伍，從而下調主體肝組織的cbl-b/cbl表達。在一些實施例中，cbl-b/cbl下調配伍包括sirna/shrnai多核苷酸，特異針對cbl-b/cbl(序列號110或159或序列號193或244)，編碼由序列號111或169或序列號194或245分別代表的胺基酸序列。在一些實施例中，該cbl-b/cbl特異的sirna/shrnai多核苷酸與選自序列號112-124(表5)或161-170(表7)和序列號195-208(表9)或246-255(表11)的核苷酸序列互補。在一些實施例中，cbl-b/cbl下調配伍包括crispr/cas9,crispr/cpf1載體，特異針對主體中的cbl-b/cbl。cbl-b/cbl特異的crispr/cas9,crispr/cpf1載體包括先導多核苷酸，選自序列號125-158(表6)或171-192(表8)和序列號209-243(表10)或256-280(表12)。在一些實施例中，本發明提供了主體hcv感染的治療方法。在一些實施例中，主體是人。在一些實施例中，治療是通過施用配伍特異性下調主體肝組織的肝細胞中cd2ap的表達，其配伍包含至少一個與選自由序列號3-20或59-76所代表序列互補的sirna/shrnai核苷酸序列。在一些實施例中，治療是通過施用crispr/cas9,crispr/cpf1載體特異性下調主體肝組織的肝細胞中cd2ap表達，其crispr/cas9,crispr/cpf1載體包括引導序列，選自由序列號21-56或77-103。在一些實施例中，治療是通過施用含有能降低cd2ap和ns5a之間相互作用的藥物而特異性下降低在主體肝組織的肝細胞中的cd2ap和ns5a之間的相互作用。在一些實施例中，本發明提供了主體糖尿病的治療方法。在一些實施例中，主體是人和狗。在一些實施例中，治療是通過施用配伍特異性下調主體肝組織的肝細胞中cd2ap的表達，其配伍包含至少一個與選自由序列號3-20或59-76所代表序列互補的sirna/shrnai核苷酸序列。在一些實施例中，治療是通過施用crispr/cas9,crispr/cpf1載體特異性下調主體肝組織的肝細胞中cd2ap表達，其crispr/cas9,crispr/cpf1載體包括引導序列，其引導序列選自序列號21-56或77-103。在一些實施例中，治療是通過施用含有此前所描述的能降低cd2ap和irs1之間相互作用的藥物而特異性下降低在主體肝組織的肝細胞中的cd2ap和irs1之間的相互作用。在一些實施例中，本發明提供了主體糖尿病的治療方法。在一些實施例中，主體是人和狗。在一些實施例中，治療是通過施用配伍特異性下調主體肝組織的肝細胞中cbl-b/cbl的表達，其配伍包含至少一個與選自由序列號112-124或161-170和序列號195-208或246-255所代表序列互補的sirna/shrnai核苷酸序列。在一些實施例中，治療是通過施用crispr/cas9,crispr/cpf1載體特異性下調主體肝組織的肝細胞中cbl-b/cbl表達，其crispr/cas9,crispr/cpf1載體包括引導序列，其引導序列選自序列號125-158或171-192和序列號209-243或256-280。在一些實施例中，治療是通過施用含有此前所描述的能降低cbl-b/cbl和irs1之間相互作用的藥物而特異性下降低在主體肝組織的肝細胞中的cbl-b/cbl和irs1之間的相互作用。在一些實施例中，本發明提供肝臟異化診斷方法。所述診斷方法包括提供來自個體的肝臟樣品，讓肝臟樣品與檢測cd2ap表達的檢測試劑接觸；因而，當在肝臟樣品中檢測到cd2ap表達時，提示肝臟異化。所述異化包括hcv感染和糖尿病。檢測cd2ap表達的方法可以是任一合適方法，包括pcr和免疫染色。在一些實施例中，本發明提供個體肝臟異化診斷試劑盒。所述試劑盒包括針對cd2ap蛋白的特異抗體或針對cd2apmrna的特異多聚核苷酸探針，和一個次級試劑，能夠檢測與cd2ap結合的抗體或來自cd2apmrna的信號。提供下面實施例的唯一目的是說明本發明的原理；它們決不旨在限制或縮小本發明的範圍。實施例1.材料與方法1.1細胞系和病毒人肝癌細胞huh7，其衍生的huh7-lunet和huh7.5.1細胞，和hek293t細胞，在含有5％co2的潮溼環境下，培養於dulbecco改良的eagle培養基(dmem)(gibco，#11965-092，美國)，補充3.17g/l碳酸氫鈉、10％胎牛血清(gibco，#10099-141)，3g/lhepes，100u/ml的青黴素和鏈黴素。攜帶hcv1b亞基因組hcv複製子pfki389neo/ns3-3』的con1細胞，衍生自huh7-lunet，保持在與huh7-lunet細胞相同的培養基，添加0.5mg/mlg418(默克，345810)(42)。感染性hcvjfh1病毒含有hcv基因型2a株基因組全長cdna序列(43)。hcvj399em病毒衍生自jfh-1病毒，在ns5a的399個胺基酸後面插入egfp基因，引入五個適應突變到jfh1基因組，增強病毒生產能力(44)。jfh1luc報告病毒由在武漢病毒研究所陳緒林教授提供(45)。為了產生病毒備份，原始hcv病毒用dmem稀釋，接種到huh7.5.1細胞，感染複數(moi)為0.1。被感染的細胞在72hpi傳代一次。然後，在感染後7或8天收集上清液，分裝並存儲在80℃.1.2質粒構建與試劑人源cd2ap(genbank#nm_012120)(序列號1；序列號2為胺基酸序列)和來自hcv基因型2a(ab047639jfh1)的ns5a(序列號1；序列號2為胺基酸序列)分別用相應的引物克隆到真核表達載體質粒prk-7ha和prk-7flag(addgene)。取自感染了hcvjfh1的huh7.5.1細胞和未感染的huh7.5.1細胞的總rna作為模板。用全長ns5a和cd2ap作為模板，通過聚合酶鏈反應(pcr)擴增截短ns5a和cd2ap。哺乳動物細胞表達質粒pcdna3.1bira(r118g)-ha(bira*)購自addgene。hcvns5a亞克隆到bira*的n端。整個ns5a-bira*-ha序列被用限制性內切酶salⅰ和notⅰ從pcdna3.1中切除並插入到phage。抗flag,ha,或β-actin小鼠單克隆抗體(mab)購自天津三江生物(天津，中國)；抗hcv核心蛋白小鼠多克隆抗體和抗cd2ap兔多克隆抗體(h-290)購自聖克魯斯生物技術購買；抗hcv2ans5a單克隆抗體(7b5和2f6)購自biofront；抗ns5a單克隆抗體9e10是由charlesrice教授提供(洛克菲勒大學)(46)。抗phospho(p)-akt(ser473)(4060),akt(4691),p-erk(9106s),erk(4695p)andpi3k-aktinhibitorly294002(9901)的兔單克隆抗體購自細胞信號技術(麻薩諸塞州、美國)；抗adrp(ab52355)的兔多克隆抗體抗adrp(ab52355)購自abcam；兔抗calnexin(rlt0613)多克隆抗體購自ruiyingbio(蘇州，中國)；hcslipidtox深紅色中性脂質染色和alexafluor標記的二抗購自invitrogen公司(carlsbad，美國)；辣根過氧化酶(hrp)標記的二抗購自antgene生物科技(武漢，中國)；小鼠igg1同型對照和hrp-鏈黴親和素蛋白購自biolegend(聖地牙哥，美國)；4』,6-diamidine-2』-phenylindoledihydro-chloride(dapi)購自羅氏(曼海姆，德國)。所有其他試劑均購自amresco(俄亥俄，美國)。1.3細胞裂解物製備和免疫印跡(wb)用冰冷的磷酸鹽緩衝液(pbs)輕輕地衝洗細胞，然後溶於裂解液(20mmtris-hcl(ph7.5)，150mmnacl,1mmedta,1mmegta、1％tritonx-100，2.5mm焦磷酸鈉，1mmβ-磷酸甘油，1mmna3vo4，1mmpmsf)(li等，2009)。bca法測定蛋白濃度。用10％sds-page電泳分開蛋白，然後轉移到硝酸纖維素膜(#9004700、billerica,美國)。用含有5％脫脂牛奶的tbst(tris緩衝生理鹽水(tbs)加上0.1％tween-20))封閉，分開的蛋白用特定的初級抗體，辣根過氧化物酶標記的第二抗體來探測。1.4免疫共沉澱(co-ip)對於外源表達質粒的co-ip，首先使用磷酸鈣法將質粒轉染到10cm培養皿中的hek293t細胞中。36小時後，用1ml裂解液將細胞裂解。將等體積的裂解液與2μg特異性的抗體或同種型對照抗體和20μlproteing瓊脂糖珠(16-266，merckmillipore)進行孵育，4℃旋轉4小時。用細胞裂解液將珠子洗滌6次後，加20μl2×sds上樣緩衝液中煮沸，用10％sds-page膠分離蛋白質。對於內源性的co-ip實驗，將hcvjfh1感染72小時或者未感染的huh7.5.1細胞直接裂解。利用特異性的抗體進行上面所述的co-ip。1.5免疫螢光染色在20mm玻璃共聚焦皿(nest)上培養細胞。感染後72小時，然後將細胞固定在4％(w/v)多聚甲醛(pfa)，室溫(rt)15分鐘。在用含有10％山羊血清加1％的bsa的pbst封閉細胞後，細胞在含有指明的初級抗體的封閉緩衝液中孵育。結合的抗體用alexafluor標記的二抗來檢測。細胞核用dapi復染。脂滴的染色用hcslipidtox深紅中性脂肪染色劑。添加抗猝滅螢光介質後，用共聚焦顯微鏡拍攝照片(perkinelmerultraviewvox共聚焦顯微鏡)。1.6rna提取和實時定量rt-pcr(qpcr)培養的細胞和培養的上清液中的總rna的提取，按照製造商的說明分別採用rna純組織試劑盒和rna純病毒試劑盒(cwbiotech，北京)。使用primescriptrt試劑盒(takarabio，日本)從1微克總rna合成cdna第一條鏈。在bio-radconnecttmqpcr儀器(cfx連接tm光學模塊)上，使用sybrgreensupermix(bio-rad，美國)定量rna。胞內hcvrna和細胞rna的量相對於gapdhrna水平進行歸化。由系列稀釋的hcvjfh-1cdna質粒製備標準曲線，參照標準曲線測定培養上清液中的hcvrna水平。1.7逆轉錄病毒的製備與轉導為了建立一個穩定的表達下調的細胞系，根據製造商的說明，將幹擾靶標基因的短髮夾rna(shrna)亞克隆到psuperretropuro質粒(oligoengine)。水皰性口炎病毒糖蛋白(vsv-g)-假型逆轉錄病毒顆粒，在293t細胞中採用磷酸鈣法製備。簡而言之，由psuperretropuro構件和包裝質粒pgag-pol和pvsv-g共轉染hek293t細胞。shrna逆轉錄病毒的備份與7.5微克/毫升聚凝胺轉導huh7.5.1細胞。表達下調細胞用2毫克/毫升嘌呤黴素(amersco)選擇至少7天。存活克隆的幹擾效果是由qpcr或免疫印跡(wb)分析證實。針對cd2apmrna的sirna/shrna序列列於表1。1.8在cd2ap下調細胞中功能拯救cd2ap為了功能性的拯救cd2ap幹擾，cd2ap下調細胞huh7.5.1(shcd2ap-6#)瞬時轉染慢病毒載體phage，表達外源擺動突變ha-cd2ap基因(sh-cd2ap6#-ha-cd2ap)，其中靶標cd2ap序列ggaaacagatgatgtgaaa(序列號1的2175-2193)變成ggagacggacgacgtaaag(序列號281)。慢病毒製備參考楊等人的文章(48)。含有空載體的慢病毒顆粒轉染shcd2ap-6#細胞作為對照。1.9親和捕獲生物素化蛋白質生物素化蛋白質在4℃進行分離，使用加以修改的以前程序(49)。簡而言之，huh7細胞穩定表達ns5abira*，在添加了50μm生物素的完全培養基中孵育24小時。五個10cm細胞培養板上長滿細胞，用上述細胞裂解液裂解。生物素化蛋白與100μl鏈黴親和素瓊脂糖珠4℃震蕩過夜後析出。然後深度洗淨鏈黴親和素瓊脂糖珠(49)。ns5a的相互作用蛋白，經由質譜分析和免疫印跡法證實。1.10hcvpp進入與hcvires依賴性翻譯實驗hcvpp的製備參照文獻中的方法(50)。將質粒pnl4.3.lucre和pcdna3.1-e1e2按照3:1比例共轉hek293t細胞。48小時後，收集培養上清，1000g離心後通過0.45μm過濾器(merckmillipore)過濾，長期使用可分裝儲存在-80℃。對於hcvpp進入效率測定，將cd2ap沉默和對照huh7.5.1細胞鋪在24孔板中，細胞密度約30％，過夜培養後加入250μlhcvpp，6h後換成新鮮培養基。48h後，用試劑盒(e1500，promega)檢測螢火蟲螢光素酶活性。對於hcvires介導的翻譯實驗：用lipofectamine2000將phcv-ires質粒轉染到cd2ap沉默和對照huh7.5.1細胞中。48小時後，用雙螢光素酶試劑盒(e1910，promega)檢測f-luc和r-luc活性，二者的比值可以代表翻譯效率(f-luc/r-luc)。1.11分離脂滴脂滴富集部分由密度梯度離心製備(51)。簡而言之，約95％融合度的細胞，在pbs中收刮，通過離心分離沉澱，1000xg，5分鐘，然後溶解於1ml低滲緩衝液(50mmhepes,1mmedta和2mmmgcl2atph7.4,1mmpmsf和混合蛋白酶抑制劑)。孵育懸浮混合物，4℃，20分鐘，超聲處理20下。細胞核通過離心去除，1000xg，4℃，5分鐘。收集1ml上清液，混合等體積的1.5m蔗糖等滲緩衝液(50mmhepes,100mmkcl,2mmmgcl2)，放置在sw55ti(beckman)離心管的底部，然後在混合物上放置3ml含有1mmpmsf等滲緩衝液。離心樣品，10000g，4℃，2小時。收集頂層ld組份，用10％的三氯乙酸沉澱(tca)，用乙醚：乙醇(1:1)洗一次，在2×sds上樣緩衝液中煮沸，sds-page分離。1.12oa刺激為了確定oa刺激引起的脂滴脂滴積累，1.5×105cells接種到共聚焦皿，完全培養基中培養16小時。然後培養細胞12小時，其無血清dmem培養液含0.5mmoa和2％bsa(w/v)；然後ld染色。1.13C型肝炎病毒滴定用1個moi的j399em病毒感染目的細胞。72h後，收集培養基中的胞外病毒。胞內病毒的收集過程如下：用pbs清洗細胞以除去殘留的細胞外病毒，將細胞在pbs中刮下來，1000g離心5min；將細胞沉澱重新懸浮於基礎dmem中，並在液氮中進行三輪反覆凍融以釋放細胞內的病毒顆粒；1000g離心5min除去細胞碎片得到細胞內的病毒。用lindenbach等描述的終點稀釋法測定病毒的滴度(52)。將胞外病毒和胞內病毒用dmem培養基進行10倍系列稀釋。用稀釋的病毒樣本感染96孔板中的huh7.5.1細胞(每個稀釋度6個復孔)。感染96h後，在螢光顯微鏡下觀察每個稀釋度中egfp陽性的孔數，根據reedandmuench公式計算病毒滴度(52)。1.14細胞活力檢測通過mts(g3581，promega)測定cd2ap沉默細胞的活力。將待測細胞鋪在96孔板中，每孔5×103個細胞。37℃培養箱中分別培養24h，48h，72h和96h。在相應的時間點每孔加入20μlmts試劑，37℃孵育1小時。讀板儀(perkinelmer，usa)測量490nm處的吸光值。1.15統計分析所有實驗至少重複3次，使用graphpadprism軟體作圖。實驗結果用雙尾的學生t檢驗統計分析。定量數據誤差顯示的是平均值±標準誤(mean±standarderror，sem)。p<0.05視為有顯著性差異，*(p<0.05)；p<0.01視為有極顯著性差異，**(p<0.01)；ns表示統計學上無顯著性差異。irs1泛素化實驗將cd2ap沉默和對照細胞在完全培養基中培養48小時，隨後進行裂解。將細胞裂解液(1ml)分別與2μg兔源的irs1抗體以及30μlproteing瓊脂糖珠在4℃孵育4小時。經過漂洗後，將珠子在30μl2×sds上樣緩衝液中煮沸，得到蛋白樣品。分別取分裝的10μl樣品進行跑膠，檢測從cd2ap沉默和對照細胞中純化的irs1的量。將二者純化的irs1的總蛋白量調節一致，從而定量irs1的泛素化水平。胰島素信號通路實驗胰島素信號通路的分子用相應的的抗體來檢測，檢測細胞裂解物中這些分子，而這些裂解物來自對照和cd2ap下調細胞huh7.5.1。rna幹擾huh7.5.1細胞接種為50％融合度，然後用cbl-b，cbl特異的小幹擾rnas(sirnas)或陰性對照sirna轉染細胞。sirna的轉染使用pepmute試劑，遵照製造商的說明(signagen，美國)。基因沉默在轉染後48小時測量。cbl-b，cbl特異的小幹擾rnas系列列於表3和5。對irs1的影響用irs1,cbl-b,cbl和肌動蛋白的特異抗體來檢測。免疫組化(ihc)為了對hcv感染小鼠的cd2ap染色，在指定的時間，收集hcv感染或模擬感染的小鼠的肝組織右頁，進行切片，厚度為5微米。為了對來自於感染或未感染hcv的病人的肝活檢標本的cd2ap染色，組織切片，厚度5微米。組織切片加熱，65℃，1小時。經過除蠟，脫水和3％過氧化氫處理10分鐘後，進行抗原回收。切片浸入10mm檸檬酸鈉緩衝液(ph6.0)，95-100℃加熱，30分鐘；然後在緩衝液中冷卻至室溫。然後封閉切片，使用正常羊血清，稀釋在0.02％pbst，室溫，1小時。切片孵育於兔抗cd2ap抗體(genetex，美國)或同型對照兔igg，室溫，1小時。hrp耦合的羊抗兔二抗用於檢測結合的初級抗體，室溫，1小時。遵照產品說明，用dab配伍顯色(maxim，中國)。切片用hematoxylin負染2分鐘。經脫水和加蓋玻片後，切片照相，使用pannormic數字切片掃描儀(3dhistech，匈牙利)。肝切片的使用經過中國科學院武漢病毒研究所審查委員會批准。批准號：wivh28201601。2.結果2.1用bioid方法在huh7細胞中發現全新ns5a相關的宿主蛋白huh7細胞轉染bioid構件ns5a-bira*-ha，在50μm外源生物素存在下培養，標記與ns5a密切相關的蛋白(圖1)。然後用鏈黴親和素-hrp檢測被外源生物素標記的細胞蛋白。與沒有生物素的細胞相比，在生物素存在下，觀察到生物素標記蛋白質的增加(圖2)。對於圖2，ns5a-bira-ha構件轉染huh7細胞。在免疫檢測ns5a或ha標籤後，確認構件的表達。然後將細胞分裂成兩份，一份經50μm生物素處理，另一份沒有處理。然後細胞裂解物進行sds-page。用鏈黴親和素-hrp進行免疫印跡，檢測生物素化的細胞蛋白。我們發現的是，與沒有生物素處理的細胞相比，生物素處理後，更多的蛋白質被生物素標記。為確定生物素標記的宿主蛋白，鏈黴親和素純化的蛋白質進行分離與考馬斯亮藍亮藍染色(圖3)。對七個特定的條帶進行質譜分析，並揭示了這些細胞蛋白的身份。有趣的是，這些蛋白質要麼與運輸胞器，如copg2,cd2ap,golga5和pace1，或者與rna生物學，如rpa34,ef2p和np1l1，相關。我們首先集中在研究cd2ap，其是一個接頭蛋白，含有sh3域，最早被發現與cd2胞內域結合(53)。cd2ap還結合肌動蛋白帽蛋白(cp)，具有高親和力，降低肌動蛋白聚合的速率(54,55)，從而在肌動蛋白纖維組織中起到了重要的作用。2.2hcvns5a蛋白與cd2ap相互作用hcv非結構蛋白ns5a具有多個富含脯氨酸的序列，特異性地結合生長因子受體結合蛋白2(grb2)接頭蛋白，其含有sh3結構域(56,57)。由於cd2ap有三個sh3結構域(35)，我們直接測試cd2ap是否確實結合ns5a。當ha標記的cd2ap與flag標籤的ns5a在hek293t細胞內過度表達，我們發現，由ns5a可以特異性地沉澱cd2ap(圖4)。為確定在hcv感染期間ns5a是否結合cd2ap，我們對hcvjfh1感染的huh7.5.1細胞或沒有感染的huh7.5.1細胞進行了免疫共沉澱(co-ip)分析。我們發現，在受感染的細胞中，抗cd2ap抗體確實與ns5a免疫共沉澱。同時，cd2ap也可以被ns5a特異性抗體沉澱(圖5)。為了進一步證明在受感染huh7.5.1細胞中cd2ap與ns5a相互作用，我們採用鼠igg亞型抗體作為ns5a抗體的對照，進行免疫共沉澱實驗。我們發現，在hcv感染的細胞中，cd2ap確實結合ns5a(圖6)。此外，通過共聚焦成像分析，我們觀察到,在受hcv-j399em感染的細胞中，cd2ap和ns5a共定位，hcv-j399em是hcv2a株，其中ns5a帶有gfp標記(圖7)。另外，我們在受jfh1感染的huh7.5.1細胞中，雙染cd2ap和ns5a，發現cd2ap和ns5a共定位(結果未顯示)。綜上所述，這些結果表明，在hcv感染細胞中cd2ap與內源性ns5a相互作用。2.3ns5a的結構域iii與cd2apsh3結構域相互作用由於cd2ap包含三個sh3結構域，我們進一步實驗確定,在cd2ap中是一個特定的sh3結構域還是所有三個sh3結構域負責結合ns5a。我們製備了各種cd2ap的截短突變體，編碼1-107aa,1-268aa,1-330aa,331-639aa,60-639aa,and1-639aa(按照序列號2)，它分別包含第一，第二和第三，所有三個sh3結構域，沒有sh3結構域但保留cd2ap的所有其他域，沒有第一個sh3結構域但保留cd2ap的所有其他域，和全長cd2ap(圖8)。我們然後在hek293t細胞中與ha標記的全長ns5a一起共表達這些cd2ap蛋白，進行免疫共沉澱實驗。如圖9所示，缺失sh3結構域的cd2ap突變體不與ns5a相互作用(圖9，參見331-639)。相反地，全長cd2ap或包含sh3結構域的cd2ap蛋白結合ns5a；當cd2a的突變體保留更多的sh3結構域時，結合被增強(圖9，比較1-107,1-268,和1-330)。第二和第三的sh3結構域參與ns5a結合，由下面的觀察進一步證實，沒有第一個sh3結構域的cd2ap仍然結合ns5a(圖9，參見60-639)。我們也鑑定了ns5a中參與和cd2ap結合的區域。ns5a包含n-末端的兩親性螺旋，錨定蛋白到胞質膜，和三個結構域(結構域i，結構域ii和結構域iii)，被兩個低複雜度序列(lcs)分開(58,59)。我們製備全長ns5a和一系列分別缺乏結構域i，ii，iii的ns5a突變體(圖10)，研究哪些結構域結合cd2ap。我們發現，刪除ns5a的第三個結構域，cd2ap無法結合ns5a(圖11)。然而，刪除其他ns5a結構域，不影響ns5a結合cd2ap，從而提示，ns5a的第三結構域和cd2ap的sh3結構域相互作用。2.4在靶向脂滴前cd2ap通過肌動蛋白依賴的方式運送ns5a為了探討cd2ap與ns5a相互作用的功能，huh7.5.1細胞穩定表達mcherry標記的全長cd2ap或缺乏所有三個sh3結構域的cd2ap突變體，然後感染hcv-j399em。通過實時圖像跟蹤，我們發現，只有全長cd2ap與gfp-ns5a共定位和與ns5a共移動，而沒有sh3結構域的cd2ap突變體不與ns5a共定位(圖12，左面板，框內的點)。對cd2ap和ns5a實況圖像的量化，進一步證實全長cd2ap與ns5a共移動(圖13)。觀察到ns5a與缺失所有三個sh3結構域的cd2ap之間沒有共定位，進一步支持了我們的結論，ns5a和cd2ap的sh3結構域相互作用。由於cd2ap點的移動依賴於肌動纖維聚合(60)，我們通過用秋水仙鹼(一種微管蛋白聚合抑制劑)或細胞鬆弛素b(肌動蛋白聚合抑制劑)處理受感染細胞，來調查cd2ap與ns5a的共移動是否依賴於肌動纖維聚合。我們發現，細胞鬆弛素b而不是秋水仙素處理顯著降低ns5a和cd2ap的共定位(圖14，左面板)。然而，在培養基中用dmso取代細胞鬆弛素b後4小時，恢復cd2ap和ns5a的共定位(圖14，右上面板)。這些結果表明，cd2ap和ns5a的共定位依賴於肌動蛋白細胞骨架。ns5a必須通過微管系統運往脂滴來組裝(12)，我們發現，細胞用秋水仙素處理後，cd2ap與ns5a複合物沒有移動(圖15)。由於秋水仙鹼處理不影響cd2ap和ns5a的共定位，但是細胞鬆弛素b處理阻止cd2ap和ns5a共定位，我們假設，依賴於肌動蛋白的cd2ap和ns5a共定位，是發生在hcv組裝之前。如果這個假設是正確的，我們預見更少的ns5a與脂滴結合。為了檢驗這一假設，我們採用con1複製系統，其中脂滴在數量上大為減少。然後我們下調cd2ap表達，用生化手段檢測ns5a與脂滴的結合是否會減輕。通過在con1複製系統中下調cd2ap(記為c4#和c6#)，我們發現在con1中ns5a水平沒有受到影響，然而，ns5a與脂滴組份的結合明顯減少(圖16)。其中calnexin和adrp，分別為er和ld標記物(圖16)。由於總的ns5a的表達水平不受影響，但當cd2ap表達下調時脂滴結合ns5a的水平減少，我們得出的結論是，cd2ap通過肌動蛋白細胞骨架運輸ns5a到脂滴。這些結果也表明，cd2ap的下調不影響hcv基因組複製而降低hcv裝配。2.5cd2ap影響ld的生物合成由於cd2ap可能在hcv的裝配和釋放發揮作用，同時我們已經表明cd2ap表達下調減少ns5a轉運到脂滴，我們隨後研究，在轉運ns5a到脂滴之外，cd2ap是否在hcv組裝中發揮其他作用。我們首先測試了下調cd2ap對脂滴生物合成的影響。下調cd2ap顯著降低了脂滴的生物合成和積累(圖17，bsa下左柱，nc和6#)。由於在非感染的條件下脂滴的生物合成是非常有限的，我們進一步探討，在oa處理下，下調cd2ap對脂滴生物合成的影響。我們發現，cd2ap下調明顯減輕脂滴的生物合成(圖17，在oa右列)。在oa處理下，對超過200細胞中的脂滴計數，證實當cd2ap下調時，每個細胞有顯著少的脂滴(圖17，黑盒，比較nc和6#，p＜0.05)。為了證明cd2ap確實影響脂滴的生物合成，我們染色經過oa或bsa處理的cd2ap拯救細胞。我們發現，與對照細胞相比，cd2ap過表達細胞顯示顯著多的脂滴(圖18)。在oa處理下，對超過200個細胞中的脂滴計數，證實當cd2ap上調時，每個細胞有顯著上調的脂滴(圖18，黑盒，比較nc和ha-cd2ap，p＜0.05)。為了排除由於核心蛋白表達水平降低而導致減少脂滴的可能性，我們隨後在cd2ap下調和對照細胞中過表達核心蛋白。我們發現，與對照細胞相比，在cd2ap下調細胞中上調核心蛋白不會顯著增加脂滴的形成(圖19，右邊兩個面板和黑框，顯示在核心蛋白過表達後，脂滴明顯減少，在p＜0.05)，從而進一步證明，當cd2ap表達下調，脂滴的生物合成與hcv核心蛋白定位於脂滴上受到阻礙。然而，在cd2ap下調細胞中增強cd2ap表達後，脂滴的水平顯著提高，同樣地核心蛋白在脂滴上的定位也顯著增加(圖20，右邊兩個面板和黑框，顯示過表達cd2ap後位於脂滴的核心蛋白顯著增加)。這些結果表明，cd2ap在細胞脂滴的生成和導向hcv組件到脂滴中起重要作用。2.6下調cd2ap抑制hcv的繁殖由於hcv基因組複製不受cd2ap和ns5a相互作用的影響，我們試圖通過沉默cd2ap來檢測這種相互作用對hcv繁殖的影響。我們製備了兩個穩定cd2ap下調細胞系(huh7.5.1-shcd2ap-4，記為4#；huh7.5.1-shcd2ap-6，記為6#)；huh7.5.1-shcd2ap陰性對照，記為nc。下調cd2ap並不影響細胞的生長。然而，細胞感染hcv-jfh172小時後，與對照細胞相比，下調cd2ap顯著降低hcvmrna水平(圖21)。免疫印跡分析證實，hcv的ns5a和核心蛋白表達顯著減少(圖22)。另外，釋放到cd2ap下調細胞培養上清的病毒rna拷貝數也明顯減少(圖23，p＜0.01)。通過使用一個帶有renila螢光素酶報告基因的報告病毒j399em+lm，進一步證實了cd2ap下調在hcv複製的影響(圖24)。為了排除此影響是由於cd2ap下調的脫靶效應的可能性，我們進行了一項拯救實驗。我們瞬時表達ha-cd2ap突變體，在cd2ap下調細胞shcd2ap-6#的靶點含有的擺動突變(記為6#-ha-cd2ap)。hcvjfh1感染後，ha-cd2ap突變體的表達(6#-ha-cd2ap+)，但不是空載體(6#-ha-cd2ap-)，拯救了細胞內hcvrna水平(圖25)。與rna水平一致，相比轉染空載體的細胞，在6#-ha-cd2ap中核心蛋白和ns5a蛋白水平也顯示部分恢復(2道與3道，圖26)。總之，這些結果表明，在huh7.5.1細胞中，下調內源性cd2ap顯著抑制hcv的繁殖。2.7下調cd2ap不妨礙hcv侵入，基因組rna複製和ires依賴性翻譯，但抑制hcv感染性顆粒的產生由於cd2ap不影響hcv亞基因組複製但在hcv繁殖中起重要作用，我們進一步研究，cd2ap影響hcv感染的的根本機制。我們首先通過轉導hcv假顆粒，探討cd2ap是否影響hcv侵入。用hcvpps轉導cd2ap穩定下調細胞。轉導48小時後後，測定螢光素酶活性，作為hcv侵入效率的指標。如圖27所示，cd2ap下調和對照細胞之間，hcvpp感染無顯著差異，提示hcv侵入不受cd2ap下調的影響。我們隨後探討，下調cd2ap是否會影響hcv內部核糖體進入位點(ires)導向的翻譯。用雙順反子報告質粒phcv-ires來監測hcvires活性。在質粒phcv-ires中，上遊renilla螢光素酶基因(rluc)的翻譯是由5』帽子結構介導的，下遊火螢螢光素酶基因(fluc)是由hcvires元件控制的。hcvires依賴性翻譯水平是針對rluc活性利用fluc活性進行歸一化計算。與對照組相比，沉默cd2ap不顯著影響hcvires依賴性翻譯(圖28，空盒子代表cd2ap相對翻譯水平，而黑框測量歸一化hcvires活性)。我們進一步評估在亞基因組複製子con1細胞中，cd2ap下調對hcv基因組rna複製的影響。下調con1細胞的cd2ap後，在cd2ap下調和對照con1細胞之間，我們發現hcvrna和蛋白質水平沒有顯著差異(圖29)，從而證明cd2ap並不直接影響hcv亞基因組複製。然後我們測試cd2ap下調是否影響hcv的裝配和釋放。穩定表達sh-cd2ap-4#,6#或sh-nchuh7.5.1細胞感染j399em，moi為1。在感染後72小時(hpi)，病毒滴度在細胞質和培養上清液均顯著降低(圖30和圖31)，從而提示cd2ap參與hcv裝配或/和釋放。2.8cd2ap調控與脂滴相關聯的多個hcv組分由於在沒有C型肝炎病毒感染的情況下cd2ap的下調緩解脂滴生物合成，我們隨後研究，當細胞在被C型肝炎病毒感染的時候是否有同樣的現象。我們用jfh1感染了cd2ap下調(4#和6#)和對照細胞(nc)，對脂滴，ns5a，或hcv核心蛋白染色。我們發現，hcv感染後，脂滴的形成在cd2ap下調的細胞中嚴重受損(圖32(a)和圖33(a)；ld面板下4#和6#)。此外，定位於脂滴的ns5a和核心蛋白明顯下降(圖32(a)和圖33(a)；合併面板下4#和6#)。cd2ap下調細胞與對照細胞相比，其ns5a或核心蛋白的陽性脂滴百分比存在顯著差異(圖32b和33b、比較4#和6#)。前面我們證明了在con1細胞中cd2ap下調不影響ns5a的表達水平，因此ns5a在脂滴上的定位減少是由於cd2ap下調後，脂滴生物合成減少從而造成轉運缺陷而造成的。在huh7.5.1細胞中下調cd2ap增加irs1和p-irs1的總體水平(圖34)。我們發現，irs1可以被蛋白酶體途徑降解。當用mg132處理2小時，irs1水平顯著性上調(圖35)。為了證實cd2ap下調影響irs1的蛋白酶依賴性的降解，我們比較了在mg132處理的條件下，對照細胞和cd2ap下調細胞的irs1水平。我們發現，mg132顯著增加對照細胞中irs1水平，但是cd2ap下調細胞中沒有增加(圖36)。此外，通過從對照和cd2ap下調細胞純化irs1，我們發現，cd2ap下調顯著降低irs1的多位泛素化(圖37)。為了弄清與cd2ap形成的蛋白複合物，我們用抗irs1抗體進行了免疫共沉澱，發現cd2ap與irs1可以共沉澱。我們用抗cbl-b和抗cbl抗體同樣進行了免疫共沉澱實驗，我們發現，irs1與cbl-b和cbl同樣可以共沉澱(圖38)。為了進一步證明cd2ap，irs1和cbl-b/cbl存在於蛋白複合物，我們雙染了cd2ap和irs1與irs1和cbl-b/cbl，發現irs1和cd2ap，irs1和cbl-b/cbl確實共定位(圖39)。為了證實cbl-b/cbl是作用於irs1的e3連接酶，我們下調huh7.5.1細胞中的cbl-b/cbl，發現irs1的顯著上調(圖40)。因此，cbl-b/cbl是作用於irs1的e3連接酶。這些結果證明，cd2ap，irs1和cbl-b/cbl存在於同一蛋白複合物。因為irs1守衛胰島素信號通路，我們檢測在cd2ap下調後，胰島素信號通路是否受到影響。我們發現，cd2ap下調增加p-akt(s473)水平，但是下調p-ampk(t172)和p-hsl(s554)的水平(圖41)。預期地，當cd2ap下調的細胞huh7.5.1中cd2ap被拯救時，p-akt水平被下調(圖42)。為了證明ampk直接負責hsl的磷酸化，我們用ampk的一個抑制劑dorsomophin處理細胞，發現dorsomophin確實降低p-ampk水平，相應地降低hsl水平(圖43)。以上結果來自腫瘤細胞系。我們然後用hcv感染小鼠模型研究我們的結果是否有體內重要性。小鼠肝和血清中的hcv滴度在感染後不同時間點用qpcr進行監測(圖44,45)。hcv滴度的趨勢與已經發表的非常類似(61)。我們隨後在來自hcv感染小鼠的肝組織中染色cd2ap，發現在感染後1,2和4個月時，cd2ap顯著上調。由於在此時間範圍之外的早或晚的時間沒有明顯cd2ap染色，提示cd2ap表達不與hcv滴度相關，而是hcv感染的結果(圖46)。有趣的是，在這個小鼠模型中，cd2ap強染色的時間段與脂肪肝的發生有很好的相關性。此外，我們研究在hcv感染的病人中cd2ap是否被上調。我們發現，hcv感染病人的肝活檢樣品，16份中有9份顯示中到強cd2ap染色，而未感染hcv病人的肝活檢樣品，12份中只有1份顯示強cd2ap染色(圖47)。最後，我們研究來自糖尿病病人的肝活檢樣品中，是否能檢測到cd2ap免疫染色。我們發現，大多數糖尿病病人的肝組織顯示強cd2ap染色。因此，在人肝活檢樣品中，cd2ap表達在糖尿病病人的肝中顯著增加(圖48)。儘管本發明參照特異的實施方式來描述，但是將被理解的是，實施例是說明性的，本發明的範圍並不局限於此。本發明的替代實施例對本發明涉及的領域的普通技術人員將變得顯而易見。這樣的替代實施例都被認為是包含在本發明的精神和範圍之內。因此，本發明的範圍由所附的權利要求被描述，由前面的描述所支持。references1.chooql,kuog,weineraj,overbylr,bradleydw,houghtonm.1989.isolationofacdnaclonederivedfromablood-bornenon-a,non-bviralhepatitisgenome.science244:359-362.2.rosenhr.2011.clinicalpractice.chronichepatitiscinfection.thenewenglandjournalofmedicine364:2429-2438.3.lohmannv,kornerf,kochj,herianu,theilmannl,bartenschlagerr.1999.replicationofsubgenomichepatitiscvirusrnasinahepatomacellline.science285:110-113.4.romero-breyi,merza,chiramela,leejy,chlandap,haselmanu,santarella-mellwigr,habermanna,hoppes,kalliss,waltherp,antonyc,krijnse-lockerj,bartenschlagerr.2012.three-dimensionalarchitectureandbiogenesisofmembranestructuresassociatedwithhepatitiscvirusreplication.plospathogens8:e1003056.5.ferrarisp,beaumonte,uzbekovr,brandd,gaillardj,blancharde,roingeardp.2013.sequentialbiogenesisofhostcellmembranerearrangementsinducedbyhepatitiscvirusinfection.cellularandmolecularlifesciences:cmls70:1297-1306.6.appeln,zayasm,millers,krijnse-lockerj,schallert,friebep,kalliss,engelu,bartenschlagerr.2008.essentialroleofdomainiiiofnonstructuralprotein5aforhepatitiscvirusinfectiousparticleassembly.plospathogens4:e1000035.7.miyanariy,atsuzawak,usudan,watashik,hishikit,zayasm,bartenschlagerr,wakitat,hijikatam,shimotohnok.2007.thelipiddropletisanimportantorganelleforhepatitiscvirusproduction.naturecellbiology9:1089-1097.8.shist,polyaksj,tuh,taylordr,gretchdr,laimm.2002.hepatitiscvirusns5acolocalizeswiththecoreproteinonlipiddropletsandinteractswithapolipoproteins.virology292:198-210.9.abidk,pazienzav,degottardia,rubbia-brandtl,conneb,pugnalep,rossic,mangiaa,negrof.2005.aninvitromodelofhepatitiscvirusgenotype3a-associatedtriglyceridesaccumulation.journalofhepatology42:744-751.10.hinsoner,cresswellp.2009.theantiviralprotein,viperin,localizestolipiddropletsviaitsn-terminalamphipathicalpha-helix.proceedingsofthenationalacademyofsciencesoftheunitedstatesofamerica106:20452-20457.11.masakit,suzukir,murakamik,aizakih,ishiik,murayamaa,datet,matsuuray,miyamurat,wakitat.2008.interactionofhepatitiscvirusnonstructuralprotein5awithcoreproteiniscriticalfortheproductionofinfectiousvirusparticles.journalofvirology82:7964-7976.12.laick,jengks,machidak,laimm.2008.associationofhepatitiscvirusreplicationcomplexeswithmicrotubulesandactinfilamentsisdependentontheinteractionofns3andns5a.journalofvirology82:8838-8848.13.eyrens,fichesgn,aloiaal,helbigkj,mccartneyem,mcerleancs,lik,aggarwala,turvillesg,beardmr.2014.dynamicimagingofthehepatitiscvirusns5aproteinduringaproductiveinfection.journalofvirology88:3636-3652.14.laic-k,saxenav,tsengc-h,jengk-s,koharam,laimm.2014.nonstructuralprotein5aisincorporatedintohepatitiscviruslow-densityparticlethroughinteractionwithcoreproteinandmicrotubulesduringintracellulartransport.plosone9:e99022.15.tilgh,moschenar,rodenm.2017.nafldanddiabetesmellitus.naturereviewsgastroenterology&hepatology14:32-42.16.anaim,funakim,ogiharat,terasakij,inukaik,katagirih,fukushimay,yazakiy,kikuchim,okay.1998.alteredexpressionlevelsandimpairedstepsinthepathwaytophosphatidylinositol3-kinaseactivationviainsulinreceptorsubstrates1and2inzuckerfattyrats.diabetes47:13-23.17.arakie,llpesma,pattim-e.1994.signallinginmicewithtargeteddisruption.nature372.18.bruningjc,winnayj,bonner-weirs,taylorsi,accilid,kahncr.1997.developmentofanovelpolygenicmodelofniddminmiceheterozygousforirandirs-1nullalleles.cell88:561-572.19.jiangzy,liny-w,clemonta,feenerep,heinkd,igarashim,yamauchit,whitemf,kinggl.1999.characterizationofselectiveresistancetoinsulinsignalinginthevasculatureofobesezucker(fa/fa)rats.thejournalofclinicalinvestigation104:447-457.20.kerouznj,d,ponss,kahncr.1997.differentialregulationofinsulinreceptorsubstrates-1and-2(irs-1andirs-2)andphosphatidylinositol3-kinaseisoformsinliverandmuscleoftheobesediabetic(ob/ob)mouse.journalofclinicalinvestigation100:3164.21.tamemotoh,kadowakit,tobek,yagit,sakurah,hayakawat,terauchiy,uekik,kaburagiy,satohs.1994.insulinresistanceandgrowthretardationinmicelackinginsulinreceptorsubstrate-1.22.withersdj,gutierrezjs,toweryh,burksdj,renjm,previss,zhangy,bernald,ponss,shulmangi,bonner-weirs,whitemf.1998.disruptionofirs-2causestype2diabetesinmice.nature391:900-904.23.stephensjm,leej,pilchpf.1997.tumornecrosisfactor-α-inducedinsulinresistancein3t3-l1adipocytesisaccompaniedbyalossofinsulinreceptorsubstrate-1andglut4expressionwithoutalossofinsulinreceptor-mediatedsignaltransduction.journalofbiologicalchemistry272:971-976.24.egawak,nakashiman,sharmapm,maegawah,nagaiy,kashiwagia,kikkawar,olefskyjm.2000.persistentactivationofphosphatidylinositol3-kinasecausesinsulinresistanceduetoacceleratedinsulin-inducedinsulinreceptorsubstrate-1degradationin3t3-l1adipocytes1.endocrinology141:1930-1935.25.sunxj,goldbergjl,qiaol,mitchelljj.1999.insulin-inducedinsulinreceptorsubstrate-1degradationismediatedbytheproteasomedegradationpathway.diabetes48:1359-1364.26.harutat,unot,kawaharaj,takanoa,egawak,sharmapm,olefskyjm,kobayashim.2000.arapamycin-sensitivepathwaydown-regulatesinsulinsignalingviaphosphorylationandproteasomaldegradationofinsulinreceptorsubstrate-1.molecularendocrinology14:783-794.27.leeav,goochjl,oesterreichs,gulerrl,yeed.2000.insulin-likegrowthfactori-induceddegradationofinsulinreceptorsubstrate1ismediatedbythe26sproteasomeandblockedbyphosphatidylinositol3′-kinaseinhibition.molecularandcellularbiology20:1489-1496.28.zhander,mitchelljj,wuj,sunxj.2002.molecularmechanismofinsulin-induceddegradationofinsulinreceptorsubstrate1.molecularandcellularbiology22:1016-1026.29.bosesk,rayr.2014.hepatitiscvirusinfectionandinsulinresistance.worldjdiabetes5:52-58.30.johnj.sambrookddwr.1989.molecularcloning:alaboratorymannual,secondedition.cshlpress.31.ausubelfm.1987.currentprotocolsinmolecularbiology32.rosenbergim.1996.proteinanalysisandpurification-benchtoptechniques33.copelandra.2013.methodsforproteinanalysis:apracticalguideforlaboratoryprotocols.34.johne.coliganbb.1999currentprotocolsinimmunology.35.kimjm,wuh,greeng,winklerca,koppjb,minerjh,unanueer,shawas.2003.cd2-associatedproteinhaploinsufficiencyislinkedtoglomerulardiseasesusceptibility.science300:1298-1300.36.kobayashis,sawanoa,nojimay,shibuyam,maruy.2004.thec-cbl/cd2apcomplexregulatesvegf-inducedendocytosisanddegradationofflt-1(vegfr-1).thefasebjournal18:929-931.37.baom,hanabuchis,facchinettiv,duq,boverl,plumasj,chaperotl,caow,qinj,suns-c.2012.cd2ap/ship1complexpositivelyregulatesplasmacytoiddendriticcellreceptorsignalingbyinhibitingthee3ubiquitinligasecbl.thejournalofimmunology189:786-792.38.calcogn,stephensor,donahuelm,tsuicc,pierchalaba.2014.cd2-associatedprotein(cd2ap)enhancescasitasblineagelymphoma-3/c(cbl-3/c)-mediatedretisoform-specificubiquitinationanddegradationviaitsamino-terminalsrchomology3domains.journalofbiologicalchemistry289:7307-7319.39.kowanetzk,szymkiewiczi,haglundk,kowanetzm,husnjakk,taylorjd,soubeyranp,engstromu,ladburyje,dikici.2003.identificationofanovelproline-argininemotifinvolvedincin85-dependentclusteringofcblanddown-regulationofepidermalgrowthfactorreceptors.journalofbiologicalchemistry278:39735-39746.40.gouti,middletong,aduj,ninkinann,drobotlb,filonenkov,matsukag,daviesam,waterfieldm,buchmanvl.2000.negativeregulationofpi3-kinasebyruk,anoveladaptorprotein.theembojournal19:4015-4025.41.hubertb,hartlebenb,kimj,schmidtsm,schermerb,keila,eggerl,lecharl,bornerc,h.2003.nephrinandcd2apassociatewithphosphoinositide3-ohkinaseandstimulateakt-dependentsignaling.molecularandcellularbiology23:4917-4928.42.opreac,ianachei,radoir,erscoius,tardeig,nicolaescuo,nicam,calistrup,rutas,ceausue.2014.alarmingincreaseintuberculosisandhepatitiscvirus(hcv)amonghivinfectedintravenousdrugusers.journaloftheinternationalaidssociety17:19625.43.chamondn,cossona,coatnoann,minopriop.2009.prolineracemasesareconservedmitogens:characterizationofatrypanosomavivaxprolineracemase.molecularandbiochemicalparasitology165:170-179.44.zehmerjk,bartzr,liup,andersonrg.2008.identificationofanoveln-terminalhydrophobicsequencethattargetsproteinstolipiddroplets.journalofcellscience121:1852-1860.45.wuy,liaoq,yangr,chenx,chenx.2011.anovelluciferaseandgfpdualreportervirusforrapidandconvenientevaluationofhepatitiscvirusreplication.virusresearch155:406-414.46.lindenbachbd,evansmj,syderaj,wolkb,tellinghuisentl,liucc,maruyamat,hynesro,burtondr,mckeatingja,ricecm.2005.completereplicationofhepatitiscvirusincellculture.science309:623-626.47.lic,yus,nakamuraf,yins,xuj,petrollaaa,singhn,tartakoffa,abbottdw,xinw,syms.2009.bindingofpro-priontofilaminadisruptscytoskeletonandcorrelateswithpoorprognosisinpancreaticcancer.thejournalofclinicalinvestigation119:2725-2736.48.yangl,gaoz,hul,wug,yangx,zhangl,zhuy,wongb-s,xinw,sym-s.2016.glycosylphosphatidylinositolanchormodificationmachinerydeficiencyisresponsiblefortheformationofpro-prionprotein(prp)inbxpc-3proteinandincreasescancercellmotility.journalofbiologicalchemistry291:3905-3917.49.rouxkj,kimdi,raidam,burkeb.2012.apromiscuousbiotinligasefusionproteinidentifiesproximalandinteractingproteinsinmammaliancells.thejournalofcellbiology196:801-810.50.xus,peir,guom,hanq,laij,wangy,wuc,zhouy,lum,chenx.2012.cytosolicphospholipasea2gammaisinvolvedinhepatitiscvirusreplicationandassembly.journalofvirology86:13025-13037.51.vogtda,camusg,herkere,websterbr,tsoucl,greenewc,yents,ottm.2013.lipiddroplet-bindingproteintip47regulateshepatitiscvirusrnareplicationthroughinteractionwiththeviralns5aprotein.plospathogens9:e1003302.52.lindenbachbd.2009.measuringhcvinfectivityproducedincellcultureandinvivo.methodsinmolecularbiology510:329-336.53.dustinml,olszowymw,holdorfad,lij,bromleys,desain,widderp,rosenbergerf,vandermerwepa,allenpm.1998.anoveladaptorproteinorchestratesreceptorpatterningandcytoskeletalpolarityint-cellcontacts.cell94:667-677.54.tangvw,brieherwm.2013.fsgs3/cd2apisabarbed-endcappingproteinthatstabilizesactinandstrengthensadherensjunctions.thejournalofcellbiology203:815-833.55.zhaoj,brucks,cemerskis,zhangl,butlerb,dania,cooperja,shawas.2013.cd2aplinkscortactinandcappingproteinatthecellperipherytofacilitateformationoflamellipodia.molecularandcellularbiology33:38-47.56.macdonalda,crowderk,streeta,mccormickc,harrism.2004.thehepatitiscvirusns5aproteinbindstomembersofthesrcfamilyoftyrosinekinasesandregulateskinaseactivity.thejournalofgeneralvirology85:721-729.57.brasaemledl,doliosg,shapirol,wangr.2004.proteomicanalysisofproteinsassociatedwithlipiddropletsofbasalandlipolyticallystimulated3t3-l1adipocytes.thejournalofbiologicalchemistry279:46835-46842.58.brassv,biecke,montserretr,b,hellingsja,blumhe,peninf,moradpourd.2002.anamino-terminalamphipathicα-helixmediatesmembraneassociationofthehepatitiscvirusnonstructuralprotein5a.journalofbiologicalchemistry277:8130-8139.59.tellinghuisentl,marcotrigianoj,gorbalenyaae,ricecm.2004.thens5aproteinofhepatitiscvirusisazincmetalloprotein.thejournalofbiologicalchemistry279:48576-48587.60.welscht,endlichn,g,doroshenkoe,simpsonjc,krizw,shawas,endlichk.2005.associationofcd2apwithdynamicactinonvesiclesinpodocytes.americanjournalofphysiology-renalphysiology289:f1134-f1143.61.chenj,zhaoy,zhangc,chenh,fengj,chix,pany,duj,guom,caoh,chenh,wangz,peir,wangq,panl,niuj,chenx,tangh.2014.persistenthepatitiscvirusinfectionsandhepatopathologicalmanifestationsinimmune-competenthumanizedmice.cellresearch24:1050-1066.sequencelisting中國科學院武漢病毒研究所cd2結合蛋白（cd2ap）和其相互作用蛋白1004.p001281patentinversion3.511920dnahomosapiens1atggttgactatattgtggagtatgactatgatgctgtacatgatgatgaattaactatt60cgagttggagaaatcatcaggaatgtgaaaaagctacaggaggaagggtggctggaagga120gaactaaatgggagaagaggaatgttccctgacaatttcgttaaggaaattaaaagagag180acggaattcaaggatgacagtttgcccatcaaacgggaaaggcatgggaatgtagcaagt240cttgtacaacgaataagcacctatggacttccagctggaggaattcagccacatccacaa300accaaaaacattaagaagaagaccaagaagcgtcagtgtaaagttctttttgagtacatt360ccacaaaatgaggatgaactggagctgaaagtgggagatattattgatattaatgaagag420gtagaagaaggctggtggagtggaaccctgaataacaagttgggactgtttccctcaaat480tttgtgaaagaattagaggtaacagatgatggtgaaactcatgaagcccaggacgattca540gaaactgttttggctgggcctacttcacctataccttctctgggaaatgtgagtgaaact600gcatctggatcagttacacagccaaagaaaattcgaggaattggatttggagacattttt660aaagaaggctctgtgaaacttcggacaagaacatccagtagtgaaacagaagagaaaaaa720ccagaaaagcccttaatcctacagtcactgggacccaaaactcagagtgtggagataaca780aaaacagataccgaaggtaaaattaaagctaaagaatattgtagaacattatttgcctat840gaaggtactaatgaagatgaacttacttttaaagagggggagataatccatttgataagt900aaggagactggagaagctggctggtggaggggcgaacttaatggtaaagaaggagtattt960ccagacaattttgctgtccagataaatgaacttgataaagactttccaaaaccaaagaaa1020ccaccacctcctgctaaggctccagctccaaagcctgaactgatagctgcagagaagaaa1080tatttttctttaaagcctgaagaaaaggatgaaaaatcaacactggaacagaaaccttct1140aaaccagcagctccacaagtcccacccaagaaacctactccacctaccaaagccagtaat1200ttactgagatcttctggaacagtgtacccaaagcgacctgaaaaaccagttcctccacca1260cctcctatagccaagattaatggggaagtttctagcatttcatcaaaatttgaaactgag1320ccagtatcaaaactaaagctagattctgaacagctgccccttagaccaaaatcagtagac1380tttgattcacttacagtaaggacctccaaagaaacagatgttgtaaattttgatgacata1440gcttcctcagaaaacttgcttcatctcactgcaaatagaccaaagatgcctggaagaagg1500ttgccgggccgtttcaatggtggacattctccaactcacagccccgaaaaaatcttgaag1560ttaccaaaagaagaagacagtgccaacctgaagccatctgaattaaaaaaagatacatgc1620tactctccaaagccatctgtgtacctttcaacaccttccagtgcttctaaagcaaataca1680actgctttcctgactccattagaaatcaaagctaaagtggaaacagatgatgtgaaaaaa1740aattccctggatgaacttagagcccagattattgaattgttgtgcattgtagaagcactg1800aaaaaggatcacgggaaagaactggaaaaactgcgaaaagatttggaagaagagaagaca1860atgagaagtaatctagagatggaaatagagaagctgaaaaaagctgtcctgtcttcttga19202639prthomosapiens2metvalasptyrilevalglutyrasptyraspalavalhisaspasp151015gluleuthrileargvalglygluileileargasnvallyslysleu202530glnglugluglytrpleugluglygluleuasnglyargargglymet354045pheproaspasnphevallysgluilelysarggluthrgluphelys505560aspaspserleuproilelysarggluarghisglyasnvalalaser65707580leuvalglnargileserthrtyrglyleuproalaglyglyilegln859095prohisproglnthrlysasnilelyslyslysthrlyslysarggln100105110cyslysvalleupheglutyrileproglnasngluaspgluleuglu115120125leulysvalglyaspileileaspileasnglugluvalgluglugly130135140trptrpserglythrleuasnasnlysleuglyleupheproserasn145150155160phevallysgluleugluvalthraspaspglygluthrhisgluala165170175glnaspaspsergluthrvalleualaglyprothrserproilepro180185190serleuglyasnvalsergluthralaserglyservalthrglnpro195200205lyslysileargglyileglypheglyaspilephelysgluglyser210215220vallysleuargthrargthrsersersergluthrgluglulyslys225230235240proglulysproleuileleuglnserleuglyprolysthrglnser245250255valgluilethrlysthraspthrgluglylysilelysalalysglu260265270tyrcysargthrleuphealatyrgluglythrasngluaspgluleu275280285thrphelysgluglygluileilehisleuileserlysgluthrgly290295300glualaglytrptrpargglygluleuasnglylysgluglyvalphe305310315320proaspasnphealavalglnileasngluleuasplysaspphepro325330335lysprolyslysproproproproalalysalaproalaprolyspro340345350gluleuilealaalaglulyslystyrpheserleulysprogluglu355360365lysaspglulysserthrleugluglnlysproserlysproalaala370375380proglnvalproprolyslysprothrproprothrlysalaserasn385390395400leuleuargserserglythrvaltyrprolysargproglulyspro405410415valproproproproproilealalysileasnglygluvalserser420425430ileserserlysphegluthrgluprovalserlysleulysleuasp435440445sergluglnleuproleuargprolysservalasppheaspserleu450455460thrvalargthrserlysgluthraspvalvalasnpheaspaspile465470475480alasersergluasnleuleuhisleuthralaasnargprolysmet485490495proglyargargleuproglyargpheasnglyglyhisserprothr500505510hisserproglulysileleulysleuprolysglugluaspserala515520525asnleulysprosergluleulyslysaspthrcystyrserprolys530535540proservaltyrleuserthrproserseralaserlysalaasnthr545550555560thralapheleuthrproleugluilelysalalysvalgluthrasp565570575aspvallyslysasnserleuaspgluleuargalaglnileileglu580585590leuleucysilevalglualaleulyslysasphisglylysgluleu595600605glulysleuarglysaspleuglugluglulysthrmetargserasn610615620leuglumetgluileglulysleulyslysalavalleuserser625630635321dnaartificialsequencesirnaforcd2ap3gctggaaggagaactaaatgg21421dnaartificialsequencesirna/shrnaforhumancd2ap4ggagaactaaatgggagaaga21521dnaartificialsequencesirna/shrnaforhumancd2ap5ggacttccagctggaggaatt21621dnaartificialsequencesirna/shrnaforhumancd2ap6ggagctgaaagtgggagatat21721dnaartificialsequencesirna/shrnaforhumancd2ap7gctgaaagtgggagatattat21821dnaartificialsequencesirna/shrnaforhumancd2ap8gctgaaagtgggagatattat21921dnaartificialsequencesirna/shrnaforhumancd2ap9gcccaggacgattcagaaact211021dnaartificialsequencesirna/shrnaforhumancd2ap10gctgggcctacttcacctata211121dnaartificialsequencesirna/shrnaforhumancd2ap11gccagtaatttactgagatct211221dnaartificialsequencesirna/shrnaforhumancd2ap12gcttcatctcactgcaaatag211321dnaartificialsequencesirna/shrnaforhumancd2ap13ggaagtttccagcagatttca211419dnaartificialsequencesirna/shrnaforhumancd2ap14agccgagggtctgggcaaa191519dnaartificialsequencesirna/shrnaforhumancd2ap15agccgagggtctgggcaaa191619dnaartificialsequencesirna/shrnaforhumancd2ap16tgaagagactggtaggaga191719dnaartificialsequencesirna/shrnaforhumancd2ap17ctaaatgggagaagaggaa191819dnaartificialsequencesirna/shrnaforhumancd2ap18aggatgaactggagctgaa191919dnaartificialsequencesirna/shrnaforhumancd2ap19ggtaacagatgatggtgaa192019dnaartificialsequencesirna/shrnaforhumancd2ap20ggaaacagatgatgtgaaa192120dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap21aaaggcgacaccgtagacta202220dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap22cgacaccgtagactaaggtg202320dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap23gtgggaaaaccgcggtcggg202420dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap24ggcgacaccgtagactaagg202520dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap25agggtgggaaaaccgcggtc202620dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap26tgggaaaaccgcggtcgggc202720dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap27gcgacaccgtagactaaggt202820dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap28cagggtgggaaaaccgcggt202920dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap29cgaccgcggttttcccaccc203020dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap30aaaaccgcggtcgggcgggc203120dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap31cgaggctaggcgggcgctcg203220dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap32gaaaaccgcggtcgggcggg203320dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap33gagggtctgggcaaaccggt203420dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap34tgggtccccaccttagtcta203520dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap35cgagggtctgggcaaaccgg203620dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap36gcgctcggggttggagccga203720dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap37tccgaggctaggcgggcgct203820dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap38ttttctaactgcgagtgcta203920dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap39ccgaggctaggcgggcgctc204020dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap40aaaccgcggtcgggcgggcg204120dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap41ttagcactcgcagttagaaa204220dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap42gctaggcgggcgctcggggt204320dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap43tccccactgcgggagcggcc204420dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap44cccgagcgcccgcctagcct204520dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap45accctggccgctcccgcagt204620dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap46cggccagggtgggaaaaccg204720dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap47cgagtgctaaggaagaggcg204820dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap48aactgcgagtgctaaggaag204920dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap49ggcgggctccgaggctaggc205020dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap50tccccaggagccacggcggc205120dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap51ctaccccgcccgcccgaccg205220dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap52gtagggccctcccgccgccg205320dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap53caccggtttgcccagaccct205420dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap54ccctggccgctcccgcagtg205520dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap55agccgagggtctgggcaaac205620dnaartificialsequencecrispr/cas9targetsequencesforhumancd2ap56tggccgctcccgcagtgggg20572082dnacanislupus57atgcattttaaaagtttgctgaaaaacctggaatggagacaaccaaccaggaggaaaaag60acacatagagaacatcagctgaaaaaggtcaaaagaactggggatggcaagctcagaaag120tgtctacaacttctccggtggagtcggatttctggtcacgggtcagttgactatattgtg180gagtatgactacgatgctgtacatgatgatgaattaactattcgggttggtgaaataatc240aggaatgtgaaaaaactacaggaggaaggatggctagaaggagagctaaatgggagaaga300ggaatgtttcctgataattttgttaaggaaattaagagagagacagaacccaaggatgat360aatttgcccattaaacgggaaagacatgggaatgtagcaagccttgtacaacgaataagc420acctatggacttccagctggaggaattcaaccacatccacaaaccaaaaacattaagaag480aagaccaagaagcgtcagtgtaaagttctctttgagtaccttccacaaaatgaggatgaa540ttggagctgaaagtgggagatattattgatattaatgatgaggtagaagaaggctggtgg600agtggaaccctgaacaacaagttgggactgtttccctcaaattttgtgaaagaattagag660gtaacagatgatggtgaaactcatgaagcccaagaggattcagaaacggtttttactggg720cctacctcacctttaccgtctccggggaatgggaatgaaactgcacctggatcagttaca780cagccaaagaaaattcgaggaattggatttggagatatttttaaagaaggctctgtgaaa840cttagaacaagaacatctggtagtgaaatagaagagaagaaaacggaaaagcccttaatt900atacagtcagtaggatccaaaacacagagtctggatgcaacaaaaacagacacggaaaat960aaaagtaaagcaaaggaatattgtagaacattatttgcctatgaaggtactaatgaagac1020gagctttcttttaaagagggagagataattcacttaataagtaaggagactggagaagct1080ggctggtggaagggtgaacttaatggtaaagaaggagtatttccagataattttgctatt1140cagatacatgaactggataaagactttccaaaaccaaagaaaccaccacctcctgctaaa1200ggtccagctccaaaacctgagctaatagctacagagaagaagtattttcctataaagcca1260gaagaaaaagatgaaaaatcagtactggaacagaaaccttctaaaccagcagctccacaa1320gtcccacctaagaagcctactccacccaccaaagccaataatttattgagatctcctggg1380acaatatacccaaagcgacctgaaaaaccagtccctccaccacctcctatagccaagatt1440aatggggaagtatctaccatttcatcaaaatttgaaactgagccattatcaaaaccaaag1500ctagattctgaacaattaccacttagaccaaaatcagtagacctagattcatttacagtt1560aggagctctaaagaaacagatattgtaaattttgatgacatagcttcctcagaaaacttg1620ctacatcttactgcaaacagaccgaagatgcctggaagaaggttgcctggacgcttcaat1680ggtggacattctccaacccaaagcccagaaaaaaccttgaagttaccaaaagaagaagat1740agtgccaacttaaagccgtctgaatttaaaaaggattcaagctactctccaaagccatct1800ctgtacctttcaacaccttcaagtgcttcgaaaccaaatacagctgcttttttaactcca1860ttagaaatcaaagctaaagtagaatcagatgatgggaaaaaaaaccccttggatgaactt1920agagctcagattattgaattgctgtgcattgtagaagcactgaaaaaggatcatgggaaa1980gaactggaaaaactacgaaaggatttggaagaggagaaggcaatgagaagtaatctagag2040gtggaaatcgagaagctgaaaaaggcagtcctgtcgtcttga208258693prtcanislupus58methisphelysserleuleulysasnleuglutrpargglnprothr151015argarglyslysthrhisarggluhisglnleulyslysvallysarg202530thrglyaspglylysleuarglyscysleuglnleuleuargtrpser354045argileserglyhisglyservalasptyrilevalglutyrasptyr505560aspalavalhisaspaspgluleuthrileargvalglygluileile65707580argasnvallyslysleuglnglugluglytrpleugluglygluleu859095asnglyargargglymetpheproaspasnphevallysgluilelys100105110arggluthrgluprolysaspaspasnleuproilelysarggluarg115120125hisglyasnvalalaserleuvalglnargileserthrtyrglyleu130135140proalaglyglyileglnprohisproglnthrlysasnilelyslys145150155160lysthrlyslysargglncyslysvalleupheglutyrleuprogln165170175asngluaspgluleugluleulysvalglyaspileileaspileasn180185190aspgluvalglugluglytrptrpserglythrleuasnasnlysleu195200205glyleupheproserasnphevallysgluleugluvalthraspasp210215220glygluthrhisglualaglngluaspsergluthrvalphethrgly225230235240prothrserproleuproserproglyasnglyasngluthralapro245250255glyservalthrglnprolyslysileargglyileglypheglyasp260265270ilephelysgluglyservallysleuargthrargthrserglyser275280285gluilegluglulyslysthrglulysproleuileileglnserval290295300glyserlysthrglnserleuaspalathrlysthraspthrgluasn305310315320lysserlysalalysglutyrcysargthrleuphealatyrglugly325330335thrasngluaspgluleuserphelysgluglygluileilehisleu340345350ileserlysgluthrglyglualaglytrptrplysglygluleuasn355360365glylysgluglyvalpheproaspasnphealaileglnilehisglu370375380leuasplysasppheprolysprolyslysproproproproalalys385390395400glyproalaprolysprogluleuilealathrglulyslystyrphe405410415proilelysprogluglulysaspglulysservalleugluglnlys420425430proserlysproalaalaproglnvalproprolyslysprothrpro435440445prothrlysalaasnasnleuleuargserproglythriletyrpro450455460lysargproglulysprovalproproproproproilealalysile465470475480asnglygluvalserthrileserserlysphegluthrgluproleu485490495serlysprolysleuaspsergluglnleuproleuargprolysser500505510valaspleuaspserphethrvalargserserlysgluthraspile515520525valasnpheaspaspilealasersergluasnleuleuhisleuthr530535540alaasnargprolysmetproglyargargleuproglyargpheasn545550555560glyglyhisserprothrglnserproglulysthrleulysleupro565570575lysglugluaspseralaasnleulysprosergluphelyslysasp580585590sersertyrserprolysproserleutyrleuserthrproserser595600605alaserlysproasnthralaalapheleuthrproleugluilelys610615620alalysvalgluseraspaspglylyslysasnproleuaspgluleu625630635640argalaglnileilegluleuleucysilevalglualaleulyslys645650655asphisglylysgluleuglulysleuarglysaspleuglugluglu660665670lysalametargserasnleugluvalgluileglulysleulyslys675680685alavalleuserser6905919dnaartificialsequencesirna/shrnasequencesforcaninecd2ap59gaggaatgtttcctgataa196019dnaartificialsequencesirna/shrnasequencesforcaninecd2ap60tcagtagacctagattcat196119dnaartificialsequencesirna/shrnasequencesforcaninecd2ap61gcgtcagtgtaaagttctc196219dnaartificialsequencesirna/shrnasequencesforcaninecd2ap62tagctacagagaagaagta196319dnaartificialsequencesirna/shrnasequencesforcaninecd2ap63agagggagagataattcac196419dnaartificialsequencesirna/shrnasequencesforcaninecd2ap64atcagtagacctagattca196519dnaartificialsequencesirna/shrnasequencesforcaninecd2ap65ggtactaatgaagacgagc196619dnaartificialsequencesirna/shrnasequencesforcaninecd2ap66agaagaagatagtgccaac196719dnaartificialsequencesirna/shrnasequencesforcaninecd2ap67ctcatgaagcccaagagga196819dnaartificialsequencesirna/shrnasequencesforcaninecd2ap68cgaataagcacctatggac196919dnaartificialsequencesirna/shrnasequencesforcaninecd2ap69ctggaatggagacaaccaa197019dnaartificialsequencesirna/shrnasequencesforcaninecd2ap70gcaagctcagaaagtgtct197119dnaartificialsequencesirna/shrnasequencesforcaninecd2ap71gctcagaaagtgtctacaa197219dnaartificialsequencesirna/shrnasequencesforcaninecd2ap72cagaaagtgtctacaactt197319dnaartificialsequencesirna/shrnasequencesforcaninecd2ap73gtctacaacttctccggtg197419dnaartificialsequencesirna/shrnasequencesforcaninecd2ap74ggagtcggatttctggtca197519dnaartificialsequencesirna/shrnasequencesforcaninecd2ap75gtcacgggtcagttgacta197619dnaartificialsequencesirna/shrnasequencesforcaninecd2ap76acgggtcagttgactatat197723dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap77aaaggcagacactcaaccgccgg237823dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap78atgtattgaagtgagacacctgg237923dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap79atgatgtgggactccatcccagg238023dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap80agggcgtgacccccaagtcctgg238123dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap81tgtattgaagtgagacacctggg238223dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap82gggcgtgacccccaagtcctggg238323dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap83ccatgcaggaagcatgatgtggg238423dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap84ggggtcacgccctgagccaaagg238523dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap85tccatgcaggaagcatgatgtgg238623dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap86attgaagtgagacacctgggtgg238723dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap87gactccatcccaggacttggggg238823dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap88gagtgtctgcctttggctcaggg238923dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap89tgggactccatcccaggacttgg239023dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap90agacacctgggtggctccggcgg239123dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap91tgagtgtctgcctttggctcagg239223dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap92ggactccatcccaggacttgggg239323dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap93gtgacccccaagtcctgggatgg239423dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap94ggcggttgagtgtctgcctttgg239523dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap95gtgagacacctgggtggctccgg239623dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap96cccacatcatgcttcctgcatgg239723dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap97gggactccatcccaggacttggg239823dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap98taacgcaactttctattttttgg239923dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap99ctcacttcaatacatttttaagg2310023dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap100ccagttaaaaagaaaatctaagg2310123dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap101ctcaaccgccggagccacccagg2310223dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap102taaagcaactttctattttttgg2310323dnaartificialsequencecrispr/cas9targetsequencesforcaninecd2ap103ccttagattttctttttaactgg231041398dnahepatitiscvirus104tccggatcctggctccgcgacgtgtgggactgggtttgcaccatcttgacagacttcaaa60aattggctgacctctaaattgttccccaagctgcccggcctccccttcatctcttgtcaa120aaggggtacaagggtgtgtgggccggcactggcatcatgaccacgcgctgcccttgcggc180gccaacatctctggcaatgtccgcctgggctctatgaggatcacagggcctaaaacctgc240atgaacacctggcaggggacctttcctatcaattgctacacggagggccagtgcgcgccg300aaaccccccacgaactacaagaccgccatctggagggtggcggcctcggagtacgcggag360gtgacgcagcatgggtcgtactcctatgtaacaggactgaccactgacaatctgaaaatt420ccttgccaactaccttctccagagtttttctcctgggtggacggtgtgcagatccatagg480tttgcacccacaccaaagccgtttttccgggatgaggtctcgttctgcgttgggcttaat540tcctatgctgtcgggtcccagcttccctgtgaacctgagcccgacgcagacgtattgagg600tccatgctaacagatccgccccacatcacggcggagactgcggcgcggcgcttggcacgg660ggatcacctccatctgaggcgagctcctcagtgagccagctatcagcaccgtcgctgcgg720gccacctgcaccacccacagcaacacctatgacgtggacatggtcgatgccaacctgctc780atggagggcggtgtggctcagacagagcctgagtccagggtgcccgttctggactttctc840gagccaatggccgaggaagagagcgaccttgagccctcaataccatcggagtgcatgctc900cccaggagcgggtttccacgggccttaccggcttgggcacggcctgactacaacccgccg960ctcgtggaatcgtggaggaggccagattaccaaccgcccaccgttgctggttgtgctctc1020cccccccccaagaaggccccgacgcctcccccaaggagacgccggacagtgggtctgagc1080gagagcaccatatcagaagccctccagcaactggccatcaagacctttggccagcccccc1140tcgagcggtgatgcaggctcgtccacgggggcgggcgccgccgaatccggcggtccgacg1200tcccctggtgagccggccccctcagagacaggttccgcctcctctatgccccccctcgag1260ggggagcctggagatccggacctggagtctgatcaggtagagcttcaacctcccccccag1320ggggggggggtagctcccggttcgggctcggggtcttggtctacttgctccgaggaggac1380gataccaccgtgtgctgc1398105466prthepatitiscvirus105serglysertrpleuargaspvaltrpasptrpvalcysthrileleu151015thraspphelysasntrpleuthrserlysleupheprolysleupro202530glyleupropheilesercysglnlysglytyrlysglyvaltrpala354045glythrglyilemetthrthrargcysprocysglyalaasnileser505560glyasnvalargleuglysermetargilethrglyprolysthrcys65707580metasnthrtrpglnglythrpheproileasncystyrthrglugly859095glncysalaprolysproprothrasntyrlysthralailetrparg100105110valalaalaserglutyralagluvalthrglnhisglysertyrser115120125tyrvalthrglyleuthrthraspasnleulysileprocysglnleu130135140proserprogluphephesertrpvalaspglyvalglnilehisarg145150155160phealaprothrprolysprophepheargaspgluvalserphecys165170175valglyleuasnsertyralavalglyserglnleuprocysglupro180185190gluproaspalaaspvalleuargsermetleuthraspproprohis195200205ilethralagluthralaalaargargleualaargglyserpropro210215220serglualaserserservalserglnleuseralaproserleuarg225230235240alathrcysthrthrhisserasnthrtyraspvalaspmetvalasp245250255alaasnleuleumetgluglyglyvalalaglnthrgluprogluser260265270argvalprovalleuasppheleugluprometalagluglugluser275280285aspleugluproserileproserglucysmetleuproargsergly290295300pheproargalaleuproalatrpalaargproasptyrasnpropro305310315320leuvalglusertrpargargproasptyrglnproprothrvalala325330335glycysalaleuproproprolyslysalaprothrproproproarg340345350argargargthrvalglyleusergluserthrileserglualaleu355360365glnglnleualailelysthrpheglyglnproproserserglyasp370375380alaglyserserthrglyalaglyalaalagluserglyglyprothr385390395400serproglygluproalaprosergluthrglyseralasersermet405410415proproleugluglygluproglyaspproaspleugluseraspgln420425430valgluleuglnproproproglnglyglyglyvalalaproglyser435440445glyserglysertrpserthrcysserglugluaspaspthrthrval450455460cyscys4651063729dnahomosapiens106atggcgagccctccggagagcgatggcttctcggacgtgcgcaaggtgggctacctgcgc60aaacccaagagcatgcacaaacgcttcttcgtactgcgcgcggccagcgaggctgggggc120ccggcgcgcctcgagtactacgagaacgagaagaagtggcggcacaagtcgagcgccccc180aaacgctcgatcccccttgagagctgcttcaacatcaacaagcgggctgactccaagaac240aagcacctggtggctctctacacccgggacgagcactttgccatcgcggcggacagcgag300gccgagcaagacagctggtaccaggctctcctacagctgcacaaccgtgctaagggccac360cacgacggagctgcggccctcggggcgggaggtggtgggggcagctgcagcggcagctcc420ggccttggtgaggctggggaggacttgagctacggtgacgtgcccccaggacccgcattc480aaagaggtctggcaagtgatcctgaagcccaagggcctgggtcagacaaagaacctgatt540ggtatctaccgcctttgcctgaccagcaagaccatcagcttcgtgaagctgaactcggag600gcagcggccgtggtgctgcagctgatgaacatcaggcgctgtggccactcggaaaacttc660ttcttcatcgaggtgggccgttctgccgtgacggggcccggggagttctggatgcaggtg720gatgactctgtggtggcccagaacatgcacgagaccatcctggaggccatgcgggccatg780agtgatgagttccgccctcgcagcaagagccagtcctcgtccaactgctctaaccccatc840agcgtccccctgcgccggcaccatctcaacaatcccccgcccagccaggtggggctgacc900cgccgatcacgcactgagagcatcaccgccacctccccggccagcatggtgggcgggaag960ccaggctccttccgtgtccgcgcctccagtgacggcgaaggcaccatgtcccgcccagcc1020tcggtggacggcagccctgtgagtcccagcaccaacagaacccacgcccaccggcatcgg1080ggcagcgcccggctgcaccccccgctcaaccacagccgctccatccccatgccggcttcc1140cgctgctcgccttcggccaccagcccggtcagtctgtcgtccagtagcaccagtggccat1200ggctccacctcggattgtctcttcccacggcgatctagtgcttcggtgtctggttccccc1260agcgatggcggtttcatctcctcggatgagtatggctccagtccctgcgatttccggagt1320tccttccgcagtgtcactccggattccctgggccacaccccaccagcccgcggtgaggag1380gagctaagcaactatatctgcatgggtggcaaggggccctccaccctgaccgcccccaac1440ggtcactacattttgtctcggggtggcaatggccaccgctgcaccccaggaacaggcttg1500ggcacgagtccagccttggctggggatgaagcagccagtgctgcagatctggataatcgg1560ttccgaaagagaactcactcggcaggcacatcccctaccattacccaccagaagaccccg1620tcccagtcctcagtggcttccattgaggagtacacagagatgatgcctgcctacccacca1680ggaggtggcagtggaggccgactgccgggacacaggcactccgccttcgtgcccacccgc1740tcctacccagaggagggtctggaaatgcaccccttggagcgtcggggggggcaccaccgc1800ccagacagctccaccctccacacggatgatggctacatgcccatgtccccaggggtggcc1860ccagtgcccagtggccgaaagggcagtggagactatatgcccatgagccccaagagcgta1920tctgccccacagcagatcatcaatcccatcagacgccatccccagagagtggaccccaat1980ggctacatgatgatgtcccccagcggtggctgctctcctgacattggaggtggccccagc2040agcagcagcagcagcagcaacgccgtcccttccgggaccagctatggaaagctgtggaca2100aacggggtagggggccaccactctcatgtcttgcctcaccccaaacccccagtggagagc2160agcggtggtaagctcttaccttgcacaggtgactacatgaacatgtcaccagtgggggac2220tccaacaccagcagcccctccgactgctactacggccctgaggacccccagcacaagcca2280gtcctctcctactactcattgccaagatcctttaagcacacccagcgccccggggagccg2340gaggagggtgcccggcatcagcacctccgcctttccactagctctggtcgccttctctat2400gctgcaacagcagatgattcttcctcttccaccagcagcgacagcctgggtgggggatac2460tgcggggctaggctggagcccagccttccacatccccaccatcaggttctgcagccccat2520ctgcctcgaaaggtggacacagctgctcagaccaatagccgcctggcccggcccacgagg2580ctgtccctgggggatcccaaggccagcaccttacctcgggcccgagagcagcagcagcag2640cagcagcccttgctgcaccctccagagcccaagagcccgggggaatatgtcaatattgaa2700tttgggagtgatcagtctggctacttgtctggcccggtggctttccacagctcaccttct2760gtcaggtgtccatcccagctccagccagctcccagagaggaagagactggcactgaggag2820tacatgaagatggacctggggccgggccggagggcagcctggcaggagagcactggggtc2880gagatgggcagactgggccctgcacctcccggggctgctagcatttgcaggcctacccgg2940gcagtgcccagcagccggggtgactacatgaccatgcagatgagttgtccccgtcagagc3000tacgtggacacctcgccagctgcccctgtaagctatgctgacatgcgaacaggcattgct3060gcagaggaggtgagcctgcccagggccaccatggctgctgcctcctcatcctcagcagcc3120tctgcttccccgactgggcctcaaggggcagcagagctggctgcccactcgtccctgctg3180gggggcccacaaggacctgggggcatgagcgccttcacccgggtgaacctcagtcctaac3240cgcaaccagagtgccaaagtgatccgtgcagacccacaagggtgccggcggaggcatagc3300tccgagactttctcctcaacacccagtgccacccgggtgggcaacacagtgccctttgga3360gcgggggcagcagtagggggcggtggcggtagcagcagcagcagcgaggatgtgaaacgc3420cacagctctgcttcctttgagaatgtgtggctgaggcctggggagcttgggggagccccc3480aaggagccagccaaactgtgtggggctgctgggggtttggagaatggtcttaactacata3540gacctggatttggtcaaggacttcaaacagtgccctcaggagtgcacccctgaaccgcag3600cctcccccacccccaccccctcatcaacccctgggcagcggtgagagcagctccacccgc3660cgctcaagtgaggatttaagcgcctatgccagcatcagtttccagaagcagccagaggac3720cgtcagtag37291071242prthomosapiens107metalaserproprogluseraspglypheseraspvalarglysval151015glytyrleuarglysprolyssermethislysargphephevalleu202530argalaalaserglualaglyglyproalaargleuglutyrtyrglu354045asnglulyslystrparghislysserseralaprolysargserile505560proleuglusercyspheasnileasnlysargalaaspserlysasn65707580lyshisleuvalalaleutyrthrargaspgluhisphealaileala859095alaaspserglualagluglnaspsertrptyrglnalaleuleugln100105110leuhisasnargalalysglyhishisaspglyalaalaalaleugly115120125alaglyglyglyglyglysercysserglyserserglyleuglyglu130135140alaglygluaspleusertyrglyaspvalproproglyproalaphe145150155160lysgluvaltrpglnvalileleulysprolysglyleuglyglnthr165170175lysasnleuileglyiletyrargleucysleuthrserlysthrile180185190serphevallysleuasnserglualaalaalavalvalleuglnleu195200205metasnileargargcysglyhissergluasnphephepheileglu210215220valglyargseralavalthrglyproglygluphetrpmetglnval225230235240aspaspservalvalalaglnasnmethisgluthrileleugluala245250255metargalametseraspglupheargproargserlysserglnser260265270serserasncysserasnproileservalproleuargarghishis275280285leuasnasnproproproserglnvalglyleuthrargargserarg290295300thrgluserilethralathrserproalasermetvalglyglylys305310315320proglyserpheargvalargalaserseraspglygluglythrmet325330335serargproalaservalaspglyserprovalserproserthrasn340345350argthrhisalahisarghisargglyseralaargleuhispropro355360365leuasnhisserargserileprometproalaserargcysserpro370375380seralathrserprovalserleuserserserserthrserglyhis385390395400glyserthrseraspcysleupheproargargserseralaserval405410415serglyserproseraspglyglypheileserseraspglutyrgly420425430serserprocysasppheargserserpheargservalthrproasp435440445serleuglyhisthrproproalaargglygluglugluleuserasn450455460tyrilecysmetglyglylysglyproserthrleuthralaproasn465470475480glyhistyrileleuserargglyglyasnglyhisargcysthrpro485490495glythrglyleuglythrserproalaleualaglyaspglualaala500505510seralaalaaspleuaspasnargphearglysargthrhisserala515520525glythrserprothrilethrhisglnlysthrproserglnserser530535540valalaserilegluglutyrthrglumetmetproalatyrpropro545550555560glyglyglyserglyglyargleuproglyhisarghisseralaphe565570575valprothrargsertyrproglugluglyleuglumethisproleu580585590gluargargglyglyhishisargproaspserserthrleuhisthr595600605aspaspglytyrmetprometserproglyvalalaprovalproser610615620glyarglysglyserglyasptyrmetprometserprolysserval625630635640seralaproglnglnileileasnproileargarghisproglnarg645650655valaspproasnglytyrmetmetmetserproserglyglycysser660665670proaspileglyglyglyproserserserserserserserasnala675680685valproserglythrsertyrglylysleutrpthrasnglyvalgly690695700glyhishisserhisvalleuprohisprolysproprovalgluser705710715720serglyglylysleuleuprocysthrglyasptyrmetasnmetser725730735provalglyaspserasnthrserserproseraspcystyrtyrgly740745750progluaspproglnhislysprovalleusertyrtyrserleupro755760765argserphelyshisthrglnargproglygluproglugluglyala770775780arghisglnhisleuargleuserthrserserglyargleuleutyr785790795800alaalathralaaspaspserserserserthrserseraspserleu805810815glyglyglytyrcysglyalaargleugluproserleuprohispro820825830hishisglnvalleuglnprohisleuproarglysvalaspthrala835840845alaglnthrasnserargleualaargprothrargleuserleugly850855860aspprolysalaserthrleuproargalaarggluglnglnglngln865870875880glnglnproleuleuhisproprogluprolysserproglyglutyr885890895valasnileglupheglyseraspglnserglytyrleuserglypro900905910valalaphehisserserproservalargcysproserglnleugln915920925proalaproarggluglugluthrglythrgluglutyrmetlysmet930935940aspleuglyproglyargargalaalatrpglngluserthrglyval945950955960glumetglyargleuglyproalaproproglyalaalaserilecys965970975argprothrargalavalproserserargglyasptyrmetthrmet980985990glnmetsercysproargglnsertyrvalaspthrserproalaala99510001005provalsertyralaaspmetargthrglyilealaalagluglu101010151020valserleuproargalathrmetalaalaalaserserserser102510301035alaalaseralaserprothrglyproglnglyalaalagluleu104010451050alaalahisserserleuleuglyglyproglnglyproglygly105510601065metseralaphethrargvalasnleuserproasnargasngln107010751080seralalysvalileargalaaspproglnglycysargargarg108510901095hissersergluthrpheserserthrproseralathrargval110011051110glyasnthrvalpropheglyalaglyalaalavalglyglygly111511201125glyglysersersersersergluaspvallysarghisserser113011351140alaserphegluasnvaltrpleuargproglygluleuglygly114511501155alaprolysgluproalalysleucysglyalaalaglyglyleu116011651170gluasnglyleuasntyrileaspleuaspleuvallysaspphe117511801185lysglncysproglnglucysthrprogluproglnpropropro119011951200proproproprohisglnproleuglyserglygluserserser120512101215thrargargsersergluaspleuseralatyralaserileser122012251230pheglnlysglnprogluasparggln123512401083723dnacanislupusmisc_feature(2356)..(2356)nisa,c,g,ort108atggcgagccctccggagaccgacggcttctcggacgtgcgcaaggtgggctacctgcgc60aaacccaagagcatgcacaagcgcttcttcgtgctgcgggcggccagcgaggcggggggc120ccggcgcgcctcgagtactacgagaacgagaagaagtggcggcacaagtcgagcgccccc180aaacgctcgatccccctcgagagctgcttcaacatcaacaagcgggcggactccaagaac240aagcacctggtggccctttacacccgggacgagcactttgccatcgcggcggacagcgag300gccgagcaggacagctggtaccaggccctcctgcagctgcacaaccgggccaagggccac360cacgacggcgcctcggcccccggggcgggaggcggcgggggcagctgcagcggcagctcg420ggcctcggggaggccggcgaggacttgagctacggggacgtgcccccgggacctgcgttc480aaggaggtctggcaggtgatcctgaaacccaagggcctggggcagacaaagaacctgatt540ggcatctaccgcctctgcctgaccagcaagaccatcagcttcgtgaagctgaactccgag600gcggcggccgtggtgctgcagctgatgaacatccgacgttgcggccactcggagaacttc660ttcttcatcgaagtgggccgttccgcagtgacgggacccggcgagttctggatgcaggtg720gatgactccgtggtggcccagaacatgcacgagaccatcctggaggccatgcgggccatg780agcgacgagttccgccctcggagtaagagccagtcctcctccaactgctccaaccccatc840agcgtccccctgcgccggcaccacctcaacaacccccctcccagccaggtggggctgacg900cgccgctcgcgcaccgagagcatcaccgccacctctccggccagcatggtgggcgggaag960cagggctccttccgtgtgcgcgcgtccagcgacggcgagggcaccatgtcccgcccggcc1020tcggtggacggcagccccgtgagcccgagcaccaccaggacccacgcgcaccggcatcgc1080ggcagctcccggctgcaccccccgctcaaccacagccgctccatccccatgccttcctct1140cgctgctcgccttccgccaccagcccggtcagcctgtcgtccagcagcaccagtggccac1200ggctccacctcggactgcctcttcccccggcgctctagtgcctctgtgtcgggttccccc1260agcgacggtggtttcatctcctctgacgagtacggctcgagtccctgcgatttccgaagt1320tccttccgcagtgtcaccccggattccctgggccacacccccccggcccgcggcgaggag1380gagctgagcaactacatctgcatgggaggcaaagggtcctccaccctcaccgcccccaac1440ggtcactacattttgcctcggggtggcaatggccaccgctacatcccgggggctggcttg1500ggcaccagcccggccctggctgcggatgaagcggccgctgcggccgacctggataaccgg1560ttccgaaagcggactcactccgcgggcacatcccctaccatttcccaccagaagaccccg1620tcccagtcttctgtggcttccattgaggagtacacggagatgatgcctgcctacccgcca1680ggaggtggcagtggaggccgactgcctggctaccggcactctgccttcgtgcccacccac1740tcctaccccgaggagggtctggaaatgcaccctctggacaggcgtgggggccaccaccgg1800ccggacgccgccgccctccacacggatgatggctacatgcccatgtccccgggagtggca1860ccggtgcccagcagccggaagggcagtggggactatatgcccatgagccccaagagcgtg1920tccgcgccgcagcagatcatcaaccccattagacgccatccccagagggtggaccccaat1980ggctacatgatgatgtccccaagcggcagctgctctcctgacattggaggtgggcccggc2040agcagcagcagcggcagcgccgccccttctgggagcagctatggcaagctgtggacaaac2100ggggtagggggccaccaccctcacgccctgccgcaccccaaactccccgtggagagcggg2160agtggcaagctcctgtcttgtaccggcgactacatgaacatgtcgccggtgggggactcc2220aacaccagcagcccctccgacggctactacggcccagaggacccccagcacaagccagtt2280ctctcctactactcattgccaaggtcctttaagcacacccagcgccctggggagctggag2340gagagcgcccggcacnagcacctccgcctctcctccagctcgggtcgtcttctctacgcc2400gcgacggcggaagattcctcctcctccaccagcagcgacagcctgggcccagggggatac2460tgtggggtcaggccggatcccggcctcccgcatatccaccatcaggtcctgcagcctcac2520ctgcctcggaaggtggacacggccgcgcagaccaacagccgcctggctcggcccacgagg2580ctgtccctgggggaccccaaggccagcaccttacctcgggttcgagagcagcagcacccg2640ccgcccctgctgcaccctccggagcccaagagccccggggaatatgtgaatattgagttc2700gggagcgatcagccgggctacttatcggggccggtggctgcccgcagctcgccttctgtc2760aggtgcccaccccagctccagccagctccccgcgaggaagagactggcaccgaggagtac2820atgaacatggacctggggcctggccggagggcagcctggcaggagggtgctggggtccag2880cccggcagggtgggccccgcgccccccggggccgctagcgtgtgcaggcccacccgggca2940gtgcccagcagccggggcgactacatgaccatgcaggtgggctgtcccggccagggctac3000gtggacacctcgccagtggcccccatcagctacgctgacatgcggacaggcattgtcgtg3060gaggaggccagcctgccgggggccacagcggccgccccctcctcggcctcggcagcctcg3120gcttcccccacggcgcctccaaaagcgggggagctggtggcccgctcctccctgctgggg3180ggcccgcagggacccgggggcatgagcgccttcacccgggtgaacctcagccccaaccgc3240aaccagagtgccaaagtgatccgcgccgacccgcaggggtgccggaggcggcatagctct3300gagaccttctcctccacgcccagtgccacccgggcgggcaacgcagtgcccttcggcggg3360ggggcggccctggggggcagcggtggcggcagcagcgcggaggatatgaaacgccacagt3420tcggcttcctttgagaacgtgtggctgaggcctggggagctcgggggagcccccaaggag3480ccggccccgcacgctggggccgccgggggtttggagaatgggcttaactacatagacctg3540gatttggtcaaggacttcaaacagtgctctcaggagcgcccccctcaaccgcagccgccc3600ccgcccccggcccctcatcagcctctgggcagcagtgagagcagttcaaccagccgctcc3660agcgaggatctaagcgcctatgccagcatcagtttccagaagcagccagaggacctccag3720tag37231091240prtcanislupusmisc_feature(786)..(786)xaacanbeanynaturallyoccurringaminoacid109metalaserproprogluthraspglypheseraspvalarglysval151015glytyrleuarglysprolyssermethislysargphephevalleu202530argalaalaserglualaglyglyproalaargleuglutyrtyrglu354045asnglulyslystrparghislysserseralaprolysargserile505560proleuglusercyspheasnileasnlysargalaaspserlysasn65707580lyshisleuvalalaleutyrthrargaspgluhisphealaileala859095alaaspserglualagluglnaspsertrptyrglnalaleuleugln100105110leuhisasnargalalysglyhishisaspglyalaseralaprogly115120125alaglyglyglyglyglysercysserglyserserglyleuglyglu130135140alaglygluaspleusertyrglyaspvalproproglyproalaphe145150155160lysgluvaltrpglnvalileleulysprolysglyleuglyglnthr165170175lysasnleuileglyiletyrargleucysleuthrserlysthrile180185190serphevallysleuasnserglualaalaalavalvalleuglnleu195200205metasnileargargcysglyhissergluasnphephepheileglu210215220valglyargseralavalthrglyproglygluphetrpmetglnval225230235240aspaspservalvalalaglnasnmethisgluthrileleugluala245250255metargalametseraspglupheargproargserlysserglnser260265270serserasncysserasnproileservalproleuargarghishis275280285leuasnasnproproproserglnvalglyleuthrargargserarg290295300thrgluserilethralathrserproalasermetvalglyglylys305310315320glnglyserpheargvalargalaserseraspglygluglythrmet325330335serargproalaservalaspglyserprovalserproserthrthr340345350argthrhisalahisarghisargglyserserargleuhispropro355360365leuasnhisserargserileprometproserserargcysserpro370375380seralathrserprovalserleuserserserserthrserglyhis385390395400glyserthrseraspcysleupheproargargserseralaserval405410415serglyserproseraspglyglypheileserseraspglutyrgly420425430serserprocysasppheargserserpheargservalthrproasp435440445serleuglyhisthrproproalaargglygluglugluleuserasn450455460tyrilecysmetglyglylysglyserserthrleuthralaproasn465470475480glyhistyrileleuproargglyglyasnglyhisargtyrilepro485490495glyalaglyleuglythrserproalaleualaalaaspglualaala500505510alaalaalaaspleuaspasnargphearglysargthrhisserala515520525glythrserprothrileserhisglnlysthrproserglnserser530535540valalaserilegluglutyrthrglumetmetproalatyrpropro545550555560glyglyglyserglyglyargleuproglytyrarghisseralaphe565570575valprothrhissertyrproglugluglyleuglumethisproleu580585590aspargargglyglyhishisargproaspalaalaalaleuhisthr595600605aspaspglytyrmetprometserproglyvalalaprovalproser610615620serarglysglyserglyasptyrmetprometserprolysserval625630635640seralaproglnglnileileasnproileargarghisproglnarg645650655valaspproasnglytyrmetmetmetserproserglysercysser660665670proaspileglyglyglyproglyserserserserglyseralaala675680685proserglysersertyrglylysleutrpthrasnglyvalglygly690695700hishisprohisalaleuprohisprolysleuprovalglusergly705710715720serglylysleuleusercysthrglyasptyrmetasnmetserpro725730735valglyaspserasnthrserserproseraspglytyrtyrglypro740745750gluaspproglnhislysprovalleusertyrtyrserleuproarg755760765serphelyshisthrglnargproglygluleuglugluseralaarg770775780hisxaahisleuargleuserserserserglyargleuleutyrala785790795800alathralagluaspserserserserthrserseraspserleugly805810815proglyglytyrcysglyvalargproaspproglyleuprohisile820825830hishisglnvalleuglnprohisleuproarglysvalaspthrala835840845alaglnthrasnserargleualaargprothrargleuserleugly850855860aspprolysalaserthrleuproargvalarggluglnglnhispro865870875880proproleuleuhisproprogluprolysserproglyglutyrval885890895asnileglupheglyseraspglnproglytyrleuserglyproval900905910alaalaargserserproservalargcysproproglnleuglnpro915920925alaproarggluglugluthrglythrgluglutyrmetasnmetasp930935940leuglyproglyargargalaalatrpglngluglyalaglyvalgln945950955960proglyargvalglyproalaproproglyalaalaservalcysarg965970975prothrargalavalproserserargglyasptyrmetthrmetgln980985990valglycysproglyglnglytyrvalaspthrserprovalalapro99510001005ilesertyralaaspmetargthrglyilevalvalglugluala101010151020serleuproglyalathralaalaalaproserseralaserala102510301035alaseralaserprothralaproprolysalaglygluleuval104010451050alaargserserleuleuglyglyproglnglyproglyglymet105510601065seralaphethrargvalasnleuserproasnargasnglnser107010751080alalysvalileargalaaspproglnglycysargargarghis108510901095sersergluthrpheserserthrproseralathrargalagly110011051110asnalavalpropheglyglyglyalaalaleuglyglysergly111511201125glyglyserseralagluaspmetlysarghisserseralaser113011351140phegluasnvaltrpleuargproglygluleuglyglyalapro114511501155lysgluproalaprohisalaglyalaalaglyglyleugluasn116011651170glyleuasntyrileaspleuaspleuvallysaspphelysgln117511801185cysserglngluargproproglnproglnpropropropropro119011951200alaprohisglnproleuglysersergluserserserthrser120512101215argsersergluaspleuseralatyralaserileserphegln122012251230lysglnprogluaspleugln123512401103033dnahomosapiens110atgggctatttgtgtgttaatttcatttggttcttgggaataacgactcaccgcgttgat60ttaaagaaagaactaaaattccagatggcaaactcaatgaatggcagaaaccctggtggt120cgaggaggaaatccccgaaaaggtcgaattttgggtattattgatgctattcaggatgca180gttggaccccctaagcaagctgccgcagatcgcaggaccgtggagaagacttggaagctc240atggacaaagtggtaagactgtgccaaaatcccaaacttcagttgaaaaatagcccacca300tatatacttgatattttgcctgatacatatcagcatttacgacttatattgagtaaatat360gatgacaaccagaaacttgcccaactcagtgagaatgagtactttaaaatctacattgat420agccttatgaaaaagtcaaaacgggcaataagactctttaaagaaggcaaggagagaatg480tatgaagaacagtcacaggacagacgaaatctcacaaaactgtcccttatcttcagtcac540atgctggcagaaatcaaagcaatctttcccaatggtcaattccagggagataactttcgt600atcacaaaagcagatgctgctgaattctggagaaagttttttggagacaaaactatcgta660ccatggaaagtattcagacagtgccttcatgaggtccaccagattagctctggcctggaa720gcaatggctctaaaatcaacaattgatttaacttgcaatgattacatttcagtttttgaa780tttgatatttttaccaggctgtttcagccttggggctctattttgcggaattggaatttc840ttagctgtgacacatccaggttacatggcatttctcacatatgatgaagttaaagcacga900ctacagaaatatagcaccaaacccggaagctatattttccggttaagttgcactcgattg960ggacagtgggccattggctatgtgactggggatgggaatatcttacagaccatacctcat1020aacaagcccttatttcaagccctgattgatggcagcagggaaggattttatctttatcct1080gatgggaggagttataatcctgatttaactggattatgtgaacctacacctcatgaccat1140ataaaagttacacaggaacaatatgaattatattgtgaaatgggctccacttttcagctc1200tgtaagatttgtgcagagaatgacaaagatgtcaagattgagccttgtgggcatttgatg1260tgcacctcttgccttacggcatggcaggagtcggatggtcagggctgccctttctgtcgt1320tgtgaaataaaaggaactgagcccataatcgtggacccctttgatccaagagatgaaggc1380tccaggtgttgcagcatcattgacccctttggcatgccgatgctagacttggacgacgat1440gatgatcgtgaggagtccttgatgatgaatcggttggcaaacgtccgaaagtgcactgac1500aggcagaactcaccagtcacatcaccaggatcctctccccttgcccagagaagaaagcca1560cagcctgacccactccagatcccacatctaagcctgccacccgtgcctcctcgcctggat1620ctaattcagaaaggcatagttagatctccctgtggcagcccaacgggttcaccaaagtct1680tctccttgcatggtgagaaaacaagataaaccactcccagcaccacctcctcccttaaga1740gatcctcctccaccgccacctgaaagacctccaccaatcccaccagacaatagactgagt1800agacacatccatcatgtggaaagcgtgccttccagagacccgccaatgcctcttgaagca1860tggtgccctcgggatgtgtttgggactaatcagcttgtgggatgtcgactcctaggggag1920ggctctccaaaacctggaatcacagcgagttcaaatgtcaatggaaggcacagtagagtg1980ggctctgacccagtgcttatgcggaaacacagacgccatgatttgcctttagaaggagct2040aaggtcttttccaatggtcaccttggaagtgaagaatatgatgttcctccccggctttct2100cctcctcctccagttaccaccctcctccctagcataaagtgtactggtccgttagcaaat2160tctctttcagagaaaacaagagacccagtagaggaagatgatgatgaatacaagattcct2220tcatcccaccctgtttccctgaattcacaaccatctcattgtcataatgtaaaacctcct2280gttcggtcttgtgataatggtcactgtatgctgaatggaacacatggtccatcttcagag2340aagaaatcaaacatccctgacttaagcatatatttaaagggagatgtttttgattcagcc2400tctgatcccgtgccattaccacctgccaggcctccaactcgggacaatccaaagcatggt2460tcttcactcaacaggacgccctctgattatgatcttctcatccctccattaggtgaagat2520gcttttgatgccctccctccatctctcccacctcccccacctcctgcaaggcatagtctc2580attgaacattcaaaacctcctggctccagtagccggccatcctcaggacaggatcttttt2640cttcttccttcagatccctttgttgatctagcaagtggccaagttcctttgcctcctgct2700agaaggttaccaggtgaaaatgtcaaaactaacagaacatcacaggactatgatcagctt2760ccttcatgttcagatggttcacaggcaccagccagaccccctaaaccacgaccgcgcagg2820actgcaccagaaattcaccacagaaaaccccatgggcctgaggcggcattggaaaatgtc2880gatgcaaaaattgcaaaactcatgggagagggttatgcctttgaagaggtgaagagagcc2940ttagagatagcccagaataatgtcgaagttgcccggagcatcctccgagaatttgccttc3000cctcctccagtatccccacgtctaaatctatag30331111010prthomosapiens111metglytyrleucysvalasnpheiletrppheleuglyilethrthr151015hisargvalaspleulyslysgluleulyspheglnmetalaasnser202530metasnglyargasnproglyglyargglyglyasnproarglysgly354045argileleuglyileileaspalaileglnaspalavalglypropro505560lysglnalaalaalaaspargargthrvalglulysthrtrplysleu65707580metasplysvalvalargleucysglnasnprolysleuglnleulys859095asnserproprotyrileleuaspileleuproaspthrtyrglnhis100105110leuargleuileleuserlystyraspaspasnglnlysleualagln115120125leusergluasnglutyrphelysiletyrileaspserleumetlys130135140lysserlysargalaileargleuphelysgluglylysgluargmet145150155160tyrglugluglnserglnaspargargasnleuthrlysleuserleu165170175ilepheserhismetleualagluilelysalailepheproasngly180185190glnpheglnglyaspasnpheargilethrlysalaaspalaalaglu195200205phetrparglysphepheglyasplysthrilevalprotrplysval210215220pheargglncysleuhisgluvalhisglnileserserglyleuglu225230235240alametalaleulysserthrileaspleuthrcysasnasptyrile245250255servalpheglupheaspilephethrargleupheglnprotrpgly260265270serileleuargasntrpasnpheleualavalthrhisproglytyr275280285metalapheleuthrtyraspgluvallysalaargleuglnlystyr290295300serthrlysproglysertyrilepheargleusercysthrargleu305310315320glyglntrpalaileglytyrvalthrglyaspglyasnileleugln325330335thrileprohisasnlysproleupheglnalaleuileaspglyser340345350arggluglyphetyrleutyrproaspglyargsertyrasnproasp355360365leuthrglyleucysgluprothrprohisasphisilelysvalthr370375380glngluglntyrgluleutyrcysglumetglyserthrpheglnleu385390395400cyslysilecysalagluasnasplysaspvallysilegluprocys405410415glyhisleumetcysthrsercysleuthralatrpglngluserasp420425430glyglnglycysprophecysargcysgluilelysglythrglupro435440445ileilevalasppropheaspproargaspgluglyserargcyscys450455460serileileasppropheglymetprometleuaspleuaspaspasp465470475480aspaspargglugluserleumetmetasnargleualaasnvalarg485490495lyscysthraspargglnasnserprovalthrserproglyserser500505510proleualaglnargarglysproglnproaspproleuglnilepro515520525hisleuserleuproprovalproproargleuaspleuileglnlys530535540glyilevalargserprocysglyserprothrglyserprolysser545550555560serprocysmetvalarglysglnasplysproleuproalapropro565570575proproleuargaspproproproproproprogluargpropropro580585590ileproproaspasnargleuserarghisilehishisvalgluser595600605valproserargaspproprometproleuglualatrpcysproarg610615620aspvalpheglythrasnglnleuvalglycysargleuleuglyglu625630635640glyserprolysproglyilethralaserserasnvalasnglyarg645650655hisserargvalglyseraspprovalleumetarglyshisargarg660665670hisaspleuproleugluglyalalysvalpheserasnglyhisleu675680685glysergluglutyraspvalproproargleuserpropropropro690695700valthrthrleuleuproserilelyscysthrglyproleualaasn705710715720serleuserglulysthrargaspprovalglugluaspaspaspglu725730735tyrlysileproserserhisprovalserleuasnserglnproser740745750hiscyshisasnvallysproprovalargsercysaspasnglyhis755760765cysmetleuasnglythrhisglyproserserglulyslysserasn770775780ileproaspleuseriletyrleulysglyaspvalpheaspserala785790795800seraspprovalproleuproproalaargproprothrargaspasn805810815prolyshisglyserserleuasnargthrproserasptyraspleu820825830leuileproproleuglygluaspalapheaspalaleuproproser835840845leuproproproproproproalaarghisserleuilegluhisser850855860lysproproglyserserserargproserserglyglnaspleuphe865870875880leuleuproseraspprophevalaspleualaserglyglnvalpro885890895leuproproalaargargleuproglygluasnvallysthrasnarg900905910thrserglnasptyraspglnleuprosercysseraspglysergln915920925alaproalaargproprolysproargproargargthralaproglu930935940ilehishisarglysprohisglyproglualaalaleugluasnval945950955960aspalalysilealalysleumetglygluglytyralaphegluglu965970975vallysargalaleugluilealaglnasnasnvalgluvalalaarg980985990serileleuarggluphealapheproproprovalserproargleu99510001005asnleu101011219dnaartificialsequencesirna/shrnaisequencesforhumancbl-b112gcctgatacatatcagcat1911319dnaartificialsequencesirna/shrnaisequencesforhumancbl-b113gcggaattggaatttctta1911419dnaartificialsequencesirna/shrnaisequencesforhumancbl-b114gcatgccgatgctagactt1911519dnaartificialsequencesirna/shrnaisequencesforhumancbl-b115gcctgatacatatcagcat1911621dnaartificialsequencesirna/shrnaisequencesforhumancbl-b116ggagagaatgtatgaagaaca2111721dnaartificialsequencesirna/shrnaisequencesforhumancbl-b117gcggaattggaatttcttagc2111821dnaartificialsequencesirna/shrnaisequencesforhumancbl-b118gcacgactacagaaatatagc2111921dnaartificialsequencesirna/shrnaisequencesforhumancbl-b119ggaatatcttacagaccatac2112021dnaartificialsequencesirna/shrnaisequencesforhumancbl-b120gcaccaaacccggaagctata2112121dnaartificialsequencesirna/shrnaisequencesforhumancbl-b121gcctggatctaattcagaaag2112221dnaartificialsequencesirna/shrnaisequencesforhumancbl-b122ggaatcacagcgagttcaaat2112321dnaartificialsequencesirna/shrnaisequencesforhumancbl-b123ggaacacatggtccatcttca2112421dnaartificialsequencesirna/shrnaisequencesforhumancbl-b124gcatagtctcattgaacattc2112520dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b125gttgcgtttccacgtctcgg2012620dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b126gaacagctcgctcccgaaga2012720dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b127attgttgcgtttccacgtct2012820dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b128agtgctgctgcggcgtcccg2012920dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b129aggaggaggagaccgctcgc2013020dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b130gaaggagcaacccagcgcgc2013120dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b131gcgcgcaggcctccgagacg2013220dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b132cgtctcggaggcctgcgcgc2013320dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b133gtcccgcggcctccccgagt2013420dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b134ctcccctcccgcccgactcg2013520dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b135gacgccgcagcagcactagc2013620dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b136gtctcggaggcctgcgcgct2013720dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b137gcggcctccccgagtcgggc2013820dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b138ccctcccgcccgactcgggg2013920dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b139cgcggcctccccgagtcggg2014020dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b140ctccccgagtcgggcgggag2014120dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b141cgggtgtggatttgtcttga2014220dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b142gcctccccgagtcgggcggg2014320dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b143tcccgcggcctccccgagtc2014420dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b144cgcccgactcggggaggccg2014520dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b145ctctcccctcccgcccgact2014620dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b146tctcccctcccgcccgactc2014720dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b147agcgatcccactcccagccg2014820dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b148tcagcgatcccactcccagc2014920dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b149cgctgggttgctccttcttc2015020dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b150gcccgactcggggaggccgc2015120dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b151gcgctgggttgctccttctt2015220dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b152cctccccgagtcgggcggga2015320dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b153tgtgtgtggggagccccggc2015420dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b154gtgtgtggggagccccggct2015520dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b155cgctggacaccccacccctg2015620dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b156gccgcagcagcactagcagg2015720dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b157cggggctccccacacacact2015820dnaartificialsequencecrispr/cas9targetsequencesforhumancbl-b158ctgggtcctgtgtgtgccac201592952dnacanislupus159atggcaaattctatgaatggcagaaaccctggtggtcgaggaggaaacccccgaaaagga60cggattttgggtatcattgatgctattcaagatgcagttggacctccgaagcaagcagca120gcagatcgcaggacggtggagaaaacttggaaactcatggacaaagtggtcagactgtgt180caaaatcccaagcttcagttgaaaaatagcccaccatatatacttgatatcttacctgat240acatatcagcatttacgacttatactgagtaaatatgatgacaaccagaaacttgcccaa300ctcagtgagaatgagtattttaaaatctacatcgatagtctaatgaaaaagtcaaagcgg360gcaataagactctttaaagaaggcaaggagaggatgtatgaagagcagtcacaggacaga420cgaaatctcacaaaactgtcccttatcttcagtcacatgctggcagaaatcaaagcaatc480tttcccaatgggcagttccagggagataactttcgtatcacgaaagcagatgctgctgaa540ttctggagaaagttttttggagacaaaactattgtaccatggaaagtattcagacagtgc600cttcatgaggttcatcaaattagctctggcctggaagcaatggctctgaaatcaacaatt660gatttaacttgtaatgattacatttcagtttttgaatttgatatttttaccaggctcttt720cagccttggggctctattttacggaattggaatttcttagctgtaacacatccaggttac780atggcatttctcacatacgatgaagttaaagcacgactgcagaaatacagcaccaaacct840ggaagctacattttccggttaagctgcaccagattgggacagtgggccattggctatgtg900acaggggatggcaatatcttacagaccataccacataacaagcccttgtttcaagccctg960attgatggcagcagggaaggattctatctttatcctgatgggaggagttataatcctgat1020ttaactggattatgtgaacccacaccacatgaccatataaaagttacgcaggaacaatat1080gaattatattgtgaaatgggctccacttttcagctctgtaaaatttgtgctgagaacgac1140aaagatgtcaagattgagccctgtgggcatttgatgtgcacctcttgccttacagcgtgg1200caggagtcggacggccaaggctgccccttttgccgctgtgaaataaaaggaacagagccc1260ataatcgtggacccctttgatccaagagatgaaggttccaggtgctgtagcatcattgac1320ccctttggaatgccaatgctggacctggatgatgacgatgaccgagaagagtccttgatg1380atgaatcggttggcaaatgttcgaaagtgcactgataggcaaaattcaccagtcacatca1440ccaggatcctctccccttgcacagagaagaaagccacatccagatcctctccagatccca1500catctgagcctgccaccagtacctcctcgcctggatctaattcagaaaggcatagttcgg1560tctccctgtggcagtcccactggttcaccaaagtcttctccttgcatggtgagaaaacaa1620gataaaccactcccagcaccgcctcctcccttaagagatcctcctccacctccccctgag1680agacctcccccgatcccacctgacaacagactgagtcgacacttccatcacgtggaaagt1740gtgccttctagagaccagccaatgcctcttgaagcctggtgccctcgggatgtgtttgga1800actaatcagtcagtgggttgtcgacaattaggggatggctctccaaagcctggaatcaca1860gcaagttcaaatgtaaatggaaggcacagtagaatgggctctgaccctgtgcttctgcga1920aaacacagacgccacgatttgcctttagaaggagccaaggtcttttccaatggtcacctg1980ggaagcgaagagtacgatgttcctccccggctttcacctcctcctccagctgccaccctt2040gtccctagcatcaagtgtactggcccgttagcaaatcccctttcagagaaaaccagagac2100ccagtcgaggaagatgatgatgaatacaagattccttcatcccatcctgtttccctgaat2160tcacaaccatctcattgccataacgtaaaacctcctcttaggtcttgtgataatggtcat2220tgtgtattgaatggaacacatggtacatcttcagaggtgaagaaatcaaacatccctgaa2280ttaggcatttatttaaagggagatgtttttgattcagcctctgatccagtgccattacca2340cctgccaggcctccaactcgggacaatccaaagcatggttcttcactcaacaggacgccc2400tctgattatgatcttctcatccctccattaggtgaagatgcttttgatgccctcccccca2460tccctcccgcctcccccacctcccgcaaggcacagcctcatcgaacactctaaacctccc2520ggctccaatagccgaccatcctcaggacaggaccttttccttcttccttcagaccccttc2580tttgatccagtaagtggtcaagtccctctgcctcctgctaggagattaccaggggaaaat2640gtcaaatccaacagaacatcacaggactatgatcagcttccttcagcttcagatggttcg2700caggcaccagcccggcctcccaagccgcgcccgcgcaggaccgcccccgaggtccagcac2760cggaagccccacgggcccgaggcagcgtcggaaaacgtggacgcgaagatcgccaaactc2820atgggggagggctacgccttcgaggaagtgaagagggcgctggagatcgcccagaacaac2880gtcgaggtggcccggagcatcctgcgcgagttcgcctacccgccgcccgtctccccgcgg2940ctgcacctctag2952160983prtcanislupus160metalaasnsermetasnglyargasnproglyglyargglyglyasn151015proarglysglyargileleuglyileileaspalaileglnaspala202530valglyproprolysglnalaalaalaaspargargthrvalglulys354045thrtrplysleumetasplysvalvalargleucysglnasnprolys505560leuglnleulysasnserproprotyrileleuaspileleuproasp65707580thrtyrglnhisleuargleuileleuserlystyraspaspasngln859095lysleualaglnleusergluasnglutyrphelysiletyrileasp100105110serleumetlyslysserlysargalaileargleuphelysglugly115120125lysgluargmettyrglugluglnserglnaspargargasnleuthr130135140lysleuserleuilepheserhismetleualagluilelysalaile145150155160pheproasnglyglnpheglnglyaspasnpheargilethrlysala165170175aspalaalagluphetrparglysphepheglyasplysthrileval180185190protrplysvalpheargglncysleuhisgluvalhisglnileser195200205serglyleuglualametalaleulysserthrileaspleuthrcys210215220asnasptyrileservalpheglupheaspilephethrargleuphe225230235240glnprotrpglyserileleuargasntrpasnpheleualavalthr245250255hisproglytyrmetalapheleuthrtyraspgluvallysalaarg260265270leuglnlystyrserthrlysproglysertyrilepheargleuser275280285cysthrargleuglyglntrpalaileglytyrvalthrglyaspgly290295300asnileleuglnthrileprohisasnlysproleupheglnalaleu305310315320ileaspglyserarggluglyphetyrleutyrproaspglyargser325330335tyrasnproaspleuthrglyleucysgluprothrprohisasphis340345350ilelysvalthrglngluglntyrgluleutyrcysglumetglyser355360365thrpheglnleucyslysilecysalagluasnasplysaspvallys370375380ilegluprocysglyhisleumetcysthrsercysleuthralatrp385390395400glngluseraspglyglnglycysprophecysargcysgluilelys405410415glythrgluproileilevalasppropheaspproargaspglugly420425430serargcyscysserileileasppropheglymetprometleuasp435440445leuaspaspaspaspaspargglugluserleumetmetasnargleu450455460alaasnvalarglyscysthraspargglnasnserprovalthrser465470475480proglyserserproleualaglnargarglysprohisproasppro485490495leuglnileprohisleuserleuproprovalproproargleuasp500505510leuileglnlysglyilevalargserprocysglyserprothrgly515520525serprolysserserprocysmetvalarglysglnasplysproleu530535540proalaproproproproleuargaspproproproproproproglu545550555560argproproproileproproaspasnargleuserarghisphehis565570575hisvalgluservalproserargaspglnprometproleugluala580585590trpcysproargaspvalpheglythrasnglnservalglycysarg595600605glnleuglyaspglyserprolysproglyilethralaserserasn610615620valasnglyarghisserargmetglyseraspprovalleuleuarg625630635640lyshisargarghisaspleuproleugluglyalalysvalpheser645650655asnglyhisleuglysergluglutyraspvalproproargleuser660665670proproproproalaalathrleuvalproserilelyscysthrgly675680685proleualaasnproleuserglulysthrargaspprovalgluglu690695700aspaspaspglutyrlysileproserserhisprovalserleuasn705710715720serglnproserhiscyshisasnvallysproproleuargsercys725730735aspasnglyhiscysvalleuasnglythrhisglythrserserglu740745750vallyslysserasnileprogluleuglyiletyrleulysglyasp755760765valpheaspseralaseraspprovalproleuproproalaargpro770775780prothrargaspasnprolyshisglyserserleuasnargthrpro785790795800serasptyraspleuleuileproproleuglygluaspalapheasp805810815alaleuproproserleuproproproproproproalaarghisser820825830leuilegluhisserlysproproglyserasnserargproserser835840845glyglnaspleupheleuleuproseraspprophepheaspproval850855860serglyglnvalproleuproproalaargargleuproglygluasn865870875880vallysserasnargthrserglnasptyraspglnleuproserala885890895seraspglyserglnalaproalaargproprolysproargproarg900905910argthralaprogluvalglnhisarglysprohisglyprogluala915920925alasergluasnvalaspalalysilealalysleumetglyglugly930935940tyralapheglugluvallysargalaleugluilealaglnasnasn945950955960valgluvalalaargserileleuarggluphealatyrpropropro965970975valserproargleuhisleu98016119dnaartificialsequencesirna/shrnasequencesforcaninecbl-b161cccaccatatatacttgat1916219dnaartificialsequencesirna/shrnasequencesforcaninecbl-b162cctgatacatatcagcatt1916319dnaartificialsequencesirna/shrnasequencesforcaninecbl-b163gcgggcaataagactcttt1916419dnaartificialsequencesirna/shrnasequencesforcaninecbl-b164gcagaaatacagcaccaaa1916519dnaartificialsequencesirna/shrnasequencesforcaninecbl-b165gcaccaaacctggaagcta1916619dnaartificialsequencesirna/shrnasequencesforcaninecbl-b166gcaatatcttacagaccat1916719dnaartificialsequencesirna/shrnasequencesforcaninecbl-b167ccacaccacatgaccatat1916819dnaartificialsequencesirna/shrnasequencesforcaninecbl-b168gcctcctcccttaagagat1916919dnaartificialsequencesirna/shrnasequencesforcaninecbl-b169ccttcatcccatcctgttt1917019dnaartificialsequencesirna/shrnasequencesforcaninecbl-b170cctctgatccagtgccatt1917123dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b171cccccgaaaaggacggattttgg2317223dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b172ccccgaaaaggacggattttggg2317323dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b173ccaaaatccgtccttttcggggg2317423dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b174cccaaaatccgtccttttcgggg2317523dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b175cgaggaggaaacccccgaaaagg2317623dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b176gggtttcctcctcgaccaccagg2317723dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b177tacccaaaatccgtccttttcgg2317823dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b178agcaagcagcagcagatcgcagg2317923dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b179acccaaaatccgtccttttcggg2318023dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b180ggtttcctcctcgaccaccaggg2318123dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b181tctgctgctgcttgcttcggagg2318223dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b182agaaaccctggtggtcgaggagg2318323dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b183ggcagaaaccctggtggtcgagg2318423dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b184agcagcagcagatcgcaggacgg2318523dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b185agcagcagatcgcaggacggtgg2318623dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b186gaggaaacccccgaaaaggacgg2318723dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b187gatgctattcaagatgcagttgg2318823dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b188tctatgaatggcagaaaccctgg2318923dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b189cgatctgctgctgcttgcttcgg2319023dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b190gcaggacggtggagaaaacttgg2319123dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b191atgaatggcagaaaccctggtgg2319223dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl-b192ggagaaaacttggaaactcatgg231932721dnahomosapiens193atggccggcaacgtgaagaagagctctggggccgggggcggcagcggctccgggggctcg60ggttcgggtggcctgattgggctcatgaaggacgccttccagccgcaccaccaccaccac120caccacctcagcccccacccgccggggacggtggacaagaagatggtggagaagtgctgg180aagctcatggacaaggtggtgcggttgtgtcagaacccaaagctggcgctaaagaatagc240ccaccttatatcttagacctgctaccagatacctaccagcatctccgtactatcttgtca300agatatgaggggaagatggagacacttggagaaaatgagtattttagggtgtttatggag360aatttgatgaagaaaactaagcaaaccataagcctcttcaaggagggaaaagaaagaatg420tatgaggagaattctcagcctaggcgaaacctaaccaaactgtccctcatcttcagccac480atgctggcagaactaaaaggaatctttccaagtggactctttcagggagacacatttcgg540attactaaagcagatgctgcggaattttggagaaaagcttttggggaaaagacaatagtc600ccttggaagagctttcgacaggctctacatgaagtgcatcccatcagttctgggctggag660gccatggctctgaaatccactattgatctgacctgcaatgattatatttcggtttttgaa720tttgacatctttacccgactctttcagccctggtcctctttgctcaggaattggaacagc780cttgctgtaactcatcctggctacatggcttttttgacgtatgacgaagtgaaagctcgg840ctccagaaattcattcacaaacctggcagttatatcttccggctgagctgtactcgtctg900ggtcagtgggctattgggtatgttactgctgatgggaacattctccagacaatccctcac960aataaacctctcttccaagcactgattgatggcttcagggaaggcttctatttgtttcct1020gatggacgaaatcagaatcctgatctgactggcttatgtgaaccaactccccaagaccat1080atcaaagtgacccaggaacaatatgaattatactgtgagatgggctccacattccaacta1140tgtaaaatatgtgctgaaaatgataaggatgtaaagattgagccctgtggacacctcatg1200tgcacatcctgtcttacatcctggcaggaatcagaaggtcagggctgtcctttctgccga1260tgtgaaattaaaggtactgaacccatcgtggtagatccgtttgatcctagagggagtggc1320agcctgttgaggcaaggagcagagggagctccctccccaaattatgatgatgatgatgat1380gaacgagctgatgatactctcttcatgatgaaggaattggctggtgccaaggtggaacgg1440ccgccttctccattctccatggccccacaagcttcccttcccccggtgccaccacgactt1500gaccttctgccgcagcgagtatgtgttccctcaagtgcttctgctcttggaactgcttct1560aaggctgcttctggctcccttcataaagacaaaccattgccagtacctcccacacttcga1620gatcttccaccaccaccgcctccagaccggccatattctgttggagcagaatcccgacct1680caaagacgccccttgccttgtacaccaggcgactgtccctccagagacaaactgccccct1740gtcccctctagccgccttggagactcatggctgccccggccaatccccaaagtaccagta1800tctgccccaagttccagtgatccctggacaggaagagaattaaccaaccggcactcactt1860ccattttcattgccctcacaaatggagcccagaccagatgtgcctaggctcggaagcacg1920ttcagtctggatacctccatgagtatgaatagcagcccattagtaggtccagagtgtgac1980caccccaaaatcaaaccttcctcatctgccaatgccatttattctctggctgccagacct2040cttcctgtgccaaaactgccacctggggagcaatgtgagggtgaagaggacacagagtac2100atgactccctcttccaggcctctacggcctttggatacatcccagagttcacgagcatgt2160gattgcgaccagcagattgatagctgtacgtatgaagcaatgtataatattcagtcccag2220gcgccatctatcaccgagagcagcacctttggtgaagggaatttggccgcagcccatgcc2280aacactggtcccgaggagtcagaaaatgaggatgatgggtatgatgtcccaaagccacct2340gtgccggccgtgctggcccgccgaactctctcagatatctctaatgccagctcctccttt2400ggctggttgtctctggatggtgatcctacaacaaatgtcactgaaggttcccaagttccc2460gagaggcctccaaaaccattcccgcggagaatcaactctgaacggaaagctggcagctgt2520cagcaaggtagtggtcctgccgcctctgctgccaccgcctcacctcagctctccagtgag2580atcgagaacctcatgagtcaggggtactcctaccaggacatccagaaagctttggtcatt2640gcccagaacaacatcgagatggccaaaaacatcctccgggaatttgtttccatttcttct2700cctgcccatgtagctacctag2721194906prthomosapiens194metalaglyasnvallyslysserserglyalaglyglyglysergly151015serglyglyserglyserglyglyleuileglyleumetlysaspala202530pheglnprohishishishishishishisleuserprohispropro354045glythrvalasplyslysmetvalglulyscystrplysleumetasp505560lysvalvalargleucysglnasnprolysleualaleulysasnser65707580proprotyrileleuaspleuleuproaspthrtyrglnhisleuarg859095thrileleuserargtyrgluglylysmetgluthrleuglygluasn100105110glutyrpheargvalphemetgluasnleumetlyslysthrlysgln115120125thrileserleuphelysgluglylysgluargmettyrglugluasn130135140serglnproargargasnleuthrlysleuserleuilepheserhis145150155160metleualagluleulysglyilepheproserglyleupheglngly165170175aspthrpheargilethrlysalaaspalaalagluphetrparglys180185190alapheglyglulysthrilevalprotrplysserpheargglnala195200205leuhisgluvalhisproileserserglyleuglualametalaleu210215220lysserthrileaspleuthrcysasnasptyrileservalpheglu225230235240pheaspilephethrargleupheglnprotrpserserleuleuarg245250255asntrpasnserleualavalthrhisproglytyrmetalapheleu260265270thrtyraspgluvallysalaargleuglnlyspheilehislyspro275280285glysertyrilepheargleusercysthrargleuglyglntrpala290295300ileglytyrvalthralaaspglyasnileleuglnthrileprohis305310315320asnlysproleupheglnalaleuileaspglyphearggluglyphe325330335tyrleupheproaspglyargasnglnasnproaspleuthrglyleu340345350cysgluprothrproglnasphisilelysvalthrglngluglntyr355360365gluleutyrcysglumetglyserthrpheglnleucyslysilecys370375380alagluasnasplysaspvallysilegluprocysglyhisleumet385390395400cysthrsercysleuthrsertrpglnglusergluglyglnglycys405410415prophecysargcysgluilelysglythrgluproilevalvalasp420425430propheaspproargglyserglyserleuleuargglnglyalaglu435440445glyalaproserproasntyraspaspaspaspaspgluargalaasp450455460aspthrleuphemetmetlysgluleualaglyalalysvalgluarg465470475480proproserprophesermetalaproglnalaserleuproproval485490495proproargleuaspleuleuproglnargvalcysvalproserser500505510alaseralaleuglythralaserlysalaalaserglyserleuhis515520525lysasplysproleuprovalproprothrleuargaspleupropro530535540proproproproaspargprotyrservalglyalagluserargpro545550555560glnargargproleuprocysthrproglyaspcysproserargasp565570575lysleuproprovalproserserargleuglyaspsertrpleupro580585590argproileprolysvalprovalseralaproserserserasppro595600605trpthrglyarggluleuthrasnarghisserleupropheserleu610615620proserglnmetgluproargproaspvalproargleuglyserthr625630635640pheserleuaspthrsermetsermetasnserserproleuvalgly645650655proglucysasphisprolysilelysproserserseralaasnala660665670iletyrserleualaalaargproleuprovalprolysleupropro675680685glygluglncysgluglyglugluaspthrglutyrmetthrproser690695700serargproleuargproleuaspthrserglnserserargalacys705710715720aspcysaspglnglnileaspsercysthrtyrglualamettyrasn725730735ileglnserglnalaproserilethrgluserserthrpheglyglu740745750glyasnleualaalaalahisalaasnthrglyproglugluserglu755760765asngluaspaspglytyraspvalprolysproprovalproalaval770775780leualaargargthrleuseraspileserasnalaserserserphe785790795800glytrpleuserleuaspglyaspprothrthrasnvalthrglugly805810815serglnvalprogluargproprolyspropheproargargileasn820825830sergluarglysalaglysercysglnglnglyserglyproalaala835840845seralaalathralaserproglnleusersergluilegluasnleu850855860metserglnglytyrsertyrglnaspileglnlysalaleuvalile865870875880alaglnasnasnileglumetalalysasnileleuargglupheval885890895serileserserproalahisvalalathr90090519519dnaartificialsequencesirna/shrnaisequencesforhumancbl195ccagacaatccctcacaat1919619dnaartificialsequencesirna/shrnaisequencesforhumancbl196ggacacctcatgtgcacat1919719dnaartificialsequencesirna/shrnaisequencesforhumancbl197ccaggcctctacggccttt1919819dnaartificialsequencesirna/shrnaisequencesforhumancbl198ccagaaagctttggtcatt1919921dnaartificialsequencesirna/shrnaisequencesforhumancbl199gcctgattgggctcatgaagg2120021dnaartificialsequencesirna/shrnaisequencesforhumancbl200gggaacattctccagacaatc2120121dnaartificialsequencesirna/shrnaisequencesforhumancbl201gcttcagggaaggcttctatt2120221dnaartificialsequencesirna/shrnaisequencesforhumancbl202gggaaggcttctatttgtttc2120321dnaartificialsequencesirna/shrnaisequencesforhumancbl203ggacacctcatgtgcacatcc2120421dnaartificialsequencesirna/shrnaisequencesforhumancbl204gcagaatcccgacctcaaaga2120521dnaartificialsequencesirna/shrnaisequencesforhumancbl205ggagcaatgtgagggtgaaga2120621dnaartificialsequencesirna/shrnaisequencesforhumancbl206gcctctacggcctttggatac2120721dnaartificialsequencesirna/shrnaisequencesforhumancbl207gctgtacgtatgaagcaatgt2120821dnaartificialsequencesirna/shrnaisequencesforhumancbl208ggtactcctaccaggacatcc2120920dnaartificialsequencecrispr/cas9targetsequencesforhumancbl209ctcggctcgactgcgagcga2021020dnaartificialsequencecrispr/cas9targetsequencesforhumancbl210gccgccgccggctatccggg2021120dnaartificialsequencecrispr/cas9targetsequencesforhumancbl211tccgcccggatagccggcgg2021220dnaartificialsequencecrispr/cas9targetsequencesforhumancbl212gctcggctcgactgcgagcg2021320dnaartificialsequencecrispr/cas9targetsequencesforhumancbl213tcgcagtcgagccgagccgg2021420dnaartificialsequencecrispr/cas9targetsequencesforhumancbl214cttcttcacgttgccggcca2021520dnaartificialsequencecrispr/cas9targetsequencesforhumancbl215cgggttcgggtggcctgatt2021620dnaartificialsequencecrispr/cas9targetsequencesforhumancbl216cgctcgcagtcgagccgagc2021720dnaartificialsequencecrispr/cas9targetsequencesforhumancbl217ccgagccggcggacccgcct2021820dnaartificialsequencecrispr/cas9targetsequencesforhumancbl218tcgggttcgggtggcctgat2021920dnaartificialsequencecrispr/cas9targetsequencesforhumancbl219gccgagccggcggacccgcc2022020dnaartificialsequencecrispr/cas9targetsequencesforhumancbl220agagctcttcttcacgttgc2022120dnaartificialsequencecrispr/cas9targetsequencesforhumancbl221gccgccgccgccggctatcc2022220dnaartificialsequencecrispr/cas9targetsequencesforhumancbl222cccaggcgggtccgccggct2022320dnaartificialsequencecrispr/cas9targetsequencesforhumancbl223cgtccttcatgagcccaatc2022420dnaartificialsequencecrispr/cas9targetsequencesforhumancbl224cggagcccaggcgggtccgc2022520dnaartificialsequencecrispr/cas9targetsequencesforhumancbl225tggcctgattgggctcatga2022620dnaartificialsequencecrispr/cas9targetsequencesforhumancbl226tcacgttgccggccatggcc2022720dnaartificialsequencecrispr/cas9targetsequencesforhumancbl227cgccgccgccgccggctatc2022820dnaartificialsequencecrispr/cas9targetsequencesforhumancbl228ggcaacgtgaagaagagctc2022920dnaartificialsequencecrispr/cas9targetsequencesforhumancbl229cggctccgggggctcgggtt2023020dnaartificialsequencecrispr/cas9targetsequencesforhumancbl230tccgggggctcgggttcggg2023120dnaartificialsequencecrispr/cas9targetsequencesforhumancbl231ggctccgggggctcgggttc2023220dnaartificialsequencecrispr/cas9targetsequencesforhumancbl232gcaacgtgaagaagagctct2023320dnaartificialsequencecrispr/cas9targetsequencesforhumancbl233gcaacgtgaagaagagctct2023420dnaartificialsequencecrispr/cas9targetsequencesforhumancbl234gccacccgaacccgagcccc2023520dnaartificialsequencecrispr/cas9targetsequencesforhumancbl235cacgttgccggccatggcct2023620dnaartificialsequencecrispr/cas9targetsequencesforhumancbl236gcccggatagccggcggcgg2023720dnaartificialsequencecrispr/cas9targetsequencesforhumancbl237gaagaagagctctggggccg2023820dnaartificialsequencecrispr/cas9targetsequencesforhumancbl238caacgtgaagaagagctctg2023920dnaartificialsequencecrispr/cas9targetsequencesforhumancbl239aagaagagctctggggccgg2024020dnaartificialsequencecrispr/cas9targetsequencesforhumancbl240gggagagaagcagggcgtga2024120dnaartificialsequencecrispr/cas9targetsequencesforhumancbl241cggcagcggctccgggggct2024220dnaartificialsequencecrispr/cas9targetsequencesforhumancbl242cctgggcagggtcggagccc2024320dnaartificialsequencecrispr/cas9targetsequencesforhumancbl243agagaagcagggcgtgaagg202442727dnacanislupusmisc_feature(142)..(142)nisa,c,g,ortmisc_feature(1876)..(1876)nisa,c,g,ort244atggccggcaacgtgaagaagagctccggggccgggggcggcggcggctccgggggctcg60ggcggcctcatcgggctcatgaaggacgccttccagccgcaccaccaccaccaccacctc120agcccccacccgcccggcaccngtgacaagaagatggtggagaagtgctggaagctcatg180gacaaggtggtgcggttgtgtcagaacccaaagctggcgctaaagaatagcccaccttat240atcttagacctgctgccagatacctaccagcatctccgcactatcttgtcaagatatgag300gggaagatggagacacttggagaaaatgagtattttagggtgttcatggagaatttgatg360aagaaaactaagcagaccataagcctcttcaaggaggggaaagaaagaatgtatgaggag420aattctcagcctaggcgaaacctaaccaaattgtccctgatcttcagccacatgctggca480gaactaaaaggaatctttccaagtggactctttcaaggagacacatttcggattactaaa540gcagatgctgcagaattttggaggaaagcttttggggaaaagacaatcgtcccttggaag600agtttccgccaggcccttcatgaagtgcatcccatcagttctgggctcgaggccatggct660ctgaaatccactattgatctgacctgcaatgattatatttctgtttttgaatttgacatc720ttcacacgactctttcagccctggtcctctttgctcaggaactggaacagtcttgctgta780actcatcctggttacatggctttcctgacgtatgatgaagtgaaagctcggctccagaag840ttcattcacaaacctggcagttacattttccggttgagctgtactcgtttgggacagtgg900gctattgggtatgtcactgctgatgggaacatcctccagacgatccctcacaataaacct960ctcttccaagccctgattgacggcttcagggaaggcttctatttgtttccagatggacgg1020aatcagaatcctgacctgacaggcctatgtgaaccaactccccaagaccacatcaaagtg1080acccaggaacaatatgaattatactgtgagatgggctccaccttccaactgtgtaaaata1140tgtgctgagaacgataaggatgtgaaaattgagccctgtggacacctcatgtgcacatcc1200tgtcttacatcctggcaggaatcagaaggccaaggctgccctttctgccgatgtgaaatt1260aaaggtactgagcccattgtggtagatccgtttgaccctcgaggaagtggcagcctactg1320aggcaaggagctgagggagctccctccccaaattatgaagatgatgacgatgaacgagct1380gatgattctctctttatgatgaaggaactggctggtgccaaggtggaacggcctccttct1440ccgttctcgatggccccacaggctcccctgcccccagtaccaccacgtcttgacctccta1500caacagcgagtgtctgttccttctagtgcttctggtcttggaactgcttctaaggtagct1560tctggctcccttcataaggacaaaccattaccaataccccccacacttcgagatcttcca1620ccaccaccccctccagaccgaccatattctgttggaacagacacccggcctcagagacgt1680cccttgccttgtacaccgggcgactgtccatccagggacaaactgccgcctgttccctct1740agccgtctcggggaatcatggctgcctcggccaatccccaaagtaccagtggttgctcca1800aactcgagtgacccctggacctctggtagagaattaaccaacaggcactcacttccattt1860tcattgccctcacaanatgaacccagaacagatgtgcctaggcttggaggcacattcaat1920gtggatacttccatgaatgtgaataacagcccactagcaagttctgagtgtgagcacccc1980aaaatcaaaccttccgcatctgccaatgccatttattctctggctgccaggcctcttcct2040gtgccaaagctgccccctggggagcagtgtgaaggtgaggaggacacagagtatatgacc2100ccctcctctagacctctagggcttccaaagccagatgggaaacggcctttggagacaacc2160cagagttcacgagcatgtgattgtgaccagcagatcgatagctgcacatatgaagcaatg2220tataatattcagtcccaagcgacaccatctgtcacagagagcagcacctttggtgaaggg2280agtctggctgcagcccacatcagcaccggccccgaggaatcagaaaatgaggaggacggg2340tatgatgtccctaagccgcccatgccagcagtgctggcccgccggactctctcagacatc2400tccaatgccagttcctcctttggctggttgtctctggaaggcgatcccaccacaaacttc2460actgagggttcccaagttcctgaaaggcctcccaaaccgttccctcggagaatcaactct2520gaacgaaaagcaggcagctgtcagcagggtggtgccgctgctgcctcaccacagctctcc2580agtgagattgagaacctcctgagccagggatactcctaccaggacattcagaaagctctg2640gtcattgcccacaacaacattgaaatggccaagaacatcctccgggaatttgtttctatc2700tcttctcccgcccacgtagccacctag2727245908prtcanislupusmisc_feature(48)..(48)xaacanbeanynaturallyoccurringaminoacidmisc_feature(626)..(626)xaacanbeanynaturallyoccurringaminoacid245metalaglyasnvallyslysserserglyalaglyglyglyglygly151015serglyglyserglyglyleuileglyleumetlysaspalaphegln202530prohishishishishishisleuserprohisproproglythrxaa354045asplyslysmetvalglulyscystrplysleumetasplysvalval505560argleucysglnasnprolysleualaleulysasnserproprotyr65707580ileleuaspleuleuproaspthrtyrglnhisleuargthrileleu859095serargtyrgluglylysmetgluthrleuglygluasnglutyrphe100105110argvalphemetgluasnleumetlyslysthrlysglnthrileser115120125leuphelysgluglylysgluargmettyrglugluasnserglnpro130135140argargasnleuthrlysleuserleuilepheserhismetleuala145150155160gluleulysglyilepheproserglyleupheglnglyaspthrphe165170175argilethrlysalaaspalaalagluphetrparglysalaphegly180185190glulysthrilevalprotrplysserpheargglnalaleuhisglu195200205valhisproileserserglyleuglualametalaleulysserthr210215220ileaspleuthrcysasnasptyrileservalpheglupheaspile225230235240phethrargleupheglnprotrpserserleuleuargasntrpasn245250255serleualavalthrhisproglytyrmetalapheleuthrtyrasp260265270gluvallysalaargleuglnlyspheilehislysproglysertyr275280285ilepheargleusercysthrargleuglyglntrpalaileglytyr290295300valthralaaspglyasnileleuglnthrileprohisasnlyspro305310315320leupheglnalaleuileaspglyphearggluglyphetyrleuphe325330335proaspglyargasnglnasnproaspleuthrglyleucysglupro340345350thrproglnasphisilelysvalthrglngluglntyrgluleutyr355360365cysglumetglyserthrpheglnleucyslysilecysalagluasn370375380asplysaspvallysilegluprocysglyhisleumetcysthrser385390395400cysleuthrsertrpglnglusergluglyglnglycysprophecys405410415argcysgluilelysglythrgluproilevalvalasppropheasp420425430proargglyserglyserleuleuargglnglyalagluglyalapro435440445serproasntyrgluaspaspaspaspgluargalaaspaspserleu450455460phemetmetlysgluleualaglyalalysvalgluargproproser465470475480prophesermetalaproglnalaproleuproprovalproproarg485490495leuaspleuleuglnglnargvalservalproserseralasergly500505510leuglythralaserlysvalalaserglyserleuhislysasplys515520525proleuproileproprothrleuargaspleupropropropropro530535540proaspargprotyrservalglythraspthrargproglnargarg545550555560proleuprocysthrproglyaspcysproserargasplysleupro565570575provalproserserargleuglyglusertrpleuproargproile580585590prolysvalprovalvalalaproasnserseraspprotrpthrser595600605glyarggluleuthrasnarghisserleupropheserleuproser610615620glnxaagluproargthraspvalproargleuglyglythrpheasn625630635640valaspthrsermetasnvalasnasnserproleualaserserglu645650655cysgluhisprolysilelysproseralaseralaasnalailetyr660665670serleualaalaargproleuprovalprolysleuproproglyglu675680685glncysgluglyglugluaspthrglutyrmetthrproserserarg690695700proleuglyleuprolysproaspglylysargproleugluthrthr705710715720glnserserargalacysaspcysaspglnglnileaspsercysthr725730735tyrglualamettyrasnileglnserglnalathrproservalthr740745750gluserserthrpheglygluglyserleualaalaalahisileser755760765thrglyprogluglusergluasnglugluaspglytyraspvalpro770775780lysproprometproalavalleualaargargthrleuseraspile785790795800serasnalaserserserpheglytrpleuserleugluglyasppro805810815thrthrasnphethrgluglyserglnvalprogluargproprolys820825830propheproargargileasnsergluarglysalaglysercysgln835840845glnglyglyalaalaalaalaserproglnleusersergluileglu850855860asnleuleuserglnglytyrsertyrglnaspileglnlysalaleu865870875880valilealahisasnasnileglumetalalysasnileleuargglu885890895phevalserileserserproalahisvalalathr90090524619dnaartificialsequencesirnasequencesforcaninecbl246ccagaagttcattcacaaa1924719dnaartificialsequencesirnasequencesforcaninecbl247ggaacatcctccagacgat1924819dnaartificialsequencesirnasequencesforcaninecbl248ccagacgatccctcacaat1924919dnaartificialsequencesirnasequencesforcaninecbl249gcttcagggaaggcttcta1925019dnaartificialsequencesirnasequencesforcaninecbl250gcaggaatcagaaggccaa1925119dnaartificialsequencesirnasequencesforcaninecbl251cctttctgccgatgtgaaa1925219dnaartificialsequencesirnasequencesforcaninecbl252gctgatgattctctcttta1925319dnaartificialsequencesirnasequencesforcaninecbl253gcttctggctcccttcata1925419dnaartificialsequencesirnasequencesforcaninecbl254gcatctgccaatgccattt1925519dnaartificialsequencesirnasequencesforcaninecbl255gctgcacatatgaagcaat1925623dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl256cccggagccgccgccgcccccgg2325723dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl257tgccgggcgggtgggggctgagg2325823dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl258cggcctcatcgggctcatgaagg2325923dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl259ggagctcttcttcacgttgccgg2326023dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl260caacgtgaagaagagctccgggg2326123dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl261ggggctcgggcggcctcatcggg2326223dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl262ggcaacgtgaagaagagctccgg2326323dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl263gcaacgtgaagaagagctccggg2326423dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl264gggggctcgggcggcctcatcgg2326523dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl265gtgaagaagagctccggggccgg2326623dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl266tgaagaagagctccggggccggg2326723dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl267cgtccttcatgagcccgatgagg2326823dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl268aagaagagctccggggccggggg2326923dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl269gaagaagagctccggggccgggg2327023dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl270gatgaggccgcccgagcccccgg2327123dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl271gtggtggtggtgcggctggaagg2327223dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl272aagagctccggggccgggggcgg2327323dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl273cacctcagcccccacccgcccgg2327423dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl274cggcggcggctccgggggctcgg2327523dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl275agctccggggccgggggcggcgg2327623dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl276gcgggtgggggctgaggtggtgg2327723dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl277tccggggccgggggcggcggcgg2327823dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl278gccgccgccgcccccggccccgg2327923dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl279cgggcgggtgggggctgaggtgg2328023dnaartificialsequencecrispr/cas9targetsequencesforcaninecbl280gccgggggcggcggcggctccgg2328119dnaartificialsequencehumancd2apwobblemutantsequence281ggagacggacgacgtaaag19當前第1頁12