一種肽文庫、其合成方法及從該文庫中篩選的活性片段的製作方法

2023-09-09 20:11:05

專利名稱：一種肽文庫、其合成方法及從該文庫中篩選的活性片段的製作方法
背景技術：
與人類相關的冠狀病毒(Severe Acute Respiratory SyndromeCoronavirus(SARS-CoV))已經被確認為新的一類病毒，人類以迄今最快的速度測定了其基因序列，主要由RNA-dependent RNApolymerase、Spike(S)、Membrane(M)、Envelope(E)、Nucleocapside(N)、Polymerase(P)等蛋白組成(1、Marra MA et al.，The genome sequence ofthe SARS-associated Coronavirus，Sciencexpress，published online atwww.sciencexpress.org，1 May，2003；2、Rota PA et al.，Characterizationof a novel coronavirus associated with severe acute respiratory syndrome，Sciencexpress，published online atwww.sciencexpress.org，1 May，2003；3、Qin E』de，et al.，A complete sequence and comparative analysis of aSARS-associated virus(Isolate BJ01)，Chinese Science Bulletin 2003，48(10)941-948；4、Peoros JS，et al.，Coronavirus as a possible cause ofsevere acute respiratory syndrome，The Lancet，published online atwww.nejm.org，April 8，2003；5、Ksiazek TG et al.，A novel coronavirusassociated with severe acute respiratory syndrom，N Engl J Med，2003，348(20)1953～1966；6、Dorsten C et al.，Identification of a novelcomavirus in patients with severe acute respiratory syndrome，N Engl JMed，published atwww.nejm.org，on April 10，2003；Anand K et al.，Coronavirus main proteinase(3CLpro)structurebasis for design ofanti-SARS drugs，Sciencexpress，published online atwww.sciencexpress.org，13 May，2003.)。其中Spike(S)蛋白、Membrane(M)蛋白和Envelope(E)蛋白組成了病毒外殼，是病毒識別、結合併進入宿主細胞的蛋白質。尤其是刺突S蛋白是關鍵性蛋白(圖1)。雖然文獻研究結果表明HCoV-229E刺突S蛋白的417-546序列是宿主受體結合的部位，但同時也指出不同的冠狀病毒通過使用不同的受體進入宿主細胞。這很可能與刺突S蛋白的變異有關(1、Marra MA etal.，The genome sequence of the SARS-associated Coronavirus，Sciencexpress，published online atwww.sciencexpress.org，1 May，2003)。Nucleocapsid(N)蛋白屬於胞漿內蛋白，處於病毒顆粒的核心部分，和Genomic RNA以結合的形式存在。病毒RNA在細胞質中複製完成後會和N蛋白結合，結合產物可以被M蛋白識別並被包裝到病毒顆粒中。所以N和M蛋白與病毒在宿主細胞中的複製具有明顯的關係。N蛋白是宿主T-細胞識別的主要位點之一(見圖1)。因而，發展合成病毒蛋白的多肽化合物化學庫，對於尋找病毒表面蛋白的多重最小多肽抗原決定簇(包括B-細胞和T-細胞抗原決定簇)，進而尋求和發展合成多肽疫苗、臨床診斷試劑和血清治療方案有極其重要的意義。同時，該化學庫還可用於篩選並發現病毒結合受體的多重配基，進而阻斷冠狀病毒進入宿主細胞，發展治療藥物(特別是對於耐藥病毒的藥物治療)有極大的參考價值。
目前，尋找抗原決定簇最為常用的方法是利用各種計算機軟體，通過已經發表的序列採用親水性、疏水性、轉角結構、HPLC滯留係數、螺旋結構、保守性等參數進行預測。我們的實驗結果表明，這種預測方法與實際測得的序列僅有30％～50％的重複性(1、ZHANG XM，LIU G and SUN MJ，Brain Research，2000，868157-164；2、ZHANGXM，LIU G and SUN MJ，Brain Research，2001，895277-282.)。另一種方法是採用組合化學技術對蛋白抗原決定簇譜的研究(「交叉重疊」多肽化合物，見表1)。
表1 HR2區的「交叉重疊」多肽1.DISGINASVV2. ISGINASVVN3. SGINASVVNI4. GINASVVNIQ5.INASVVNIQK6. NASVVNIQKE7. ASVVNIQKEI8. SVVNIQKEID9.VVNIQKEIDR
10.VNIQKEIDRL11. NIQKEIDRLN12. IQKEIDRLNE13. QKEIDRLNEV14.KEIDRLNEVA15. EIDRLNEVAK16. IDRLNEVAKN17. DRLNEVAKNL18.RLNEVAKNLN19. LNEVAKNLNE20. NEVAKNLNES21. EVAKNLNESL22.VAKNLNESLI23. AKNLNESLID24. KNLNESLIDL25. NLNESLIDLQ26.LNESLIDLQE27. NESLIDLQEL28. ESLIDLQELG29. SLIDLQELGK30.LIDLQELGKY31. IDLQELGKYE組合化學是用於新藥合成和篩選的一種全新的方法，始於90年代初，被譽為是對傳統藥物化學合成和篩選的一場「革命性突破」。它打破了逐一合成、逐一篩選的模式，以合成和篩選化合物庫的形式尋找和優化先導化合物，從而大大加快了發現藥物先導化合物的速度。
組合化學把化學合成、電腦設計、計算機技術結為一體，能同時產生許多種結構相關但有序變化的化合物，然後用高度靈敏的生物學方法對這些化合物同時進行篩選，從中確定具有生物活性的物質，再經結構測定，以期找到全新的先導化合物。組合化學以其高效、微量、高度自動化的特點而很快受到世人的矚目並被許多大的製藥公司採納，成為近年來用以發現新藥先導化合物的一種重要手段。該法的特點是以一定數目的胺基酸殘基肽(比如1到50個胺基酸殘基)為合成「交叉重疊」片段，將蛋白序列通過逐個錯位(或者間隔錯位，包括從2到合成肽片段的胺基酸的總位數)的方式全部合成出來，然後進行抗原-抗體反應(或者其它生物目的的篩選反應等，如篩選發現T-細胞免疫抗原決定簇，以及SARS-CoV的受體配基等)，一次便可以得到所有最短的抗原決定簇，進而繪成抗原決定簇譜，或者其它最小有效活性片段的多肽化合物以及它們的圖譜。
目前，通過組合化學技術在化合物合成領域已經成功用於小分子物質的合成，但卻沒有關於合成大分子物質如肽的報導。

發明內容
本發明人通過應用物理編碼法成功合成了一種「交叉重疊」多肽化學庫，並使該多肽化學庫與SARS-CoV陽性血清反應，成功篩選得到了BJ01號冠狀病毒變種的抗原決定簇，從而完成了本發明。
本發明一方面涉及一種「交叉重疊」肽庫，其中每一個肽的通式為Z-XnXn+1Xn+2Xn+3Xn+4Xn+5Xn+6Xn+7Xn+8Xn+9-Y其中X代表任何一種L-構型的天然胺基酸殘基，n是指第Xn胺基酸在蛋白質序列中的天然排列位置，n為整數，例如第1，2，3，4....n；Y代表NH2或者OH，Z代表氫或者Ac，其中Ac為乙醯基。
所述的蛋白質序列的多肽優選為與人類相關的冠狀病毒SARS-CoV相關的S、M、E和N蛋白的多肽序列。
所述的與人類相關的冠狀病毒SARS-CoV優選為SARS-CoVBJ01號、SARS-CoVBJ02號、SARS-CoVBJ03號、SARS-CoVBJ04號、SARS-CoVCUHK-W1、SARS-CoVGZ01、SARS-CoVHKU-39849、SARS-CoVTaiwan、SARS-CoVTor2、SARS-CoVUrbani、SARS-CoVVietnam。所述的與人類相關的冠狀病毒SARS-CoV最優選為SARS-CoVBJ01號。
本發明另一方面涉及一種選自如SEQ ID NO1至SEQ ID NO101所示的胺基酸序列的任意一種或其結合的胺基酸序列。
本發明再一方面涉及一種如SEQ ID NO102至103所示的胺基酸序列的任意一種或其結合的胺基酸序列。
本發明還涉及一種如SEQ ID NO104所示的胺基酸序列。
本發明還涉及一種如SEQ ID NO105至112所示的胺基酸序列的任意一種或其結合的胺基酸序列。
本發明還涉及具有抗SARS-CoV陽性血清抗原決定簇(B-細胞抗原決定簇)、T-細胞抗原決定簇、以及由此發現B-細胞抗原決定簇和T-細胞抗原決定簇作用的、含有SEQ ID NO1至SEQ ID NO112任意一種或其結合的共價或者非共價化合物。
本發明還涉及含有如SEQ ID NO1至112所示的胺基酸序列的任意一種或其結合的胺基酸序列在用於製備治療SARS-CoV引起的疾病的藥物中的應用。
本發明進一步涉及通過封閉SEQ ID NO1至SEQ ID NO112所示的胺基酸序列從而阻止SARS-CoV病毒進入宿主的阻斷劑。
本發明還涉及一種採用「混-分」物理編碼法合成含有至少一個具有2～50個胺基酸殘基的肽文庫的方法，其包括以下步驟1)選擇合適的射頻子編碼確定胺基酸序列的多肽片段；2)混合併對多肽母鏈氨基端進行脫Fmoc保護；3)混合併進行充分的洗滌；4)解碼並按所要連接的特定胺基酸進行分組；5)將特定胺基酸以3～10倍摩爾過量的形式加入到已經分組的反應瓶中，進而連接到步驟2)的多肽母鏈；以及6)重複步驟2)和5)直至達到所需長度的片段；7)最後，進行解碼並將多肽化合物從樹脂上裂解下來。
所述的多肽為具有2～50個、優選3-34個胺基酸殘基的多肽。
該方法所述的保護策略包括Fmoc/t-Bu或者Boc/Bz1保護，優選為Fmoc/tBu。
本發明的優點在於採用固相合成技術、射頻編碼技術在短時間內合成了大量的「交叉重疊」多肽化合物，並首次合成了與SARS-CoV相關的S、M、E和N蛋白的全部「交叉重疊」多肽。發明中，首次使用SARS-CoV感染的人的陽性血清對化學庫進行了篩選，並繪製了B-細胞抗原決定簇譜。


圖1描述了Spike蛋白及其結構。
圖2為合成多肽隨機抽樣LC-MS分析結果。
圖3顯示合成NH2-NAQALNTLVKQLSSNFGAISSVLNDILSRLDKVE-CONH2HPLC-MS的分析結果。
圖4顯示S蛋白篩選結果。
圖5顯示M、E和N蛋白篩選結果。
11～30號多肽ELISA篩選抗SARS-CoV陽性血清結果，其中縱坐標代表OD值，橫坐標代表合成多肽序列編號。
具體實施例方式
組合化學研究分三個階段分子多樣性化合物庫的合成；群集篩選(進行活性檢測)；活性分子的結構測定。
1、肽庫的建立要建立肽文庫必須事先考慮許多方面如何設計模板分子；選擇合適的構建單元；確定何種合成方案；如何完成半自動或者自動合成等。
本發明中的主要目的是尋找針對SARS-CoV病毒的人的B-細胞線型抗原決定簇，並繪製抗原決定簇譜。選擇逐位錯位的合成方式可以直接找到最小的抗原決定簇。因而，線型10肽被選擇為摸板分子。蛋白質以天然L-構型胺基酸組成，因而，本化學庫的構建單元自然選擇為20種天然L-構型胺基酸。高通量地合成方法一般採用固相合成技術，特點是中間體的純化僅採用簡單的洗滌和過濾操作即可完成。同時，反應中可以使用大大過量的構建單元來保證反應進行完全，並易半自動化或者自動化。因而，我們選擇了固相合成技術，在樹脂上完成了全部多肽化合物的合成。使用20種天然L-構型胺基酸為構建單元來合成數千個多肽化合物時，相同的反應條件可以在多肽編碼的情況下同步進行，如相同的脫保護步驟、不同的多肽序列與相同的保護胺基酸進行反應步驟等。這樣就可以完成大規模、快速地合成。其中編碼方式有多種，如化學編碼、物理編碼等。物理編碼的特點是無「化學汙染」，無須多餘的化學解碼步驟等。因而，我們選擇了IRORI編碼-解碼的分類技術。一次可操作數千個化合物的合成，本實驗中我們選擇了一次合成600～700個多肽化合物的規模。
2、群集篩選總的來講，篩選可分為隨機篩選和定向篩選。但無論是隨機篩選還是定向篩選，都要考慮選定篩選模式為固相篩選或用何法(細胞功能篩選、受體篩選、抗體篩選等)進行篩選，用何種指示劑(同位素標記、螢光標記、染料染色)等。具體的篩選方法有三種固相篩選、液相篩選和兩者的結合。本發明中使用的ELISA篩選方法屬於定向液相篩選，生物反應是抗原-抗體結合實驗。人抗SARS-CoV陽性血清中含有人抗SARS-CoV多克隆抗體(包括IgG和IgM)。當包被在96孔反應板中的多肽化合物與人抗SARS-CoV多克隆抗體結合後，經過仔細的洗滌即可洗去為結合多餘的、甚至是非特異的結合抗體和血清。此時，加入用辣根過氧化酶標記的抗IgG或者抗IgM抗體就會完成二次結合，並將辣根過氧化酶保留在體系內。該辣根過氧化酶在一定的條件下可以催化水解對應的底物，並可以在特定的波長下測得吸收值。此吸收值的大小與人抗SARS-CoV多克隆抗體結合的強弱和多少相關。
3、確認活性分子結構編碼技術已應用於解析組合庫中高活性化合物的結構。最早提出此技術的是Brenner和Lemer，Nicolaou等提出了射頻編碼合成儀突破了以往的編碼形式，它是建立在射頻信號及應用多功能微反應儀的半導體記憶裝置基礎上的。
本發明中使用的射頻編碼技術可以直接將裂解到96孔板中多肽化合物編碼對應孔號。因而，篩選得到的活性孔經過與編碼子序號對比即可給出多肽的序列。
本實施例中以篩選抗SARS-CoV陽性血清，尋找並繪製B-細胞抗原決定簇譜為目標，篩選了本化學庫。篩選並繪製T-細胞抗原決定簇譜和SARS-CoV融合阻斷劑正在進行中。本實施例中選擇了SARS-CoV的BJ01號序列，其組成成分的胺基酸序列如下Spike(S)蛋白如SEQ ID NO113所示。
Membrane(M)蛋白如SEQ ID NO114所示。
Envelope(E)蛋白如SEQ ID NO115所示。
Nucleocapsid(N)蛋白如SEQ ID NO116所示。
實施例1設計合成Spike(S)蛋白從1至600個胺基酸序列中每相鄰的10個胺基酸序列的「交叉重疊」序列，如下表所示。從右至左為合成次序，代表合成步驟號碼為阿拉伯數字1到10。
表2 所合成的600個以10個胺基酸為長度的序列編號 109 8 7 6 5 4 3 2 11)M F I F L L F L T L2)F I F L L F L T L T3)I F L L F L T L T S4)F L L F L T L T S G5)L L F L T L T S G S6)L F L T L T S G S D7)F L T L T S G S D L8)L T L T S G S D L D9)T L T S G S D L D R10)L T S G S D L D R C11)T S G S D L D R C T
12)SGSDLDRCTT13)GSDLDRCTTF14)SDLDRCTTFD15)DLDRCTTFDD16)LDRCTTFDDV17)DRCTTFDDVQ18)RCTTFDDVQA19)CTTFDDVQAP20)TTFDDVQAPN21)TFDDVQAPNY22)FDDVQAPNYT23)DDVQAPNYTQ24)DVQAPNYTQH25)VQAPNYTQHT26)QAPNYTQHTS27)APNYTQHTSS28)PNYTQHTSSM29)NYTQHTSSMR30)YTQHTSSMRG31)TQHTSSMRGV32)QHTSSMRGVY33)HTSSMRGVYY34)TSSMRGVYYP35)SSMRGVYYPD36)SMRGVYYPDE37)MRGVYYPDEI38)RGVYYPDEIF39)GVYYPDEIFR40)VYYPDEIFRS41)YYPDEIFRSD42)YPDEIFRSDT43)PDEIFRSDTL44)DEIFRSDTLY45)EIFRSDTLYL46)IFRSDTLYLT47)FRSDTLYLTQ48)RSDTLYLTQD49)SDTLYLTQDL50)DTLYLTQDLF51)TLYLTQDLFL52)LYLTQDLFLP53)YLTQDLFLPF54)LTQDLFLPFY55)TQDLFLPFYS56)QDLFLPFYSN57)DLFLPFYSNV58)LFLPFYSNVT59)FLPFYSNVTG60)LPFYSNVTGF61)PFYSNVTGFH
62)FYSNVTGFHT63)YSNVTGFHTI64)SNVTGFHTIN65)NVTGFHTINH66)VTGFHTINHT67)TGFHTINHTF68)GFHTINHTFD69)FHTINHTFDN70)HTINHTFDNP71)TINHTFDNPV72)INHTFDNPVI73)NHTFDNPVIP74)HTFDNPVIPF75)TFDNPVIPFK76)FDNPVIPFKD77)DNPVIPFKDG78)NPVIPFKDGI79)PVIPFKDGIY80)VIPFKDGIYF81)IPFKDGIYFA82)PFKDGIYFAA83)FKDGIYFAAT84)KDGIYFAATE85)DGIYFAATEK86)GIYFAATEKS87)IYFAATEKSN88)YFAATEKSNV89)FAATEKSNVV90)AATEKSNVVR91)ATEKSNVVRG92)TEKSNVVRGW93)EKSNVVRGWV94)KSNVVRGWVF95)SNVVRGWVFG96)NVVRGWVFGS97)VVRGWVFGST98)VRGWVFGSTM99)RGWVFGSTMN100)GWVFGSTMNN101)WVFGSTMNNK102)VFGSTMNNKS103)FGSTMNNKSQ104)GSTMNNKSQS105)STMNNKSQSV106)TMNNKSQSVI107)MNNKSQSVII108)NNKSQSVIII109)NKSQSVIIIN110)KSQSVIIINN111)SQSVIIINNS
112)QSVIIINNST113)SVIIINNSTN114)VIIINNSTNV115)IIINNSTNVV116)IINNSTNVVI117)INNSTNVVIR118)NNSTNVVIRA119)NSTNVVIRAC120)STNVVIRACN121)TNVVIRACNF122)NVVIRACNFE123)VVIRACNFEL124)VIRACNFELC125)IRACNFELCD126)RACNFELCDN127)ACNFELCDNP128)CNFELCDNPF129)NFELCDNPFF130)FELCDNPFFA131)ELCDNPFFAV132)LCDNPFFAVS133)CDNPFFAVSK134)DNPFFAVSKP135)NPFFAVSKPM136)PFFAVSKPMG137)FFAVSKPMGT138)FAVSKPMGTQ139)AVSKPMGTQT140)VSKPMGTQTH141)SKPMGTQTHT142)KPMGTQTHTM143)PMGTQTHTMI144)MGTQTHTMIF145)GTQTHTMIFD146)TQTHTMIFDN147)QTHTMIFDNA148)THTMIFDNAF149)HTMIFDNAFN150)TMIFDNAFNC151)MIFDNAFNCT152)IFDNAFNCTF153)FDNAFNCTFE154)DNAFNCTFEY155)NAFNCTFEYI156)AFNCTFEYIS157)FNCTFEYISD158)NCTFEYISDA159)CTFEYISDAF160)TFEYISDAFS161)FEYISDAFSL
162)EYISDAFSLD163)YISDAFSLDV164)ISDAFSLDVS165)SDAFSLDVSE166)DAFSLDVSEK167)AFSLDVSEKS168)FSLDVSEKSG169)SLDVSEKSGN170)LDVSEKSGNF171)DVSEKSGNFK172)VSEKSGNFKH173)SEKSGNFKHL174)EKSGNFKHLR175)KSGNFKHLRE176)SGNFKHLREF177)GNFKHLREFV178)NFKHLREFVF179)FKHLREFVFK180)KHLREFVFKN181)HLREFVFKNK182)LREFVFKNKD183)REFVFKNKDG184)EFVFKNKDGF185)FVFKNKDGFL186)VFKNKDGFLY187)FKNKDGFLYV188)KNKDGFLYVY189)NKDGFLYVYK190)KDGFLYVYKG191)DGFLYVYKGY192)GFLYVYKGYQ193)FLYVYKGYQP194)LYVYKGYQPI195)YVYKGYQPID196)VYKGYQPIDV197)YKGYQPIDVV198)KGYQPIDVVR199)GYQPIDVVRD200)YQPIDVVRDL201)QPIDVVRDLP202)PIDVVRDLPS203)IDVVRDLPSG204)DVVRDLPSGF205)VVRDLPSGFN206)VRDLPSGFNT207)RDLPSGFNTL208)DLPSGFNTLK209)LPSGFNTLKP210)PSGFNTLKPI211)SGFNTLKPIF
212)GFNTLKPIFK213)FNTLKPIFKL214)NTLKPIFKLP215)TLKPIFKLPL216)LKPIFKLPLG217)KPIFKLPLGI218)PIFKLPLGIN219)IFKLPLGINI220)FKLPLGINIT221)KLPLGINITN222)LPLGINITNF223)PLGINITNFR224)LGINITNFRA225)GINITNFRAI226)INITNFRAIL227)NITNFRAILT228)ITNFRAILTA229)TNFRAILTAF230)NFRAILTAFS231)FRAILTAFSP232)RAILTAFSPA233)AILTAFSPAQ234)ILTAFSPAQD235)LTAFSPAQDT236)TAFSPAQDTW237)AFSPAQDTWG238)FSPAQDTWGT239)SPAQDTWGTS240)PAQDTWGTSA241)AQDTWGTSAA242)QDTWGTSAAA243)DTWGTSAAAY244)TWGTSAAAYF245)WGTSAAAYFV246)GTSAAAYFVG247)TSAAAYFVGY248)SAAAYFVGYL249)AAAYFVGYLK250)AAYFVGYLKP251)AYFVGYLKPT252)YFVGYLKPTT253)FVGYLKPTTF254)VGYLKPTTFM255)GYLKPTTFML256)YLKPTTFMLK257)LKPTTFMLKY258)KPTTFMLKYD259)PTTFMLKYDE260)TTFMLKYDEN261)TFMLKYDENG
262)FMLKYDENGT263)MLKYDENGTI264)LKYDENGTIT265)KYDENGTITD266)YDENGTITDA267)DENGTITDAV268)ENGTITDAVD269)NGTITDAVDC270)GTITDAVDCS271)TITDAVDCSQ272)ITDAVDCSQN273)TDAVDCSQNP274)DAVDCSQNPL275)AVDCSQNPLA276)VDCSQNPLAE277)DCSQNPLAEL278)CSQNPLAELK279)SQNPLAELKC280)QNPLAELKCS281)NPLAELKCSV282)PLAELKCSVK283)LAELKCSVKS284)AELKCSVKSF285)ELKCSVKSFE286)LKCSVKSFEI287)KCSVKSFEID288)CSVKSFEIDK289)SVKSFEIDKG290)VKSFEIDKGI291)KSFEIDKGIY292)SFEIDKGIYQ293)FEIDKGIYQT294)EIDKGIYQTS295)IDKGIYQTSN296)DKGIYQTSNF297)KGIYQTSNFR298)GIYQTSNFRV299)IYQTSNFRVV300)YQTSNFRVVP301)QTSNFRVVPS302)TSNFRVVPSG303)SNFRVVPSGD304)NFRVVPSGDV305)FRVVPSGDVV306)RVVPSGDVVR307)VVPSGDVVRF308)VPSGDVVRFP309)PSGDVVRFPN310)SGDVVRFPNI311)GDVVRFPNIT
312)DVVRFPNITN313)VVRFPNITNL314)VRFPNITNLC315)RFPNITNLCP316)FPNITNLCPF317)PNITNLCPFG318)NITNLCPFGE319)ITNLCPFGEV320)TNLCPFGEVF321)NLCPFGEVFN322)LCPFGEVFNA323)CPFGEVFNAT324)PFGEVFNATK325)FGEVFNATKF326)GEVFNATKFP327)EVFNATKFPS328)VFNATKFPSV329)FNATKFPSVY330)NATKFPSVYA331)ATKFPSVYAW332)TKFPSVYAWE333)KFPSVYAWER334)FPSVYAWERK335)PSVYAWERKK336)SVYAWERKKI337)VYAWERKKIS338)YAWERKKISN339)AWERKKISNC340)WERKKISNCV341)ERKKISNCVA342)RKKISNCVAD343)KKISNCVADY344)KISNCVADYS345)ISNCVADYSV346)SNCVADYSVL347)NCVADYSVLY348)CVADYSVLYN349)VADYSVLYNS350)ADYSVLYNST351)DYSVLYNSTF352)YSVLYNSTFF353)SVLYNSTFFS354)VLYNSTFFST355)LYNSTFFSTF356)YNSTFFSTFK357)NSTFFSTFKC358)STFFSTFKCY359)TFFSTFKCYG360)FFSTFKCYGV361)FSTFKCYGVS
362)STFKCYGVSA363)TFKCYGVSAT364)FKCYGVSATK365)KCYGVSATKL366)CYGVSATKLN367)YGVSATKLND368)GVSATKLNDL369)VSATKLNDLC370)SATKLNDLCF371)ATKLNDLCFS372)TKLNDLCFSN373)KLNDLCFSNV374)LNDLCFSNVY375)NDLCFSNVYA376)DLCFSNVYAD377)LCFSNVYADS378)CFSNVYADSF379)FSNVYADSFV380)SNVYADSFVV381)NVYADSFVVK382)VYADSFVVKG383)YADSFVVKGD384)ADSFVVKGDD385)DSFVVKGDDV386)SFVVKGDDVR387)FVVKGDDVRQ388)VVKGDDVRQI389)VKGDDVRQIA390)KGDDVRQIAP391)GDDVRQIAPG392)DDVRQIAPGQ393)DVRQIAPGQT394)VRQIAPGQTG395)RQIAPGQTGV396)QIAPGQTGVI397)IAPGQTGVIA398)APGQTGVIAD399)PGQTGVIADY400)GQTGVIADYN401)QTGVIADYNY402)TGVIADYNYK403)GVIADYNYKL404)VIADYNYKLP405)IADYNYKLPD406)ADYNYKLPDD407)DYNYKLPDDF408)YNYKLPDDFM409)NYKLPDDFMG410)YKLPDDFMGC411)KLPDDFMGCV
412)LPDDFMGCVL413)PDDFMGCVLA414)DDFMGCVLAW415)DFMGCVLAWN416)FMGCVLAWNT417)MGCVLAWNTR418)GCVLAWNTRN419)CVLAWNTRNI420)VLAWNTRNID421)LAWNTRNIDA422)AWNTRNIDAT423)WNTRNIDATS424)NTRNIDATST425)TRNIDATSTG426)RNIDATSTGN427)NIDATSTGNY428)IDATSTGNYN429)DATSTGNYNY430)ATSTGNYNYK431)TSTGNYNYKY432)STGNYNYKYR433)TGNYNYKYRY434)GNYNYKYRYL435)NYNYKYRYLR436)YNYKYRYLRH437)NYKYRYLRHG438)YKYRYLRHGK439)KYRYLRHGKL440)YRYLRHGKLR441)RYLRHGKLRP442)YLRHGKLRPF443)LRHGKLRPFE444)RHGKLRPFER445)HGKLRPFERD446)GKLRPFERDI447)KLRPFERDIS448)LRPFERDISN449)RPFERDISNV450)PFERDISNVP451)FERDISNVPF452)ERDISNVPFS453)RDISNVPFSP454)DISNVPFSPD455)ISNVPFSPDG456)SNVPFSPDGK457)NVPFSPDGKP458)VPFSPDGKPC459)PFSPDGKPCT460)FSPDGKPCTP461)SPDGKPCTPP
462)PDGKPCTPPA463)DGKPCTPPAL464)GKPCTPPALN465)KPCTPPALNC466)PCTPPALNCY467)CTPPALNCYW468)TPPALNCYWP469)PPALNCYWPL470)PALNCYWPLN471)ALNCYWPLND472)LNCYWPLNDY473)NCYWPLNDYG474)CYWPLNDYGF475)YWPLNDYGFY476)WPLNDYGFYT477)PLNDYGFYTT478)LNDYGFYTTT479)NDYGFYTTTG480)DYGFYTTTGI481)YGFYTTTGIG482)GFYTTTGIGY483)FYTTTGIGYQ484)YTTTGIGYQP485)TTTGIGYQPY486)TTGIGYQPYR487)TGIGYQPYRV488)GIGYQPYRVV489)IGYQPYRVVV490)GYQPYRVVVL491)YQPYRVVVLS492)QPYRVVVLSF493)PYRVVVLSFE494)YRVVVLSFEL495)RVVVLSFELL496)VVVLSFELLN497)VVLSFELLNA498)VLSFELLNAP499)LSFELLNAPA500)SFELLNAPAT501)FELLNAPATV502)ELLNAPATVC503)LLNAPATVCG504)LNAPATVCGP505)NAPATVCGPK506)APATVCGPKL507)PATVCGPKLS508)ATVCGPKLST509)TVCGPKLSTD510)VCGPKLSTDL511)CGPKLSTDLI
512)GPKLSTDLIK513)PKLSTDLIKN514)KLSTDLIKNQ515)LSTDLIKNQC516)STDLIKNQCV517)TDLIKNQCVN518)DLIKNQCVNF519)LIKNQCVNFN520)IKNQCVNFNF521)KNQCVNFNFN522)NQCVNFNFNG523)QCVNFNFNGL524)CVNFNFNGLT525)VNFNFNGLTG526)NFNFNGLTGT527)FNFNGLTGTG528)NFNGLTGTGV529)FNGLTGTGVL530)NGLTGTGVLT531)GLTGTGVLTP532)LTGTGVLTPS533)TGTGVLTPSS534)GTGVLTPSSK535)TGVLTPSSKR536)GVLTPSSKRF537)VLTPSSKRFQ538)LTPSSKRFQP539)TPSSKRFQPF540)PSSKRFQPFQ541)SSKRFQPFQQ542)SKRFQPFQQF543)KRFQPFQQFG544)RFQPFQQFGR545)FQPFQQFGRD546)QPFQQFGRDV547)PFQQFGRDVS548)FQQFGRDVSD549)QQFGRDVSDF550)QFGRDVSDFT551)FGRDVSDFTD552)GRDVSDFTDS553)RDVSDFTDSV554)DVSDFTDSVR555)VSDFTDSVRD556)SDFTDSVRDP557)DFTDSVRDPK558)FTDSVRDPKT559)TDSVRDPKTS560)DSVRDPKTSE561)SVRDPKTSEI
562)VRDPKTSEIL563)RDPKTSEILD564)DPKTSEILDI565)PKTSEILDIS566)KTSEILDISP567)TSEILDISPC568)SEILDISPCS569)EILDISPCSF570)ILDISPCSFG571)LDISPCSFGG572)DISPCSFGGV573)ISPCSFGGVS574)SPCSFGGVSV575)PCSFGGVSVI576)CSFGGVSVIT577)SFGGVSVITP578)FGGVSVITPG579)GGVSVITPGT580)GVSVITPGTN581)VSVITPGTNA582)SVITPGTNAS583)VITPGTNASS584)ITPGTNASSE585)TPGTNASSEV586)PGTNASSEVA587)GTNASSEVAV588)TNASSEVAVL589)NASSEVAVLY590)ASSEVAVLYQ591)SSEVAVLYQD592)SEVAVLYQDV593)EVAVLYQDVN594)VAVLYQDVNC595)AVLYQDVNCT596)VLYQDVNCTD597)LYQDVNCTDV598)YQDVNCTDVS599)QDVNCTDVST600)DVNCTDVSTASpike(S)蛋白從1至600個胺基酸序列中每相鄰的10個胺基酸序列的合成操作步驟如下A.多肽脫Fmoc保護/胺基酸殘基的連接步驟1.均分樹脂採用IRORI均分樹脂裝置，在每一個Microkan中(共600個)加入15mg Rink Amide樹脂；
2.均分IRORI射頻子採用IRORI均分射頻子裝置，在每一個Microkan中加入一個射頻子，並用IRORI加蓋器加蓋；3.脫Fmoc保護集中所有待反應的Microkan，置於一個2L錐形瓶中，加入20％哌啶/DMF振搖，15min×2次；4.洗滌樹脂DMF 3min×6，DCM 3min×3，晾乾；5.IRORI射頻子編碼(或解碼)採用IRORI Sorting程序及相應配件編碼。按照多肽化學庫的序列對射頻子編碼(或解碼)，每一特定編碼代表一個特定序列，並以此將Microkan分配至不同胺基酸反應瓶中；6.洗滌樹脂用重蒸DMF潤洗待反應的樹脂；7.Fmoc-保護胺基酸的連接準確稱取計算量的Fmoc保護胺基酸、HOBt、HBTU(或DIC或BOP)，並用重蒸DMF溶解。將Microkan加入特定胺基酸反應瓶中，振搖反應3h；8.茚三酮檢測取事先設定好的對照組序列，用茚三酮法檢測反應情況。如果呈陰性反應，表明反應完全，進行步驟9的操作；如果呈陽性反應，表明反應不完全，需繼續反應；如果延長時間或改用縮合能力更強的縮合劑後，仍不能完全反應，則採用步驟11的方法封閉後，進行步驟9的操作；9.洗滌樹脂DMF 3min×3，DCM 3min×1；10.脫Fmoc保護/連接根據序列要求，重複步驟3-9的操作，直至反應結束；11.封閉採用乙醯化多肽的氨基端，15％Ac2O/CH2Cl2，30min；12.洗滌樹脂DCM 3min×2，晾乾。
B.多肽裂解步驟1.配製裂解液根據序列特點，從選取並配製適當的裂解液；2.裂解每一個Microkan使用1mL裂解液在室溫下裂解2h；3.洗滌再用1mL裂解液浸泡洗滌Microkan 5min；4.晾乾或離心乾燥合併兩次裂解液，離心濃縮至少量；
5.沉澱將事先用冰水浴充分冷卻的甲基叔丁基醚/石油醚(v/v3/1)加入到殘餘物中，放入冰水浴中冷卻20min使析晶完全；6.離心離心沉澱多肽化合物，10min後取出，小心傾去上清夜；7.洗滌多肽加入冷卻的甲基叔丁基醚/石油醚，充分洗滌後，離心，傾去上清夜；8.重複步驟7的操作一次；9.晾乾將多肽殘餘物在室溫下揮幹，直至完全乾燥；10.檢測抽檢部分多肽樣品，用10％HOAc/H2O或30％CH3CN/H2O或60％CH3CN/H2O溶解，並用HPLC-MS檢測純度。
C.以下表3-表12為第1步至第10步的投料D.從肽庫中隨機抽取的多肽化合物的純度以及正確的分子量見表13
表3合成1～600號「交叉重疊」多肽第一步投料計算結果S-1-步驟-1 FW 總數N(mmol)AA-m(mg)HBTU(mg)HOBt(mg)DIPEA(mmol) DIPEA(uL)AFmoc-Ala 311.330 1.08 336.2 401.4 165.4 2.16 370CFmoc-Cys(Trt)585.717 0.612 358.4 227.5 93.71.22 210DFmoc-Asp(OtBu) 411.542 1.512 622.2 562.0 231.5 3.02 518EFmoc-Glu(OtBu) 425.515 0.54 229.8 200.7 82.71.08 185FFmoc-Phe 387.449 1.764 683.4 655.6 270.1 3.53 604GFmoc-Gly 297.337 1.332 396.0 495.1 204.0 2.66 456HFmoc-His(Trt)619.76 0.216 133.9 80.333.10.43 74IFmoc-Ile 353.429 1.044 368.9 388.0 159.9 2.09 357KFmoc-Lys(Boc)468.530 1.08 506.0 401.4 165.4 2.16 370LFmoc-Leu 353.438 1.368 483.5 508.4 209.5 2.74 468MFmoc-Met 371.56 0.216 80.280.333.10.43 74NFmoc-Asn(Trt)596.744 1.584 945.2 588.7 242.6 3.17 542PFmoc-Pro 337.435 1.26 425.1 468.3 192.9 2.52 431QFmoc-Gln(Trt)610.717 0.612 373.7 227.5 93.71.22 210RFmoc-Arg(Pmc)662.821 0.756 501.1 281.0 115.8 1.51 259SFmoc-Ser(tBu)383.448 1.728 662.5 642.2 264.6 3.46 592TFmoc-Thr(tBu)397.551 1.836 729.8 682.4 281.1 3.67 629VFmoc-Val 339.449 1.764 598.7 655.6 270.1 3.53 604WFmoc-Trp 426.55 0.18 76.866.927.60.36 62YFmoc-Tyr(tBu)459.631 1.116 512.9 414.8 170.9 2.23 382
表4合成1～600號「交叉重疊」多肽第二步使用試劑投料計算結果S-1-步驟-2 FW 個數控制組總數N(mmol)AA-m(mg) DIC(mg) DIC(uL)HOBt(mg)A Fmoc-Ala 311.329 1 30 1.08 336.2136.3 168165.4C Fmoc-Cys(Trt)585.717 0 17 0.61 2 358.477.295 93.7D Fmoc-Asp(OtBu) 411.542 0 42 1.512 622.2190.8 236231.5E Fmoc-Glu(OtBu) 425.515 1 16 0.576 245.172.790 88.2F Fmoc-Phe 387.449 0 49 1.764 683.4222.6 275270.1G Fmoc-Gly 297.337 2 39 1.404 417.4177.2 219215.0H Fmoc-His(Trt)619.76 0 6 0.216 133.927.334 33.1I Fmoc-Ile 353.429 0 29 1.044 368.9131.8 163159.9K Fmoc-Lys(Boc)468.530 1 31 1.116 522.8140.8 174170.9L Fmoc-Leu 353.438 1 39 1.404 496.2177.2 219215.0M Fmoc-Met 371.56 0 6 0.216 80.2 27.334 33.1N Fmoc-Asn(Trt)596.744 1 45 1.62 966.7204.4 252248.1P Fmoc-Pro 337.435 0 35 1.26 425.1159.0 196192.9Q Fmoc-Gln(Trt)610.717 0 17 0.612 373.777.295 93.7R Fmoc-Arg(Pmc)662.821 0 21 0.756 501.195.4118115.8S Fmoc-Ser(tBu)383.448 1 49 1.764 676.3222.6 275270.1T Fmoc-Thr(tBu)397.552 1 53 1.908 758.4240.8 297292.2V Fmoc-Val 339.449 1 50 1.8610.9227.2 280275.6W Fmoc-Trp 426.55 0 5 0.18 76.8 22.728 27.6Y Fmoc-Tyr(tBu)459.631 1 32 1.152 529.5145.4 179176.4
表5合成1～600號「交叉重疊」多肽第三步投料計算結果S-1-步驟3FW 個數 控制組 總數N(mmo1)AA-m(mg) DIC(mg)DIC(uL)HOBt(mg)A Fmoc-Ala 311.329 029 1.044 325.0131.8 163159.9C Fmoc-Cys(Trt)585.717 118 0.648 379.581.8 10199.2D Fmoc-Asp(OtBu) 411.542 042 1.512 622.2190.8 236231.5E Fmoc-Glu(OtBu) 425.515 015 0.54 229.868.1 84 82.7F Fmoc-Phe 387.449 251 1.836 711.3231.7 286281.1G Fmoc-Gly 297.337 138 1.368 406.7172.6 213209.5H Fmoc-His(Trt)619.76 06 0.216 133.927.3 34 33.1I Fmoc-Ile 353.429 231 1.116 394.4140.8 174170.9K Fmoc-Lys(Boc)468.530 030 1.08 506.0136.3 168165.4L Fmoc-Leu 353.439 140 1.44 508.9181.7 224220.5M Fmoc-Met 371.56 06 0.216 80.2 27.3 34 33.1N Fmoc-Asn(Trt)596.744 044 1.584 945.2199.9 247242.6P Fmoc-Pro 337.435 136 1.296 437.3163.6 202198.5Q Fmoc-Gln(Trt)610.717 017 0.612 373.777.2 95 93.7R Fmoc-Arg(Pmc)662.821 021 0.756 501.195.4 118115.8S Fmoc-Ser(tBu)383.448 250 1.8690.1227.2 280275.6T Fmoc-Thr(tBu)397.551 152 1.872 744.1236.2 292286.7V Fmoc-Val 339.449 049 1.764 598.7222.6 275270.1W Fmoc-Trp 426.55 05 0.18 76.8 22.7 28 27.6Y Fmoc-Tyr(tBu)459.631 031 1.116 512.9140.8 174170.9
表6合成1～600號「交叉重疊」多肽第四步投料計算結果S-1-步驟-4 FW 個數 控制組 總數N(mmol) AA-m(mg) DIC(mg)DIC(uL)HOBt(mg)A Fmoc-Ala311.329029 1.044325.0131.8 163159.9C Fmoc-Cys(Trt) 585.717017 0.612358.477.2 95 93.7D Fmoc-Asp(OtBu) 411.542042 1.512622.2190.8 236231.5E Fmoc-Glu(OtBu) 425.515015 0.54 229.868.1 84 82.7F Fmoc-Phe387.450050 1.8 697.3227.2 280275.6G Fmoc-Gly297.337037 1.332396.0168.1 208204.0H Fmoc-His(Trt) 619.76 06 0.216133.927.3 34 33.1I Fmoc-Ile353.429130 1.08 381.7136.3 168165.4K Fmoc-Lys(Boc) 468.530030 1.08 506.0136.3 168165.4L Fmoc-Leu353.439241 1.476521.6186.3 230226.0M Fmoc-Met371.56 06 0.21680.2 27.3 34 33.1N Fmoc-Asn(Trt) 596.744145 1.62 966.7204.4 252248.1P Fmoc-Pro337.435237 1.332449.4168.1 208204.0Q Fmoc-Gln(Trt) 610.717017 0.612373.777.2 95 93.7R Fmoc-Arg(Pmc) 662.821021 0.756501.195.4 118115.8S Fmoc-Ser(tBu) 383.447148 1.728662.5218.1 269264.6T Fmoc-Thr(tBu) 397.551051 1.836729.8231.7 286281.1V Fmoc-Val339.449352 1.872635.4236.2 292286.7W Fmoc-Trp426.55 05 0.18 76.8 22.7 28 27.6Y Fmoc-Tyr(tBu) 459.631132 1.152529.5145.4 179176.4
表7合成1～600號「交叉重疊」多肽第五步投料計算結果S-1-步驟-5 FW 個數控制組 總數N(mmo1)AA-m(mg)DIC(mg)DIC(uL)HOBt(mg)A Fmoc-Ala 311.329 130 1.08 336.2 136.3 168165.4C Fmoc-Cys(Trt)585.717 118 0.648 379.5 81.8 10199.2D Fmoc-Asp(OtBu) 411.542 244 1.584 651.8 199.9 247242.6E Fmoc-Glu(OtBu) 425.515 015 0.54 229.8 68.1 84 82.7F Fmoc-Phe 387.450 353 1.908 739.2 240.8 297292.2G Fmoc-Gly 297.337 037 1.332 396.0 168.1 208204.0H Fmoc-His(Trt)619.76 06 0.216 133.9 27.3 34 33.1I Fmoc-Ile 353.429 130 1.08 381.7 136.3 168165.4K Fmoc-Lys(Boc)468.530 030 1.08 506.0 136.3 168165.4L Fmoc-Leu 353.440 141 1.476 521.6 186.3 230226.0M Fmoc-Met 371.56 06 0.216 80.227.3 34 33.1N Fmoc-Asn(Trt)596.744 044 1.584 945.2 199.9 247242.6P Fmoc-Pro 337.435 035 1.26 425.1 159.0 196192.9Q Fmoc-Gln(Trt)610.717 017 0.612 373.7 77.2 95 93.7R Fmoc-Arg(Pmc)662.821 021 0.756 501.1 95.4 118115.8S Fmoc-Ser(tBu)383.447 047 1.692 648.7 213.5 264259.1T Fmoc-Thr(tBu)397.551 253 1.908 758.4 240.8 297292.2V Fmoc-Val 339.448 048 1.728 586.5 218.1 269264.6W Fmoc-Trp 426.55 05 0.18 76.822.7 28 27.6Y Fmoc-Tyr(tBu)459.631 031 1.116 512.9 140.8 174170.9
表8合成1～600號「交叉重疊」多肽第六步投料計算結果S-1-步驟-6 FW 個數 控制組 總數N(mmol)AA-m(mg) DIC(mg)DIC(uL)HOBt(mg)A Fmoc-Ala 311.329433 1.188 369.8149.9 185181.9C Fmoc-Cys(Trt)585.717017 0.612 358.477.2 95 93.7D Fmoc-Asp(OtBu) 411.541041 1.476 607.4186.3 230226.0E Fmoc-Glu(OtBu) 425.515015 0.54 229.868.1 84 82.7F Fmoc-Phe 387.450050 1.8697.3227.2 280275.6G Fmoc-Gly 297.337037 1.332 396.0168.1 208204.0H Fmoc-His(Trt)619.76 06 0.216 133.927.3 34 33.1I Fmoc-Ile 353.429029 1.044 368.9131.8 163159.9K Fmoc-Lys(Boc)468.530131 1.116 522.8140.8 174170.9L Fmoc-Leu 353.441243 1.548 547.1195.4 241237.0M Fmoc-Met 371.56 06 0.216 80.2 27.3 34 33.1N Fmoc-Asn(Trt)596.744044 1.584 945.2199.9 247242.6P Fmoc-Pro 337.435035 1.26 425.1159.0 196192.9Q Fmoc-Gln(Trt)610.717017 0.612 373.777.2 95 93.7R Fmoc-Arg(Pmc)662.821021 0.756 501.195.4 118115.8S Fmoc-Ser(tBu)383.447047 1.692 648.7213.5 264259.1T Fmoc-Thr(tBu)397.551253 1.908 758.4240.8 297292.2V Fmoc-Val 339.448149 1.764 598.7222.6 275270.1W Fmoc-Tm 426.55 05 0.18 76.8 22.7 28 27.6Y Fmoc-Tyr(tBu)459.631132 1.152 529.5145.4 179176.4
表9合成1～600號「交叉重疊」多肽第七步投料計算結果S-1-步驟-7FW 個數 控制組 總數N(mmol)AA-m(mg)DIC(mg)DIC(uL)HOBt(mg)A Fmoc-Ala 311.329231 1.116 347.4 140.8 174170.9C Fmoc-Cys(Trt) 585.717118 0.648 379.5 81.8 10199.2D Fmoc-Asp(OtBu)411.541041 1.476 607.4 186.3 230226.0E Fmoc-Glu(OtBu)425.515015 0.54 229.8 68.1 84 82.7F Fmoc-Phe 387.451152 1.872 725.2 236.2 292286.7G Fmoc-Gly 297.337037 1.332 396.0 168.1 208204.0H Fmoc-His(Trt) 619.76 06 0.216 133.9 27.3 34 33.1I Fmoc-Ile 353.429029 1.044 368.9 131.8 163159.9K Fmoc-Lys(Boc) 468.530030 1.08 506.0 136.3 168165.4L Fmoc-Leu 353.441142 1.512 534.3 190.8 236231.5M Fmoc-Met 371.56 06 0.216 80.227.3 34 33.1N Fmoc-Asn(Trt) 596.744044 1.584 945.2 199.9 247242.6P Fmoc-Pro 337.435136 1.296 437.3 163.6 202198.5Q Fmoc-Gln(Trt) 610.717017 0.612 373.7 77.2 95 93.7R Fmoc-Arg(Pmc) 662.821122 0.792 524.9 100.0 123121.3S Fmoc-Ser(tBu) 383.447249 1.764 676.3 222.6 275270.1T Fmoc-Thr(tBu) 397.550151 1.836 729.8 231.7 286281.1V Fmoc-Val 339.448149 1.764 598.7 222.6 275270.1W Fmoc-Trp 426.55 05 0.18 76.822.7 28 27.6Y Fmoc-Tyr(tBu) 459.631031 1.116 512.9 140.8 174170.9
表10合成1～600號「交叉重疊」多肽第八步投料計算結果S-1-步驟-8 FW 個數 控制組 總數N(mmol) AA-m(mg) DIC(mg) DIC(uL)HOBt(mg)A Fmoc-Ala 311.329231 1.116347.4140.8 174170.9C Fmoc-Cys(Trt)585.716016 0.576337.472.7 90 88.2D Fmoc-Asp(OtBu) 411.541041 1.476607.4186.3 230226.0E Fmoc-Glu(OtBu) 425.515015 0.54 229.868.1 84 82.7F Fmoc-Phe 387.451152 1.872725.2236.2 292286.7G Fmoc-Gly 297.337138 1.368406.7172.6 213209.5H Fmoc-His(Trt)619.76 06 0.216133.927.3 34 33.1I Fmoc-Ile 353.430232 1.152407.1145.4 179176.4K Fmoc-Lys(Boc)468.530030 1.08 506.0136.3 168165.4L Fmoc-Leu 353.441243 1.548547.1195.4 241237.0M Fmoc-Met 371.56 06 0.21680.2 27.3 34 33.1N Fmoc-Asn(Trt)596.744145 1.62 966.7204.4 252248.1P Fmoc-Pro 337.435035 1.26 425.1159.0 196192.9Q Fmoc-Gln(Trt)610.717118 0.648395.781.8 10199.2R Fmoc-Arg(Pmc)662.821021 0.756501.195.4 118115.8S Fmoc-Ser(tBu)383.447148 1.728662.5218.1 269264.6T Fmoc-Thr(tBu)397.550050 1.8 715.5227.2 280275.6V Fmoc-Val 339.448048 1.728586.5218.1 269264.6W Fmoc-Trp 426.55 05 0.18 76.8 22.7 28 27.6Y Fmoc-Tyr(tBu)459.631031 1.116512.9140.8 174170.9
表11合成1～600號「交叉重疊」多肽第九步投料計算結果S-1-步驟-9 FW 個數 控制組 總數 N(mmol)AA-m(mg) HOBt(mg)BOP(mg)DIPEA(mg)A Fmoc-Ala 311.3291301.8560.3 275.6 780.2 465.3C Fmoc-Cys(Trt)585.7160160.96 562.3 147.0 416.1 248.1D Fmoc-Asp(OtBu) 411.5410412.46 1012.3 376.7 1066.3 635.9E Fmoc-Glu(OtBu) 425.5150150.9383.0 137.8 390.1 232.6F Fmoc-Phe 387.4520523.12 1208.7 477.8 1352.4 806.5G Fmoc-Gly 297.3370372.22 660.0 339.9 962.3 573.8H Fmoc-His(Trt)619.76 06 0.36 223.1 55.1156.0 93.1I Fmoc-Ile 353.4302321.92 678.5 294.0 832.2 496.3K Fmoc-Lys(Boc)468.5301311.86 871.4 284.8 806.2 480.8L Fmoc-Leu 353.4410412.46 869.4 376.7 1066.3 635.9M Fmoc-Met 371.56 06 0.36 133.7 55.1156.0 93.1N Fmoc-Asn(Trt)596.7431442.64 1575.3 404.3 1144.3 682.4P Fmoc-Pro 337.4351362.16 728.8 330.8 936.3 558.3Q Fmoc-Gln(Trt)610.7170171.02 622.9 156.2 442.1 263.6R Fmoc-Arg(Pmc)662.8210211.26 835.1 192.9 546.2 325.7S Fmoc-Ser(tBu)383.4471482.88 1104.2 441.0 1248.3 744.4T Fmoc-Thr(tBu)397.5502523.12 1240.2 477.8 1352.4 806.5V Fmoc-Val 339.4482503 1018.2 459.4 1300.4 775.4W Fmoc-Trp 426.55 05 0.3128.0 45.9130.0 77.5Y Fmoc-Tyr(tBu)459.6310311.86 854.9 284.8 806.2 480.8
表12合成1～600號「交叉重疊」多肽第十步投料計算結果S-1-步驟-10 FW 個數 控制組 總數 N(mmol)AA-m(mg) HOBt(mg)BOP(mg) DIEA(mg)A Fmoc-Ala 311.329 1301.8560.3275.6 780.2 465.3C Fmoc-Cys(Trt)585.716 0160.96 562.3147.0 416.1 248.1D Fmoc-Asp(OtBu) 411.541 1422.52 1037.0 385.9 1092.3651.4E Fmoc-Glu(OtBu) 425.515 0150.9383.0137.8 390.1 232.6F Fmoc-Phe 387.452 1533.18 1231.9 487.0 1378.4822.0G Fmoc-Gly 297.337 2392.34 695.7358.3 1014.3604.8H Fmoc-His(Trt)619.76 06 0.36 223.155.1156.0 93.1I Fmoc-Ile 353.430 0301.8636.1275.6 780.2 465.3K Fmoc-Lys(Boc)468.530 2321.92 899.5294.0 832.2 496.3L Fmoc-Leu 353.441 1422.52 890.6385.9 1092.3651.4M Fmoc-Met 371.57 07 0.42 156.064.3182.1 108.6N Fmoc-Asn(Trt)596.743 0432.58 1539.5 395.1 1118.3666.9P Fmoc-Pro 337.435 1362.16 728.8330.8 936.3 558.3Q Fmoc-Gln(Trt)610.717 0171.02 622.9156.2 442.1 263.6R Fmoc-Arg(Pmc)662.821 0211.26 835.1192.9 546.2 325.7S Fmoc-Ser(tBu)383.447 1482.88 1104.2 441.0 1248.3744.4T Fmoc-Thr(tBu)397.550 0503 1192.5 459.4 1300.4775.4V Fmoc-Val 339.447 1482.88 977.5441.0 1248.3744.4W Fmoc-Trp 426.55 05 0.3128.045.9130.0 77.5Y Fmoc-Tyr(tBu) 459.631 0311.86 854.9284.8 806.2 480.8
表13合成肽庫中隨機抽取的多肽化合物的純度以及正確的分子量

實施例2設計合成Spike(S)蛋白601至1246位胺基酸的每10位序列的「交叉重疊」序列如下。從右至左為合成次序，代表合成步驟號碼為阿拉伯數字1到10。
表14 Spike(S)蛋白中所合成的646個以10個胺基酸為長度的序列編號10 987654321601)VNCTDVSTAI602)NCTDVSTAIH603)CTDVSTAIHA604)TDVSTAIHAD605)DVSTAIHADQ606)VSTAIHADQL607)STAIHADQLT608)TAIHADQLTP609)AIHADQLTPA610)IHADQLTPAW611)HADQLTPAWR612)ADQLTPAWRI613)DQLTPAWRIY614)QLTPAWRIYS615)LTPAWRIYST616)TPAWRIYSTG617)PAWRIYSTGN618)AWRIYSTGNN619)WRIYSTGNNV620)RIYSTGNNVF621)IYSTGNNVFQ622)YSTGNNVFQT623)STGNNVFQTQ624)TGNNVFQTQA625)GNNVFQTQAG626)NNVFQTQAGC627)NVFQTQAGCL
628)VFQTQAGCLI629)FQTQAGCLIG630)QTQAGCLIGA631)TQAGCLIGAE632)QAGCLIGAEH633)AGCLIGAEHV634)GCLIGAEHVD635)CLIGAEHVDT636)LIGAEHVDTS637)IGAEHVDTSY638)GAEHVDTSYE639)AEHVDTSYEC640)EHVDTSYECD641)HVDTSYECDI642)VDTSYECDIP643)DTSYECDIPI644)TSYECDIPIG645)SYECDIPIGA646)YECDIPIGAG647)ECDIPIGAGI648)CDIPIGAGIC649)DIPIGAGICA650)IPIGAGICAS651)PIGAGICASY652)IGAGICASYH653)GAGICASYHT654)AGICASYHTV655)GICASYHTVS656)ICASYHTVSL657)CASYHTVSLL658)ASYHTVSLLR659)SYHTVSLLRS660)YHTVSLLRST661)HTVSLLRSTS662)TVSLLRSTSQ663)VSLLRSTSQK664)SLLRSTSQKS665)LLRSTSQKSI666)LRSTSQKSIV667)RSTSQKSIVA668)STSQKSIVAY669)TSQKSIVAYT670)SQKSIVAYTM671)QKSIVAYTMS672)KSIVAYTMSL673)SIVAYTMSLG674)IVAYTMSLGA675)VAYTMSLGAD676)AYTMSLGADS677)YTMSLGADSS
678)TMSLGADSSI679)MSLGADSSIA680)SLGADSSIAY681)LGADSSIAYS682)GADSSIAYSN683)ADSSIAYSNN684)DSSIAYSNNT685)SSIAYSNNTI686)SIAYSNNTIA687)IAYSNNTIAI688)AYSNNTIAIP689)YSNNTIAIPT690)SNNTIAIPTN691)NNTIAIPTNF692)NTIAIPTNFS693)TIAIPTNFSI694)IAIPTNFSIS695)AIPTNFSISI696)IPTNFSISIT697)PTNFSISITT698)TNFSISITTE699)NFSISITTEV700)FSISITTEVM701)SISITTEVMP702)ISITTEVMPV703)SITTEVMPVS704)ITTEVMPVSM705)TTEVMPVSMA706)TEVMPVSMAK707)EVMPVSMAKT708)VMPVSMAKTS709)MPVSMAKTSV710)PVSMAKTSVD711)VSMAKTSVDC712)SMAKTSVDCN713)MAKTSVDCNM714)AKTSVDCNMY715)KTSVDCNMYI716)TSVDCNMYIC717)SVDCNMYICG718)VDCNMYICGD719)DCNMYICGDS720)CNMYICGDST721)NMYICGDSTE722)MYICGDSTEC723)YICGDSTECA724)ICGDSTECAN725)CGDSTECANL726)GDSTECANLL727)DSTECANLLL
728)STECANLLLQ729)TECANLLLQY730)ECANLLLQYG731)CANLLLQYGS732)ANLLLQYGSF733)NLLLQYGSFC734)LLLQYGSFCT735)LLQYGSFCTQ736)LQYGSFCTQL737)QYGSFCTQLN738)YGSFCTQLNR739)GSFCTQLNRA740)SFCTQLNRAL741)FCTQLNRALS742)CTQLNRALSG743)TQLNRALSGI744)QLNRALSGIA745)LNRALSGIAA746)NRALSGIAAE747)RALSGIAAEQ748)ALSGIAAEQD749)LSGIAAEQDR750)SGIAAEQDRN751)GIAAEQDRNT752)IAAEQDRNTR753)AAEQDRNTRE754)AEQDRNTREV755)EQDRNTREVF756)QDRNTREVFA757)DRNTREVFAQ758)RNTREVFAQV759)NTREVFAQVK760)TREVFAQVKQ761)REVFAQVKQM762)EVFAQVKQMY763)VFAQVKQMYK764)FAQVKQMYKT765)AQVKQMYKTP766)QVKQMYKTPT767)VKQMYKTPTL768)KQMYKTPTLK769)QMYKTPTLKY770)MYKTPTLKYF771)YKTPTLKYFG772)KTPTLKYFGG773)TPTLKYFGGF774)PTLKYFGGFN775)TLKYFGGFNF776)LKYFGGFNFS777)KYFGGFNFSQ
778)YFGGFNFSQI779)FGGFNFSQIL780)GGFNFSQILP781)GFNFSQILPD782)FNFSQILPDP783)NFSQILPDPL784)FSQILPDPLK785)SQILPDPLKP786)QILPDPLKPT787)ILPDPLKPTK788)LPDPLKPTKR789)PDPLKPTKRS790)DPLKPTKRSF791)PLKPTKRSFI792)LKPTKRSFIE793)KPTKRSFIED794)PTKRSFIEDL795)TKRSFIEDLL796)KRSFIEDLLF797)RSFIEDLLFN798)SFIEDLLFNK799)FIEDLLFNKV800)IEDLLFNKVT801)EDLLFNKVTL802)DLLFNKVTLA803)LLFNKVTLAD804)LFNKVTLADA805)FNKVTLADAG806)NKVTLADAGF807)KVTLADAGFM808)VTLADAGFMK809)TLADAGFMKQ810)LADAGFMKQY811)ADAGFMKQYG812)DAGFMKQYGE813)AGFMKQYGEC814)GFMKQYGECL815)FMKQYGECLG816)MKQYGECLGD817)KQYGECLGDI818)QYGECLGDIN819)YGECLGDINA820)GECLGDINAR821)ECLGDINARD822)CLGDINARDL823)LGDINARDLI824)GDINARDLIC825)DINARDLICA826)INARDLICAQ827)NARDLICAQK
828)ARDLICAQKF829)RDLICAQKFN830)DLICAQKFNG831)LICAQKFNGL832)ICAQKFNGLT833)CAQKFNGLTV834)AQKFNGLTVL835)QKFNGLTVLP836)KFNGLTVLPP837)FNGLTVLPPL838)NGLTVLPPLL839)GLTVLPPLLT840)LTVLPPLLTD841)TVLPPLLTDD842)VLPPLLTDDM843)LPPLLTDDMI844)PPLLTDDMIA845)PLLTDDMIAA846)LLTDDMIAAY847)LTDDMIAAYT848)TDDMIAAYTA849)DDMIAAYTAA850)DMIAAYTAAL851)MIAAYTAALV852)IAAYTAALVS853)AAYTAALVSG854)AYTAALVSGT855)YTAALVSGTA856)TAALVSGTAT857)AALVSGTATA858)ALVSGTATAG859)LVSGTATAGW860)VSGTATAGWT861)SGTATAGWTF862)GTATAGWTFG863)TATAGWTFGA864)ATAGWTFGAG865)TAGWTFGAGA866)AGWTFGAGAA867)GWTFGAGAAL868)WTFGAGAALQ869)TFGAGAALQI870)FGAGAALQIP871)GAGAALQIPF872)AGAALQIPFA873)GAALQIPFAM874)AALQIPFAMQ875)ALQIPFAMQM876)LQIPFAMQMA877)QIPFAMQMAY
878)IPFAMQMAYR879)PFAMQMAYRF880)FAMQMAYRFN881)AMQMAYRFNG882)MQMAYRFNGI883)QMAYRFNGIG884)MAYRFNGIGV885)AYRFNGIGVT886)YRFNGIGVTQ887)RFNGIGVTQN888)FNGIGVTQNV889)NGIGVTQNVL890)GIGVTQNVLY891)IGVTQNVLYE892)GVTQNVLYEN893)VTQNVLYENQ894)TQNVLYENQK895)QNVLYENQKQ896)NVLYENQKQI897)VLYENQKQIA898)LYENQKQIAN899)YENQKQIANQ900)ENQKQIANQF901)NQKQIANQFN902)QKQIANQFNK903)KQIANQFNKA904)QIANQFNKAI905)IANQFNKAIS906)ANQFNKAISQ907)NQFNKAISQI908)QFNKAISQIQ909)FNKAISQIQE910)NKAISQIQES911)KAISQIQESL912)AISQIQESLT913)ISQIQESLTT914)SQIQESLTTT915)QIQESLTTTS916)IQESLTTTST917)QESLTTTSTA918)ESLTTTSTAL919)SLTTTSTALG920)LTTTSTALGK921)TTTSTALGKL922)TTSTALGKLQ923)TSTALGKLQD924)STALGKLQDV925)TALGKLQDVV926)ALGKLQDVVN927)LGKLQDVVNQ
928)GKLQDVVNQN929)KLQDVVNQNA930)LQDVVNQNAQ931)QDVVNQNAQA932)DVVNQNAQAL933)VVNQNAQALN934)VNQNAQALNT935)NQNAQALNTL936)QNAQALNTLV937)NAQALNTLVK938)AQALNTLVKQ939)QALNTLVKQL940)ALNTLVKQLS941)LNTLVKQLSS942)NTLVKQLSSN943)TLVKQLSSNF944)LVKQLSSNFG945)VKQLSSNFGA946)KQLSSNFGAI947)QLSSNFGAIS948)LSSNFGAISS949)SSNFGAISSV950)SNFGAISSVL951)NFGAISSVLN952)FGAISSVLND953)GAISSVLNDI954)AISSVLNDIL955)ISSVLNDILS956)SSVLNDILSR957)SVLNDILSRL958)VLNDILSRLD959)LNDILSRLDK960)NDILSRLDKV961)DILSRLDKVE962)ILSRLDKVEA963)LSRLDKVEAE964)SRLDKVEAEV965)RLDKVEAEVQ966)LDKVEAEVQI967)DKVEAEVQID968)KVEAEVQIDR969)VEAEVQIDRL970)EAEVQIDRLI971)AEVQIDRLIT972)EVQIDRLITG973)VQIDRLITGR974)QIDRLITGRL975)IDRLITGRLQ976)DRLITGRLQS977)RLITGRLQSL
978)LITGRLQSLQ979)ITGRLQSLQT980)TGRLQSLQTY981)GRLQSLQTYV982)RLQSLQTYVT983)LQSLQTYVTQ984)QSLQTYVTQQ985)SLQTYVTQQL986)LQTYVTQQLI987)QTYVTQQLIR988)TYVTQQLIRA989)YVTQQLIRAA990)VTQQLIRAAE991)TQQLIRAAEI992)QQLIRAAEIR993)QLIRAAEIRA994)LIRAAEIRAS995)IRAAEIRASA996)RAAEIRASAN997)AAEIRASANL998)AEIRASANLA999)EIRASANLAA1000)IRASANLAAT1001)RASANLAATK1002)ASANLAATKM1003)SANLAATKMS1004)ANLAATKMSE1005)NLAATKMSEC1006)LAATKMSECV1007)AATKMSECVL1008)ATKMSECVLG1009)TKMSECVLGQ1010)KMSECVLGQS1011)MSECVLGQSK1012)SECVLGQSKR1013)ECVLGQSKRV1014)CVLGQSKRVD1015)VLGQSKRVDF1016)LGQSKRVDFC1017)GQSKRVDFCG1018)QSKRVpFCGK1019)SKRVDFCGKG1020)KRVDFCGKGY1021)RVDFCGKGYH1022)VDFCGKGYHL1023)DFCGKGYHLM1024)FCGKGYHLMS1025)CGKGYHLMSF1026)GKGYHLMSFP1027)KGYHLMSFPQ
1028)GYHLMSFPQA1029)YHLMSFPQAA1030)HLMSFPQAAP1031)LMSFPQAAPH1032)MSFPQAAPHG1033)SFPQAAPHGV1034)FPQAAPHGVV1035)PQAAPHGVVF1036)QAAPHGVVFL1037)AAPHGVVFLH1038)APHGVVFLHV1039)PHGVVFLHVT1040)HGVVFLHVTY1041)GVVFLHVTYV1042)VVFLHVTYVP1043)VFLHVTYVPS1044)FLHVTYVPSQ1045)LHVTYVPSQE1046)HVTYVPSQER1047)VTYVPSQERN1048)TYVPSQERNF1049)YVPSQERNFT1050)VPSQERNFTT1051)PSQERNFTTA1052)SQERNFTTAP1053)QERNFTTAPA1054)ERNFTTAPAI1055)RNFTTAPAIC1056)NFTTAPAICH1057)FTTAPAICHE1058)TTAPAICHEG1059)TAPAICHEGK1060)APAICHEGKA1061)PAICHEGKAY1062)AICHEGKAYF1063)ICHEGKAYFP1064)CHEGKAYFPR1065)HEGKAYFPRE1066)EGKAYFPREG1067)GKAYFPREGV1068)KAYFPREGVF1069)AYFPREGVFV1070)YFPREGVFVF1071)FPREGVFVFN1072)PREGVFVFNG1073)REGVFVFNGT1074)EGVFVFNGTS1075)GVFVFNGTSW1076)VFVFNGTSWF1077)FVFNGTSWFI
1078)VFNGTSWFIT1079)FNGTSWFITQ1080)NGTSWFITQR1081)GTSWFITQRN1082)TSWFITQRNF1083)SWFITQRNFF1084)WFITQRNFFS1085)FITQRNFFSP1086)ITQRNFFSPQ1087)TQRNFFSPQI1088)QRNFFSPQII1089)RNFFSPQIIT1090)NFFSPQIITT1091)FFSPQIITTD1092)FSPQIITTDN1093)SPQIITTDNT1094)PQIITTDNTF1095)QIITTDNTFV1096)IITTDNTFVS1097)ITTDNTFVSG1098)TTDNTFVSGN1099)TDNTFVSGNC1100)DNTFVSGNCD1101)NTFVSGNCDV1102)TFVSGNCDVV1103)FVSGNCDVVI1104)VSGNCDVVIG1105)SGNCDVVIGI1106)GNCDVVIGII1107)NCDVVIGIIN1108)CDVVIGIINN1109)DVVIGIINNT1110)VVIGIINNTV1111)VIGIINNTVY1112)IGIINNTVYD1113)GIINNTVYDP1114)IINNTVYDPL1115)INNTVYDPLQ1116)NNTVYDPLQP1117)NTVYDPLQPE1118)TVYDPLQPEL1119)VYDPLQPELD1120)YDPLQPELDS1121)DPLQPELDSF1122)PLQPELDSFK1123)LQPELDSFKE1124)QPELDSFKEE1125)PELDSFKEEL1126)ELDSFKEELD1127)LDSFKEELDK
1128)DSFKEELDKY1129)SFKEELDKYF1130)FKEELDKYFK1131)KEELDKYFKN1132)EELDKYFKNH1133)ELDKYFKNHT1134)LDKYFKNHTS1135)DKYFKNHTSP1136)KYFKNHTSPD1137)YFKNHTSPDV1138)FKNHTSPDVD1139)KNHTSPDVDL1140)NHTSPDVDLG1141)HTSPDVDLGD1142)TSPDVDLGDI1143)SPDVDLGDIS1144)PDVDLGDISG1145)DVDLGDISGI1146)VDLGDISGIN1147)DLGDISGINA1148)LGDISGINAS1149)GDISGINASV1150)DISGINASVV1151)ISGINASVVN1152)SGINASVVNI1153)GINASVVNIQ1154)INASVVNIQK1155)NASVVNIQKE1156)ASVVNIQKEI1157)SVVNIQKEID1158)VVNIQKEIDR1159)VNIQKEIDRL1160)NIQKEIDRLN1161)IQKEIDRLNE1162)QKEIDRLNEV1163)KEIDRLNEVA1164)EIDRLNEVAK1165)IDRLNEVAKN1166)DRLNEVAKNL1167)RLNEVAKNLN1168)LNEVAKNLNE1169)NEVAKNLNES1170)EVAKNLNESL1171)VAKNLNESLI1172)AKNLNESLID1173)KNLNESLIDL1174)NLNESLIDLQ1175)LNESLIDLQE1176)NESLIDLQEL1177)ESLIDLQELG
1178)SLIDLQELGK1179)LIDLQELGKY1180)IDLQELGKYE1181)DLQELGKYEQ1182)LQELGKYEQY1183)QELGKYEQYI1184)ELGKYEQYIK1185)LGKYEQYIKW1186)GKYEQYIKWP1187)KYEQYIKWPW1188)YEQYIKWPWY1189)EQYIKWPWYV1190)QYIKWPWYVW1191)YIKWPWYVWL1192)IKWPWYVWLG1193)KWPWYVWLGF1194)WPWYVWLGFI1195)PWYVWLGFIA1196)WYVWLGFIAG1197)YVWLGFIAGL1198)VWLGFIAGLI1199)WLGFIAGLIA1200)LGFIAGLIAI1201)GFIAGLIAIV1202)FIAGLIAIVM1203)IAGLIAIVMV1204)AGLIAIVMVT1205)GLIAIVMVTI1206)LIAIVMVTIL1207)IAIVMVTILL1208)AIVMVTILLC1209)IVMVTILLCC1210)VMVTILLCCM1211)MVTILLCCMT1212)VTILLCCMTS1213)TILLCCMTSC1214)ILLCCMTSCC1215)LLCCMTSCCS1216)LCCMTSCCSC1217)CCMTSCCSCL1218)CMTSCCSCLK1219)MTSCCSCLKG1220)TSCCSCLKGA1221)SCCSCLKGAC1222)CCSCLKGACS1223)CSCLKGACSC1224)SCLKGACSCG1225)CLKGACSCGS1226)LKGACSCGSC1227)KGACSCGSCC
1228)GACSCGSCCK1229)ACSCGSCCKF1230)CSCGSCCKFD1231)SCGSCCKFDE1232)CGSCCKFDED1233)GSCCKFDEDD1234)SCCKFDEDDS1235)CCKFDEDDSE1236)CKFDEDDSEP1237)KFDEDDSEPV1238)FDEDDSEPVL1239)DEDDSEPVLK1240)EDDSEPVLKG1241)DDSEPVLKGV1242)DSEPVLKGVK1243)SEPVLKGVKL1244)EPVLKGVKLH1245)PVLKGVKLHY1246)VLKGVKLHYT其合成方法同實施例1，所不同的是從第1步至第10步的投料。以下表15至表24為第1步至第10步的投料
表15合成Spike(S)蛋白601～1246號「交叉重疊」多肽第一步時使用試劑投料計算結果S-2-步驟-1FW 個數 控制組 總數 N(mmol)AA-m(mg) HOBt(mg)BOP(mg) DIEA(mg)A Fmoc-Ala 311.3551 562.016 627.6 308.7 847.5521.1C Fmoc-Cys(Trt) 585.7232 250.9527.1 137.8 378.3232.6D Fmoc-Asp(OtBu)411.5330 331.188 488.9 181.9 499.4307.1E Fmoc-Glu(OtBu)425.5271 281.008 428.9 154.4 423.7260.5F Fmoc-Phe 387.4311 321.152 446.3 176.4 484.3297.8G Fmoc-Gly 297.3400 401.44 428.1 220.5 605.3372.2H Fmoc-His(Trt) 619.78 0 8 0.288 178.5 44.1121.174.4I Fmoc-Ile 353.4472 491.764 623.4 270.1 741.5456.0K Fmoc-Lys(Boc) 468.5280 281.008 472.2 154.4 423.7260.5L Fmoc-Leu 353.4571 582.088 737.9 319.7 877.7539.7M Fmoc-Met 371.5131 140.504 187.2 77.2211.9130.3N Fmoc-Asn 354.4380 381.368 484.8 209.5 575.1353.6P Fmoc-Pro 337.4220 220.792 267.2 121.3 332.9204.7Q Fmoc-Gln 368.4380 381.368 504.0 209.5 575.1353.6R Fmoc-Arg(Pbf) 648.8182 200.72 467.1 110.3 302.7186.1S Fmoc-Ser(tBu) 383.4490 491.764 676.3 270.1 741.5456.0T Fmoc-Thr(tBu) 397.5493 521.872 744.1 286.7 786.9483.9V Fmoc-Val 339.4420 421.512 513.2 231.5 635.6390.8W Fmoc-Trp 426.56 0 6 0.216 92.1 33.190.8 55.8Y Fmoc-Tyr(tBu) 459.6220 220.792 364.0 121.3 332.9204.7
表16合成Spike(S)蛋白601～1246號「交叉重疊」多肽第二步時使用試劑投料計算結果S-2-步驟-2 FW 個數 控制組 總數N(mmol)AA-m(mg)DIC(mg)HOBt(mg)AFmoc-Ala 311.3552 57 2.052 638.8 259.0 314.2CFmoc-Cys(Trt)585.7221 23 0.828 485.0 104.5 126.8DFmoc-Asp(OtBu) 411.5320 32 1.152 474.0 145.4 176.4EFmoc-Glu(OtBu) 425.5271 28 1.008 428.9 127.2 154.4FFmoc-Phe 387.4311 32 1.152 446.3 145.4 176.4GFmoc-Gly 297.3411 42 1.512 449.5 190.8 231.5HFmoc-His(Trt)619.79 09 0.324 200.8 40.9 49.6IFmoc-Ile 353.4471 48 1.728 610.7 218.1 264.6KFmoc-Lys(Boc)468.5300 30 1.08 506.0 136.3 165.4LFmoc-Leu 353.4582 60 2.16 763.3 272.6 330.8MFmoc-Met 371.5131 14 0.504 187.2 63.6 77.2NFmoc-Asn 354.4370 37 1.332 472.1 168.1 204.0PFmoc-Pro 337.4221 23 0.828 279.4 104.5 126.8QFmoc-Gln 368.4381 39 1.404 517.2 177.2 215.0RFmoc-Arg(Pbf)648.8180 18 0.648 420.4 81.8 99.2SFmoc-Ser(tBu)383.4481 49 1.764 676.3 222.6 270.1TFmoc-Thr(tBu)397.5470 47 1.692 672.6 213.5 259.1VFmoc-Val 339.4420 42 1.512 513.2 190.8 231.5WFmoc-Trp 426.56 0 6 0.216 92.127.3 33.1YFmoc-Tyr(tBu)459.6231 24 0.864 397.1 109.0 132.3
表17合成Spike(S)蛋白601～1246號「交叉重疊」多肽第三步時使用試劑投料計算結果S-2-步驟-3 FW 個數 控制組 總數 N(mmol)AA-m(mg) DIC(mg) HOBt(mg)AFmoc-Ala 311.3553 582.088 650.0 263.5319.7CFmoc-Cys(Trt)585.7220 220.792 463.9 100.0121.3DFmoc-Asp(OtBu) 411.5322 341.224 503.7 154.5187.4EFmoc-Glu(OtBu) 425.5271 281.008 428.9 127.2154.4FFmoc-Phe 387.4310 311.116 432.3 140.8170.9GFmoc-Gly 297.3410 411.476 438.8 186.3226.0HFmoc-His(Trt)619.79 0 9 0.324 200.8 40.9 49.6IFmoc-Ile 353.4470 471.692 598.0 213.5259.1KFmoc-Lys(Boc)468.5300 301.08 506.0 136.3165.4LFmoc-Leu 353.4581 592.124 750.6 268.0325.2MFmoc-Met 371.5131 140.504 187.2 63.6 77.2NFmoc-Asn 354.4372 391.404 497.6 177.2215.0PFmoc-Pro 337.4220 220.792 267.2 100.0121.3QFmoc-Gln 368.4381 391.404 517.2 177.2215.0RFmoc-Arg(Pbf)648.8180 180.648 420.4 81.8 99.2SFmoc-Ser(tBu)383.4482 501.8690.1 227.2275.6TFmoc-Thu(tBu)397.5480 481.728 686.9 218.1264.6VFmoc-Val 339.4421 431.548 525.4 195.4237.0WFmoc-Trp 426.56 0 6 0.216 92.1 27.3 33.1YFmoc-Tyt(tBu)459.6220 220.792 364.0 100.0121.3
表18合成Spike(S)蛋白601～1246號「交叉重疊」多肽第四步時使用試劑投料計算結果S-2-步驟-4FW 個數 控制組 總數N(mmol) AA-m(mg)DIC(mg) HOBt(mg)AFmoc-Ala 311.3551 56 2.016627.6 254.4308.7CFmoc-Cys(Trt) 585.7220 22 0.792463.9 100.0121.3DFmoc-Asp(OtBu)411.5320 32 1.152474.0 145.4176.4EFmoc-Glu(OtBu)425.5271 28 1.008428.9 127.2154.4FFmoc-Phe 387.4310 31 1.116432.3 140.8170.9GFmoc-Gly 297.3411 42 1.512449.5 190.8231.5HFmoc-His(Trt) 619.78 0 8 0.288178.5 36.3 44.1IFmoc-Ile 353.4473 50 1.8 636.1 227.2275.6KFmoc-Lys(Boc) 468.5300 30 1.08 506.0 136.3165.4LFmoc-Leu 353.4583 61 2.196776.1 277.1336.3MFmoc-Met 371.5130 13 0.468173.9 59.1 71.7NFmoc-Asn 354.4370 37 1.332472.1 168.1204.0PFmoc-Pro 337.4220 22 0.792267.2 100.0121.3QFmoc-Gln 368.4380 38 1.368504.0 172.6209.5RFmoc-Arg(Pbf) 648.8181 19 0.684443.8 86.3 104.7SFmoc-Ser(tBu) 383.4491 50 1.8 690.1 227.2275.6TFmoc-Thr(tBu) 397.5482 50 1.8 715.5 227.2275.6VFmoc-Val 339.4421 43 1.548525.4 195.4237.0WFmoc-Trp 426.56 0 6 0.21692.127.3 33.1YFmoc-Tyr(tBu) 459.6220 22 0.792364.0 100.0121.3
表19合成Spike(S)蛋白601～1246號「交叉重疊」多肽第五步時使用試劑投料計算結果S-2-步驟-5 FW 個數 控制組 總數 N(mmol) AA-m(mg) DIC(mg) HOBt(mg)A Fmoc-Ala 311.3552 572.052638.8 259.0314.2C Fmoc-Cys(Trt)585.7220 220.792463.9 100.0121.3D Fmoc-Asp(OtBu) 411.5320 321.152474.0 145.4176.4E Fmoc-Glu(OtBu) 425.5270 270.972413.6 122.7148.8F Fmoc-Phe 387.4311 321.152446.3 145.4176.4G Fmoc-Gly 297.3411 421.512449.5 190.8231.5H Fmoc-His(Trt)619.78 0 8 0.288178.5 36.3 44.1I Fmoc-Ile 353.4471 481.728610.7 218.1264.6K Fmoc-Lys(Boc)468.5300 301.08 506.0 136.3165.4L Fmoc-Leu 353.4571 582.088737.9 263.5319.7M Fmoc-Met 371.5130 130.468173.9 59.1 71.7N Fmoc-Asn 354.4371 381.368484.8 172.6209.5P Fmoc-Pro 337.4221 230.828279.4 104.5126.8Q Fmoc-Gln 368.4382 401.44 530.5 181.7220.5R Fmoc-Arg(Pbf)648.8180 180.648420.4 81.8 99.2S Fmoc-Ser(tBu)383.4490 491.764676.3 222.6270.1T Fmoc-Thr(tBu)397.5482 501.8 715.5 227.2275.6V Fmoc-Val 339.4431 441.584537.6 199.9242.6W Fmoc-Trp 426.56 0 6 0.21692.1 27.3 33.1Y Fmoc-Tyr(tBu)459.6221 230.828380.5 104.5126.8
表20合成Spike(S)蛋白601～1246號「交叉重疊」多肽第六步時使用試劑投料計算結果S-2-步驟-6 FW 個數 控制組 總數 N(mmol) AA-m(mg)HOBt(mg)HBTu(mg)DIEA(mg)A Fmoc-Ala 311.3551 562.016627.6 308.7 726.3 521.1C Fmoc-Cys(Trt)585.7220 220.792463.9 121.3 285.3 204.7D Fmoc-Asp(OtBu) 411.5331 341.224503.7 187.4 441.0 316.4E Fmoc-Glu(OtBu) 425.5270 270.972413.6 148.8 350.2 251.2F Fmoc-Phe 387.4310 311.116432.3 170.9 402.1 288.5G Fmoc-Gly 297.3410 411.476438.8 226.0 531.8 381.5H Fmoc-His(Trt)619.78 0 8 0.288178.5 44.1103.8 74.4I Fmoc-Ile 353.4473 501.8 636.1 275.6 648.5 465.3K Fmoc-Lys(Boc)468.5293 321.152539.7 176.4 415.1 297.8L Fmoc-Leu 353.4571 582.088737.9 319.7 752.3 539.7M Fmoc-Met 371.5130 130.468173.9 71.7168.6 121.0N Fmoc-Asn 354.4370 371.332472.1 204.0 479.9 344.3P Fmoc-Pro 337.4222 240.864291.5 132.3 311.3 223.3Q Fmoc-Gln 368.4380 381.368504.0 209.5 492.9 353.6R Fmoc-Arg(Pbf)648.8180 180.648420.4 99.2233.5 167.5S Fmoc-Ser(tBu)383.4492 511.836703.9 281.1 661.5 474.6T Fmoc-Thr(tBu)397.5480 481.728686.9 264.6 622.6 446.7V Fmoc-Val 339.4431 441.584537.6 242.6 570.7 409.4W Fmoc-Trp 426.56 0 6 0.21692.133.177.855.8Y Fmoc-Tyr(tBu)459.6220 220.792364.0 121.3 285.3 204.7
表21合成Spike(S)蛋白601～1246號「交叉重疊」多肽第七步時使用試劑投料計算結果S-2-步驟-7 FW 個數 控制組總數N(mmol) AA-m(mg) HOBt(mg) HBTu(mg) DIEA(mg)A Fmoc-Ala 311.3553 583.48 1083.3532.9 1253.8 899.5C Fmoc-Cys(Trt)585.7220 221.32 773.1 202.1 475.6 341.2D Fmoc-Asp(OtBu) 411.5331 342.04 839.5 312.4 735.0 527.3E Fmoc-Glu(OtBu) 425.5270 271.62 689.3 248.1 583.7 418.7F Fmoc-Phe 387.4310 311.86 720.6 284.8 670.1 480.8G Fmoc-Gly 297.3410 412.46 731.4 376.7 886.3 635.9H Fmoc-His(Trt)619.78 0 8 0.48 297.5 73.5 172.9 124.1I Fmoc-Ile 353.4472 492.94 1039.0450.2 1059.2 759.9K Fmoc-Lys(Boc)468.5290 291.74 815.2 266.4 626.9 449.8L Fmoc-Leu 353.4572 593.54 1251.0542.1 1275.4 915.0M Fmoc-Met 371.5131 140.84 312.1 128.6 302.6 217.1N Fmoc-Ash 354.4371 382.28 808.0 349.1 821.5 589.3P Fmoc-Pro 337.4221 231.38 465.6 211.3 497.2 356.7Q Fmoc-Gln 368.4381 392.34 862.1 358.3 843.1 604.8R Fmoc-Arg(Pbf)648.8180 181.08 700.7 165.4 389.1 279.2S Fmoc-Ser(tBu)383.4490 492.94 1127.2450.2 1059.2 759.9T Fmoc-Thr(tBu)397.5491 5031192.5459.4 1080.9 775.4V Fmoc-Val 339.4421 432.58 875.7 395.1 929.5 666.9W Fmoc-Trp 426.56 0 6 0.36 153.5 55.1 29.7 93.1Y Fmoc-Tyr(tBu)459.6220 221.32 606.7 202.1 475.6 341.2
表22合成Spike(S)蛋白601～1246號「交叉重疊」多肽第八步時使用試劑投料計算結果S-2-步驟-8 FW 個數 控制組 總數 N(mmol) AA-m(mg)HOBt(mg)HBTu(mg)DIEA(mg)A Fmoc-Ala 311.3552 573.42 1064.6 523.7 1232.2 884.0C Fmoc-Cys(Trt)585.7230 231.38 808.3 211.3 497.2 356.7D Fmoc-Asp(OtBu) 411.5332 352.1 864.2 321.6 756.6 542.8E Fmoc-Glu(OtBu) 425.5270 271.62 689.3 248.1 583.7 418.7F Fmoc-Phe 387.4310 311.86 720.6 284.8 670.1 480.8G Fmoc-Gly 297.3401 412.46 731.4 376.7 886.3 635.9H Fmoc-His(Trt)619.78 0 8 0.48 297.5 73.5172.9 124.1I Fmoc-Ile 353.4473 503 1060.2 459.4 1080.9 775.4K Fmoc-Lys(Boc)468.5290 291.74 815.2 266.4 626.9 449.8L Fmoc-Leu 353.4571 583.48 1229.8 532.9 1253.8 899.5M Fmoc-Met 371.5130 130.78 289.8 119.4 281.0 201.6N Fmoc-Asn 354.4370 372.22 786.8 339.9 799.8 573.8P Fmoc-Pro 337.4220 221.32 445.4 202.1 475.6 341.2Q Fmoc-Gln 368.4381 392.34 862.1 358.3 843.1 604.8R Fmoc-Arg(Pbf)648.8180 181.08 700.7 165.4 389.1 279.2S Fmoc-Ser(tBu)383.4491 503 1150.2 459.4 1080.9 775.4T Fmoc-Thr(tBu)397.5491 503 1192.5 459.4 1080.9 775.4V Fmoc-Val 339.4422 442.64 896.0 404.3 951.2 682.4W Fmoc-Trp 426.56 0 6 0.36 153.5 55.1129.7 93.1Y Fmoc-Tyr(tBu)459.6220 221.32 606.7 202.1 475.6 341.2
表23合成Spike(S)蛋白601～1246號「交叉重疊」多肽第九步時使用試劑投料計算結果S-2-步驟-9 FW 個數 控制組 總數 N(mmol) AA-m(mg) HOBt(mg)HBTu(mg)DIEA(mg)A Fmoc-Ala 311.3551 563.36 1046.0 514.5 1210.6 868.5C Fmoc-Cys(Trt)585.7232 251.5 878.6 229.7 540.4 387.7D Fmoc-Asp(OtBu) 411.5330 331.98 814.8 303.2 713.4 511.8E Fmoc-Glu(OtBu) 425.5271 281.68 714.8 257.3 605.3 434.2F Fmoc-Phe 387.4311 321.92 743.8 294.0 691.8 496.3G Fmoc-Gly 297.3400 402.4 713.5 367.5 864.7 620.4H Fmoc-His(Trt)619.78 0 8 0.48 297.5 73.5172.9 124.1I Fmoc-Ile 353.4472 492.94 1039.0 450.2 1059.2 759.9K Fmoc-Lys(Boc)468.5280 281.68 787.1 257.3 605.3 434.2L Fmoc-Leu 353.4571 583.48 1229.8 532.9 1253.8 899.5M Fmoc-Met 371.5131 140.84 312.1 128.6 302.6 217.1N Fmoc-Asn 354.4380 382.28 808.0 349.1 821.5 589.3P Fmoc-Pro 337.4220 221.32 445.4 202.1 475.6 341.2Q Fmoc-Gln 368.4380 382.28 840.0 349.1 821.5 589.3R Fmoc-Arg(Pbf)648.8182 201.2 778.6 183.8 432.3 310.2S Fmoc-Ser(tBu)383.4490 492.94 1127.2 450.2 1059.2 759.9T Fmoc-Thr(tBu)397.5493 523.12 1240.2 477.8 1124.1 806.5V Fmoc-Val 339.4420 422.52 855.3 385.9 907.9 651.4W Fmoc-Trp 426.56 0 6 0.36 153.5 55.1129.7 93.1Y Fmoc-Tyr(tBu)459.6220 221.32 606.7 202.1 475.6 341.2
表24合成Spike(S)蛋白601～1246號「交叉重疊」多肽第十步時使用試劑投料計算結果S-2-步驟-10 FW 個數 控制組 總數 N(mmol) AA-m(mg) HOBt(mg)HBTu(mg) DIEA(mg)A Fmoc-Ala 311.3553 583.48 1083.3 532.9 1253.8 899.5C Fmoc-Cys(Trt)585.7230 231.38 808.3 211.3 497.2 356.7D Fmoc-Asp(OtBu) 411.5332 352.1 864.2 321.6 756.6 542.8E Fmoc-Glu(OtBu) 425.5272 291.74 740.4 266.4 626.9 449.8F Fmoc-Phe 387.4310 311.86 720.6 284.8 670.1 480.8G Fmoc-Gly 297.3401 412.46 731.4 376.7 886.3 635.9H Fmoc-His(Trt)619.78 0 8 0.48 297.5 73.5172.9 124.1I Fmoc-Ile 353.4471 482.88 1017.8 441.0 1037.6 744.4K Fmoc-Lys(Boc)468.5280 281.68 787.1 257.3 605.3 434.2L Fmoc-Leu 353.4560 563.36 1187.4 514.5 1210.6 868.5M Fmoc-Met 371.5130 130.78 289.8 119.4 281.0 201.6N Fmoc-Asn 354.4384 422.52 893.1 385.9 907.9 651.4P Fmoc-Pro 337.4220 221.32 445.4 202.1 475.6 341.2Q Fmoc-Gln 368.4380 382.28 840.0 349.1 821.5 589.3R Fmoc-Arg(Pbf)648.8180 181.08 700.7 165.4 389.1 279.2S Fmoc-Ser(tBu)383.4490 492.94 1127.2 450.2 1059.2 759.9T Fmoc-Thr(tBu)397.5490 492.94 1168.7 450.2 1059.2 759.9V Fmoc-Val 339.4431 442.64 896.0 404.3 951.2 682.4W Fmoc-Trp 426.56 0 6 0.36 153.5 55.1129.7 93.1Y Fmoc-Tyr(tBu)459.6220 221.32 606.7 202.1 475.6 341.2
合成Spike(S)蛋白601～1246號合成操作步驟多肽脫Fmoc保護/胺基酸殘基的連接步驟1.均分樹脂採用IRORI均分樹脂裝置，在每一個Microkan中(共646個)加入15mg Rink Amide樹脂；2.均分IRORI射頻子採用IRORI均分射頻子裝置，在每一個Microkan中加入一個射頻子，並用IRORI加蓋器加蓋；3.脫Fmoc保護集中所有待反應的Microkan，置於一個2L錐形瓶中，加入20％哌啶/DMF振搖，15min×2次；4.洗滌樹脂DMF 3min×6，DCM 3min×3，晾乾；5.IRORI射頻子編碼(或解碼)採用IRORI Sorting程序及相應配件編碼。按照多肽化學庫的序列對射頻子編碼(或解碼)，每一特定編碼代表一個特定序列，並以此將Microkan分配至不同胺基酸反應瓶中；6.洗滌樹脂用重蒸DMF潤洗待反應的樹脂；7.Fmoc-保護胺基酸的連接準確稱取計算量的Fmoc保護胺基酸、HOBt、HBTU(或DIC或BOP)，並用重蒸DMF溶解。將Microkan加入特定胺基酸反應瓶中，振搖反應3h；8.茚三酮檢測取事先設定好的對照組序列，用茚三酮法檢測反應情況。如果呈陰性反應，表明反應完全，進行步驟9的操作；如果呈陽性反應，表明反應不完全，需繼續反應；如果延長時間或改用縮合能力更強的縮合劑後，仍不能完全反應，則採用步驟11的方法封閉後，進行步驟9的操作；9.洗滌樹脂DMF 3min×3，DCM 3min×1；10.脫Fmoc保護/連接根據序列要求，重複步驟3-9的操作，直至反應結束；11.封閉採用乙醯化多肽的氨基端，15％Ac2O/CH2Cl2，30min；12.洗滌樹脂DCM 3min×2，晾乾。
多肽裂解步驟1.配製裂解液根據序列特點，從選取並配製適當的裂解液；2.裂解每一個Microkan使用1mL裂解液在室溫下裂解2h；3.洗滌再用1mL裂解液浸泡洗滌Microkan 5min；4.晾乾或離心乾燥合併兩次裂解液，離心濃縮至少量；
5.沉澱將事先用冰水浴充分冷卻的甲基叔丁基醚/石油醚(v/v3/1)加入到殘餘物中，放入冰水浴中冷卻20min使析晶完全；6.離心離心沉澱多肽化合物(3000rpm以上)，10min後取出，小心傾去上清夜；7.洗滌多肽加入冷卻的甲基叔丁基醚/石油醚，充分洗滌後，離心，傾去上清夜；8.重複步驟7的操作一次；9.晾乾將多肽殘餘物在室溫下揮幹，直至完全乾燥；10.檢測抽檢部分多肽樣品，用10％HOAc/H2O或30％CH3CN/H2O或60％CH3CN/H2O溶解，並用HPLC-MS檢測純度。
實施例3設計合成Membrane(M)蛋白、Envelop(E)蛋白和Nucleocapside(N)蛋白全部胺基酸序列的每10位胺基酸「交叉重疊」序列如下。從右至左為合成次序，代表合成步驟號碼為阿拉伯數字1到10。
M-蛋白表25 所合成的M-蛋白212個以10個胺基酸為長度的序列編號 109876543211. M ADNGTITVE2. A DNGTITVEE3. D NGTITVEEL4. N GTITVEELK5. G TITVEELKQ6. T ITVEELKQL7. I TVEELKQLL8. T VEELKQLLE9. V EELKQLLEQ10. E ELKQLLEQW11. E LKQLLEQWN12. L KQLLEQWNL13. K QLLEQWNLV14. Q LLEQWNLVI15. L LEQWNLVIG16. L EQWNLVIGF17. E QWNLVIGFL18. Q WNLVIGFLF19. W NLVIGFLFL
20.NLVIGFLFLA21.LVIGFLFLAW22.VIGFLFLAWI23.IGFLFLAWIM24.GFLFLAWIML25.FLFLAWIMLL26.LFLAWIMLLQ27.FLAWIMLLQF28.LAWIMLLQFA29.AWIMLLQFAY30.WIMLLQFAYS31.IMLLQFAYSN32.MLLQFAYSNR33.LLQFAYSNRN34.LQFAYSNRNR35.QFAYSNRNRF36.FAYSNRNRFL37.AYSNRNRFLY38.YSNRNRFLYI39.SNRNRFLYII40.NRNRFLYIIK41.RNRFLYIIKL42.NRFLYIIKLV43.RFLYIIKLVF44.FLYIIKLVFL45.LYIIKLVFLW46.YIIKLVFLWL47.IIKLVFLWLL48.IKLVFLWLLW49.KLVFLWLLWP50.LVFLWLLWPV51.VFLWLLWPVT52.FLWLLWPVTL53.LWLLWPVTLA54.WLLWPVTLAC55.LLWPVTLACF56.LWPVTLACFV57.WPVTLACFVL58.PVTLACFVLA59.VTLACFVLAA60.TLACFVLAAV61.LACFVLAAVY62.ACFVLAAVYR63.CFVLAAVYRI64.FVLAAVYRIN65.VLAAVYRINW66.LAAVYRINWV67.AAVYRINWVT68.AVYRINWVTG69.VYRINWVTGG
70.YRINWVTGGI71.RINWVTGGIA72.INWVTGGIAI73.NWVTGGIAIA74.WVTGGIAIAM75.VTGGIAIAMA76.TGGIAIAMAC77.GGIAIAMACI78.GIAIAMACIV79.IAIAMACIVG80.AIAMACIVGL81.IAMACIVGLM82.AMACIVGLMW83.MACIVGLMWL84.ACIVGLMWLS85.CIVGLMWLSY86.IVGLMWLSYF87.VGLMWLSYFV88.GLMWLSYFVA89.LMWLSYFVAS90.MWLSYFVASF91.WLSYFVASFR92.LSYFVASFRL93.SYFVASFRLF94.YFVASFRLFA95.FVASFRLFAR96.VASFRLFART97.ASFRLFARTR98.SFRLFARTRS99.FRLFARTRSM100.RLFARTRSMW101.LFARTRSMWS102.FARTRSMWSF103.ARTRSMWSFN104.RTRSMWSFNP105.TRSMWSFNPE106.RSMWSFNPET107.SMWSFNPETN108.MWSFNPETNI109.WSFNPETNIL110.SFNPETNILL111.FNPETNILLN112.NPETNILLNV113.PETNILLNVP114.ETNILLNVPL115.TNILLNVPLR116.NILLNVPLRG117.ILLNVPLRGT118.LLNVPLRGTI119.LNVPLRGTIV
120.NVPLRGTIVT121.VPLRGTIVTR122.PLRGTIVTRP123.LRGTIVTRPL124.RGTIVTRPLM125.GTIVTRPLME126.TIVTRPLMES127.IVTRPLMESE128.VTRPLMESEL129.TRPLMESELV130.RPLMESELVI131.PLMESELVIG132.LMESELVIGA133.MESELVIGAV134.ESELVIGAVI135.SELVIGAVII136.ELVIGAVIIR137.LVIGAVIIRG138.VIGAVIIRGH139.IGAVIIRGHL140.GAVIIRGHLR141.AVIIRGHLRM142.VIIRGHLRMA143.IIRGHLRMAG144.IRGHLRMAGH145.RGHLRMAGHS146.GHLRMAGHSL147.HLRMAGHSLG148.LRMAGHSLGR149.RMAGHSLGRC150.MAGHSLGRCD151.AGHSLGRCDI152.GHSLGRCDIK153.HSLGRCDIKD154.SLGRCDIKDL155.LGRCDIKDLP156.GRCDIKDLPK157.RCDIKDLPKE158.CDIKDLPKEI159.DIKDLPKEIT160.IKDLPKEITV161.KDLPKEITVA162.DLPKEITVAT163.LPKEITVATS164.PKEITVATSR165.KEITVATSRT166.EITVATSRTL167.ITVATSRTLS168.TVATSRTLSY169.VATSRTLSYY
170.ATSRTLSYYK171.TSRTLSYYKL172.SRTLSYYKLG173.RTLSYYKLGA174.TLSYYKLGAS175.LSYYKLGASQ176.SYYKLGASQR177.YYKLGASQRV178.YKLGASQRVG179.KLGASQRVGT180.LGASQRVGTD181.GASQRVGTDS182.ASQRVGTDSG183.SQRVGTDSGF184.QRVGTDSGFA185.RVGTDSGFAA186.VGTDSGFAAY187.GTDSGFAAYN188.TDSGFAAYNR189.DSGFAAYNRY190.SGFAAYNRYR191.GFAAYNRYRI192.FAAYNRYRIG193.AAYNRYRIGN194.AYNRYRIGNY195.YNRYRIGNYK196.NRYRIGNYKL197.RYRIGNYKLN198.YRIGNYKLNT199.RIGNYKLNTD200.IGNYKLNTDH201.GNYKLNTDHA202.NYKLNTDHAG203.YKLNTDHAGS204.KLNTDHAGSN205.LNTDHAGSND206.NTDHAGSNDN207.TDHAGSNDNI208.DHAGSNDNIA209.HAGSNDNIAL210.AGSNDNIALL211.GSNDNIALLV212.SNDNIALLVQ表26所合成的E-蛋白67個以10個胺基酸為長度的序列213.MYSFVSEETG214.YSFVSEETGT215.SFVSEETGTL216.FVSEETGTLI
217.VSEETGTLIV218.SEETGTLIVN219.EETGTLIVNS220.ETGTLIVNSV221.TGTLIVNSVL222.GTLIVNSVLL223.TLIVNSVLLF224.LIVNSVLLFL225.IVNSVLLFLA226.VNSVLLFLAF227.NSVLLFLAFV228.SVLLFLAFVV229.VLLFLAFVVF230.LLFLAFVVFL231.LFLAFVVFLL232.FLAFVVFLLV233.LAFVVFLLVT234.AFVVFLLVTL235.FVVFLLVTLA236.VVFLLVTLAI237.VFLLVTLAIL238.FLLVTLAILT239.LLVTLAILTA240.LVTLAILTAL241.VTLAILTALR242.TLAILTALRL243.LAILTALRLC244.AILTALRLCA245.ILTALRLCAY246.LTALRLCAYC247.TALRLCAYCC248.ALRLCAYCCN249.LRLCAYCCNI250.RLCAYCCNIV251.LCAYCCNIVN252.CAYCCNIVNV253.AYCCNIVNVS254.YCCNIVNVSL255.CCNIVNVSLV256.CNIVNVSLVK257.NIVNVSLVKP258.IVNVSLVKPT259.VNVSLVKPTV260.NVSLVKPTVY261.VSLVKPTVYV262.SLVKPTVYVY263.LVKPTVYVYS264.VKPTVYVYSR265.KPTVYVYSRV266.PTVYVYSRVK
267.TVYVYSRVKN268.VYVYSRVKNL269.YVYSRVKNLN270.VYSRVKNLNS271.YSRVKNLNSS272.SRVKNLNSSE273.RVKNLNSSEG274.VKNLNSSEGV275.KNLNSSEGVP276.NLNSSEGVPD277.LNSSEGVPDL278.NSSEGVPDLL279.SSEGVPDLLV表27所合成的N-蛋白的413個以10個胺基酸為長度的序列280.MSDNGPQSNQ281.SDNGPQSNQR282.DNGPQSNQRS283.NGPQSNQRSA284.GPQSNQRSAP285.PQSNQRSAPR286.QSNQRSAPRI287.SNQRSAPRIT288.NQRSAPRITF289.QRSAPRITFG290.RSAPRITFGG291.SAPRITFGGP292.APRITFGGPT293.PRITFGGPTD294.RITFGGPTDS295.ITFGGPTDST296.TFGGPTDSTD297.FGGPTDSTDN298.GGPTDSTDNN299.GPTDSTDNNQ300.PTDSTDNNQN301.TDSTDNNQNG302.DSTDNNQNGG303.STDNNQNGGR304.TDNNQNGGRN305.DNNQNGGRNG306.NNQNGGRNGA307.NQNGGRNGAR308.QNGGRNGARP309.NGGRNGARPK310.GGRNGARPKQ311.GRNGARPKQR312.RNGARPKQRR
313.NGARPKQRRP314.GARPKQRRPQ315.ARPKQRRPQG316.RPKQRRPQGL317.PKQRRPQGLP318.KQRRPQGLPN319.QRRPQGLPNN320.RRPQGLPNNT321.RPQGLPNNTA322.PQGLPNNTAS323.QGLPNNTASW324.GLPNNTASWF325.LPNNTASWFT326.PNNTASWFTA327.NNTASWFTAL328.NTASWFTALT329.TASWFTALTQ330.ASWFTALTQH331.SWFTALTQHG332.WFTALTQHGK333.FTALTQHGKE334.TALTQHGKEE335.ALTQHGKEEL336.LTQHGKEELR337.TQHGKEELRF338.QHGKEELRFP339.HGKEELRFPR340.GKEELRFPRG341.KEELRFPRGQ342.EELRFPRGQG343.ELRFPRGQGV344.LRFPRGQGVP345.RFPRGQGVPI346.FPRGQGVPIN347.PRGQGVPINT348.RGQGVPINTN349.GQGVPINTNS350.QGVPINTNSG351.GVPINTNSGP352.VPINTNSGPD353.PINTNSGPDD354.INTNSGPDDQ355.NTNSGPDDQI356.TNSGPDDQIG357.NSGPDDQIGY358.SGPDDQIGYY359.GPDDQIGYYR360.PDDQIGYYRR361.DDQIGYYRRA362.DQIGYYRRAT
363.QIGYYRRATR364.IGYYRRATRR365.GYYRRATRRV366.YYRRATRRVR367.YRRATRRVRG368.RRATRRVRGG369.RATRRVRGGD370.ATRRVRGGDG371.TRRVRGGDGK372.RRVRGGDGKM373.RVRGGDGKMK374.VRGGDGKMKE375.RGGDGKMKEL376.GGDGKMKELS377.GDGKMKELSP378.DGKMKELSPR379.GKMKELSPRW380.KMKELSPRWY381.MKELSPRWYF382.KELSPRWYFY383.ELSPRWYFYY384.LSPRWYFYYL385.SPRWYFYYLG386.PRWYFYYLGT387.RWYFYYLGTG388.WYFYYLGTGP389.YFYYLGTGPE390.FYYLGTGPEA391.YYLGTGPEAS392.YLGTGPEASL393.LGTGPEASLP394.GTGPEASLPY395.TGPEASLPYG396.GPEASLPYGA397.PEASLPYGAN398.EASLPYGANK399.ASLPYGANKE400.SLPYGANKEG401.LPYGANKEGI402.PYGANKEGIV403.YGANKEGIVW404.GANKEGIVWV405.ANKEGIVWVA406.NKEGIVWVAT407.KEGIVWVATE408.EGIVWVATEG409.GIVWVATEGA410.IVWVATEGAL411.VWVATEGALN412.WVATEGALNT
413.VATEGALNTP414.ATEGALNTPK415.TEGALNTPKD416.EGALNTPKDH417.GALNTPKDHI418.ALNTPKDHIG419.LNTPKDHIGT420.NTPKDHIGTR421.TPKDHIGTRN422.PKDHIGTRNP423.KDHIGTRNPN424.DHIGTRNPNN425.HIGTRNPNNN426.IGTRNPNNNA427.GTRNPNNNAA428.TRNPNNNAAT429.RNPNNNAATV430.NPNNNAATVL431.PNNNAATVLQ432.NNNAATVLQL433.NNAATVLQLP434.NAATVLQLPQ435.AATVLQLPQG436.ATVLQLPQGT437.TVLQLPQGTT438.VLQLPQGTTL439.LQLPQGTTLP440.QLPQGTTLPK441.LPQGTTLPKG442.PQGTTLPKGF443.QGTTLPKGFY444.GTTLPKGFYA445.TTLPKGFYAE446.TLPKGFYAEG447.LPKGFYAEGS448.PKGFYAEGSR449.KGFYAEGSRG450.GFYAEGSRGG451.FYAEGSRGGS452.YAEGSRGGSQ453.AEGSRGGSQA454.EGSRGGSQAS455.GSRGGSQASS456.SRGGSQASSR457.RGGSQASSRS458.GGSQASSRSS459.GSQASSRSSS460.SQASSRSSSR461.QASSRSSSRS462.ASSRSSSRSR
463.SSRSSSRSRG464.SRSSSRSRGN465.RSSSRSRGNS466.SSSRSRGNSR467.SSRSRGNSRN468.SRSRGNSRNS469.RSRGNSRNST470.SRGNSRNSTP471.RGNSRNSTPG472.GNSRNSTPGS473.NSRNSTPGSS474.SRNSTPGSSR475.RNSTPGSSRG476.NSTPGSSRGN477.STPGSSRGNS478.TPGSSRGNSP479.PGSSRGNSPA480.GSSRGNSPAR481.SSRGNSPARM482.SRGNSPARMA483.RGNSPARMAS484.GNSPARMASG485.NSPARMASGG486.SPARMASGGG487.PARMASGGGE488.ARMASGGGET489.RMASGGGETA490.MASGGGETAL491.ASGGGETALA492.SGGGETALAL493.GGGETALALL494.GGETALALLL495.GETALALLLL496.ETALALLLLD497.TALALLLLDR498.ALALLLLDRL499.LALLLLDRLN500.ALLLLDRLNQ501.LLLLDRLNQL502.LLLDRLNQLE503.LLDRLNQLES504.LDRLNQLESK505.DRLNQLESKV506.RLNQLESKVS507.LNQLESKVSGS08.NQLESKVSGK509.QLESKVSGKG510.LESKVSGKGQ511.ESKVSGKGQQ512.SKVSGKGQQQ
513.KVSGKGQQQQ514.VSGKGQQQQG515.SGKGQQQQGQ516.GKGQQQQGQT517.KGQQQQGQTV518.GQQQQGQTVT519.QQQQGQTVTK520.QQQGQTVTKK521.QQGQTVTKKS522.QGQTVTKKSA523.GQTVTKKSAA524.QTVTKKSAAE525.TVTKKSAAEA526.VTKKSAAEAS527.TKKSAAEASK528.KKSAAEASKK529.KSAAEASKKP530.SAAEASKKPR531.AAEASKKPRQ532.AEASKKPRQK533.EASKKPRQKR534.ASKKPRQKRT535.SKKPRQKRTA536.KKPRQKRTAT537.KPRQKRTATK538.PRQKRTATKQ539.RQKRTATKQY540.QKRTATKQYN541.KRTATKQYNV542.RTATKQYNVT543.TATKQYNVTQ544.ATKQYNVTQA545.TKQYNVTQAF546.KQYNVTQAFG547.QYNVTQAFGR548.YNVTQAFGRR549.NVTQAFGRRG550.VTQAFGRRGP551.TQAFGRRGPE552.QAFGRRGPEQ553.AFGRRGPEQT554.FGRRGPEQTQ555.GRRGPEQTQG556.RRGPEQTQGN557.RGPEQTQGNF558.GPEQTQGNFG559.PEQTQGNFGD560.EQTQGNFGDQ561.QTQGNFGDQD562.TQGNFGDQDL
563.QGNFGDQDLI564.GNFGDQDLIR565.NFGDQDLIRQ566.FGDQDLIRQG567.GDQDLIRQGT568.DQDLIRQGTD569.QDLIRQGTDY570.DLIRQGTDYK571.LIRQGTDYKH572.IRQGTDYKHW573.RQGTDYKHWP574.QGTDYKHWPQ575.GTDYKHWPQI576.TDYKHWPQIA577.DYKHWPQIAQ578.YKHWPQIAQF579.KHWPQIAQFA580.HWPQIAQFAP581.WPQIAQFAPS582.PQIAQFAPSA583.QIAQFAPSAS584.IAQFAPSASA585.AQFAPSASAF586.QFAPSASAFF587.FAPSASAFFG588.APSASAFFGM589.PSASAFFGMS590.SASAFFGMSR591.ASAFFGMSRI592.SAFFGMSRIG593.AFFGMSRIGM594.FFGMSRIGME595.FGMSRIGMEV596.GMSRIGMEVT597.MSRIGMEVTP598.SRIGMEVTPS599.RIGMEVTPSG600.IGMEVTPSGT601.GMEVTPSGTW602.MEVTPSGTWL603.EVTPSGTWLT604.VTPSGTWLTY605.TPSGTWLTYH606.PSGTWLTYHG607.SGTWLTYHGA608.GTWLTYHGAI609.TWLTYHGAIK610.WLTYHGAIKL611.LTYHGAIKLD612.TYHGAIKLDD
613.YHGAIKLDDK614.HGAIKLDDKD615.GAIKLDDKDP616.AIKLDDKDPQ617.IKLDDKDPQF618.KLDDKDPQFK619.LDDKDPQFKD620.DDKDPQFKDN621.DKDPQFKDNV622.KDPQFKDNVI623.DPQFKDNVIL624.PQFKDNVILL625.QFKDNVILLN626.FKDNVILLNK627.KDNVILLNKH628.DNVILLNKHI629.NVILLNKHID630.VILLNKHIDA631.ILLNKHIDAY632.LLNKHIDAYK633.LNKHIDAYKT634.NKHIDAYKTF635.KHIDAYKTFP636.HIDAYKTFPP637.IDAYKTFPPT638.DAYKTFPPTE639.AYKTFPPTEP640.YKTFPPTEPK641.KTFPPTEPKK642.TFPPTEPKKD643.FPPTEPKKDK644.PPTEPKKDKK645.PTEPKKDKKK646.TEPKKDKKKK647.EPKKDKKKKT648.PKKDKKKKTD649.KKDKKKKTDE650.KDKKKKTDEA651.DKKKKTDEAQ652.KKKKTDEAQP653.KKKTDEAQPL654.KKTDEAQPLP655.KTDEAQPLPQ656.TDEAQPLPQR657.DEAQPLPQRQ658.EAQPLPQRQK659.AQPLPQRQKK660.QPLPQRQKKQ661.PLPQRQKKQP662.LPQRQKKQPT
663.PQRQKKQPTV664.QRQKKQPTVT665.RQKKQPTVTL666.QKKQPTVTLL667.KKQPTVTLLP668.KQPTVTLLPA669.QPTVTLLPAA670.PTVTLLPAAD671.TVTLLPAADM672.VTLLPAADMD673.TLLPAADMDD674.LLPAADMDDF675.LPAADMDDFS676.PAADMDDFSR677.AADMDDFSRQ678.ADMDDFSRQL679.DMDDFSRQLQ680.MDDFSRQLQN681.DDFSRQLQNS682.DFSRQLQNSM683.FSRQLQNSMS684.SRQLQNSMSG685.RQLQNSMSGA686.QLQNSMSGAS687.LQNSMSGASA688.QNSMSGASAD689.NSMSGASADS690.SMSGASADST691.MSGASADSTQ692.SGASADSTQA以下表28至表37為上述1～692號「交叉重疊」多肽第1-10步使用的試劑投料
表28合成上述1～692號「交叉重疊」多肽第一步時使用試劑投料計算結果MEN-步驟-1 FW 個數 控制組 總數 N(mmol) AA-m(mg) HOBt(mg)HBTu(mg) DIEA(mg)AFmoc-Ala 311.3561572.088650.0 319.7 752.3 539.7CFmoc-Cys(Trt)585.76 06 0.216126.5 33.177.8 55.8DFmoc-Asp(OtBu) 411.5270270.972400.0 148.8 350.2 251.2EFmoc-Glu(OtBu) 425.5220220.792337.0 121.3 285.3 204.7FFmoc-Phe 387.4270270.972376.6 148.8 350.2 251.2GFmoc-Gly 297.3601612.232663.6 341.8 804.2 576.9HFmoc-His(Trt)619.78 08 0.288178.5 44.1103.8 74.4IFmoc-Ile 353.4310311.116394.4 170.9 402.1 288.5KFmoc-Lys(Boc)468.5370371.332624.0 204.0 479.9 344.3LFmoc-Leu 353.4713742.772979.6 424.5 998.7 716.5MFmoc-Met 371.5122140.576214.0 88.2207.5 148.9NFmoc-Asn 354.4401411.512535.9 231.5 544.8 390.8PFmoc-Pro 337.4371381.404473.7 215.0 505.8 362.9QFmoc-Gln 368.4381391.44 530.5 220.5 518.8 372.2RFmoc-Arg(Pbf)648.8480481.7281121.1 264.6 622.6 446.7SFmoc-Ser(tBu)383.4500501.8 690.1 275.6 648.5 465.3TFmoc-Thr(tBu)397.5481491.8 715.5 275.6 648.5 465.3VFmoc-Val 339.4391401.476501.0 226.0 531.8 381.5.
WFmoc-Trp 426.5120120.432184.2 66.2155.6 111.7YFmoc-Tyr(tBu)459.6230230.828380.5 126.8 298.3 214.0
表29合成上述1～692號「交叉重疊」多肽第二步時使用試劑投料計算結果MEN-步驟-2 FW 個數 控制組 總數 N(mmol)AA-m(mg) HOBt(mg) BOP(mg) DIEA(mg)A Fmoc-Ala 311.3551562.052 638.8 314.2 862.6530.4C Fmoc-Cys(Trt)585.76 06 0.216 126.5 33.1 90.8 55.8D Fmoc-Asp(OtBu) 411.5270270.972 400.0 148.8 408.6251.2E Fmoc-Glu(OtBu) 425.5220220.792 337.0 121.3 332.9204.7F Fmoc-Phe 387.4270270.972 376.6 148.8 408.6251.2G Fmoc-Gly 297.3601612.232 663.6 341.8 938.3576.9H Fmoc-His(Trt)619.78 08 0.288 178.5 44.1 121.174.4I Fmoc-Ile 353.4312331.26 445.3 192.9 529.7325.7K Fmoc-Lys(Boc)468.5371381.404 657.8 215.0 590.2362.9L Fmoc-Leu 353.4712732.7954.2 413.5 1135.0 697.9M Fmoc-Met 371.5121130.504 187.2 77.2 211.9130.3N Fmoc-Asn 354.4411421.548 548.6 237.0 650.7400.1P Fmoc-Pro 337.4370371.332 449.4 204.0 559.9344.3Q Fmoc-Gln 368.4370371.332 490.7 204.0 559.9344.3R Fmoc-Arg(Pbf)648.8482501.872 1214.6 286.7 786.9483.9S Fmoc-Ser(tBu)383.4500501.8690.1 275.6 756.7465.3T Fmoc-Thr(tBu)397.5490491.764 701.2 270.1 741.5456.0V Fmoc-Val 339.4391401.476 501.0 226.0 620.5381.5W Fmoc-Trp 426.5120120.432 184.2 66.2 181.6111.7Y Fmoc-Tyr(tBu)459.6230230.828 380.5 126.8 348.1214.0
表30合成上述1～692號「交叉重疊」多肽第三步時使用試劑投料計算結果MEN-步驟-3 FW 個數 控制組 總數 N(mmol) AA-m(mg) HOBt(mg)BOP(mg)DIEA(mg)A Fmoc-Ala 311.3551562.052638.8314.2 862.6 530.4C Fmoc-Cys(Trt)585.76 06 0.216126.533.190.8 55.8D Fmoc-Asp(OtBu) 411.5271281.044429.6159.9 438.9 269.9E Fmoc-Glu(OtBu) 425.5230230.828352.3126.8 348.1 214.0F Fmoc-Phe 387.4271281.044404.4159.9 438.9 269.9G Fmoc-Gly 297.3600602.16 642.2330.8 908.0 558.3H Fmoc-His(Trt)619.78 08 0.288178.544.1121.1 74.4I Fmoc-Ile 353.4313341.332470.7204.0 559.9 344.3K Fmoc-Lys(Boc)468.5370371.332624.0204.0 559.9 344.3L Fmoc-Leu 353.4701712.592916.0396.9 1089.6 670.0M Fmoc-Met 371.5120120.432160.566.2181.6 111.7N Fmoc-Asn 354.4410411.476523.1226.0 620.5 381.5P Fmoc-Pro 337.4373401.548522.3237.0 650.7 400.1Q Fmoc-Gln 368.4360361.296477.4198.5 544.8 335.0R Fmoc-Arg(Pbf)648.8481491.8 1167.8 275.6 756.7 465.3S Fmoc-Ser(tBu)383.4510511.836703.9281.1 771.8 474.6T Fmoc-Thr(tBu)397.5501511.872744.1286.7 786.9 483.9V Fmoc-Val 339.4380381.368464.3209.5 575.1 353.6W Fmoc-Trp 426.5120120.432184.266.2181.6 111.7Y Fmoc-Tyr(tBu)459.6230230.828380.5126.8 348.1 214.0
表31合成上述1～692號「交叉重疊」多肽第四步時使用試劑投料計算結果MEN-步驟-4 FW 個數 控制組 總數 N(mmol) AA-m(mg) HOBt(mg)BOP(mg) DIEA(mg)A Fmoc-Ala 311.3551562.052638.8314.2 862.6530.4C Fmoc-Cys(Trt)585.76 06 0.216126.533.190.8 55.8D Fmoc-Asp(OtBu) 411.5270270.972400.0148.8 408.6251.2E Fmoc-Glu(OtBu) 425.5240240.864367.6132.3 363.2223.3F Fmoc-Phe 387.4270270.972376.6148.8 408.6251.2G Fmoc-Gly 297.3601612.232663.6341.8 938.3576.9H Fmoc-His(Trt)619.78 08 0.288178.544.1121.174.4I Fmoc-Ile 353.4321331.224432.6187.4 514.5316.4K Fmoc-Lys(Boc)468.5370371.332624.0204.0 559.9344.3L Fmoc-Leu 353.4684722.736966.9419.0 1150.1 707.2M Fmoc-Met 371.5120120.432160.566.2181.6111.7N Fmoc-Asn 354.4410411.476523.1226.0 620.5381.5P Fmoc-Pro 337.4370371.332449.4204.0 559.9344.3Q Fmoc-Gln 368.4370371.332490.7204.0 559.9344.3R Fmoc-Arg(Pbf)648.8482501.8721214.6 286.7 786.9483.9S Fmoc-Ser(tBu)383.4510511.836703.9281.1 771.8474.6T Fmoc-Thr(tBu)397.5490491.764701.2270.1 741.5456.0V Fmoc-Val 339.4380381.368464.3209.5 575.1353.6W Fmoc-Trp 426.5122140.576245.788.2242.1148.9Y Fmoc-Tyr(tBu)459.6231240.9 413.6137.8 378.3232.6
表32合成上述1～692號「交叉重疊」多肽第五步時使用試劑投料計算結果MEN-步驟-5 FW 個數 控制組 總數 N(mmol) AA-m(mg) HOBt(mg)HBTu(mg) DIEA(mg)A Fmoc-Ala 311.3552572.124661.2325.2 765.3 549.0C Fmoc-Cys(Trt)585.76 06 0.216126.533.177.8 55.8D Fmoc-Asp(OtBu) 411.5260260.936385.2143.3 337.2 241.9E Fmoc-Glu(OtBu) 425.5240240.864367.6132.3 311.3 223.3F Fmoc-Phe 387.4271281.044404.4159.9 376.1 269.9G Fmoc-Gly 297.3600602.16 642.2330.8 778.2 558.3H Fmoc-His(Trt)619.78 08 0.288178.544.1103.8 74.4I Fmoc-Ile 353.4322341.296458.0198.5 466.9 335.0K Fmoc-Lys(Boc)468.5370371.332624.0204.0 479.9 344.3L Fmoc-Leu 353.4673702.628928.7402.4 946.8 679.3M Fmoc-Met 371.5120120.432160.566.2155.6 111.7N Fmoc-Asn 354.4410411.476523.1226.0 531.8 381.5P Fmoc-Pro 337.4380381.368461.6209.5 492.9 353.6Q Fmoc-Gln 368.4370371.332490.7204.0 479.9 344.3R Fmoc-Arg(Pbf)648.8480481.7281121.1 264.6 622.6 446.7S Fmoc-Ser(tBu)383.4510511.836703.9281.1 661.5 474.6T Fmoc-Thr(tBu)397.5502521.944772.7297.7 700.4 502.5V Fmoc-Val 339.4382401.512513.2231.5 544.8 390.8W Fmoc-Trp 426.5120120.432184.266.2155.6 111.7Y Fmoc-Tyr(tBu)459.6230230.828380.5126.8 298.3 214.0
表33合成上述1～692號「交叉重疊」多肽第六步時使用試劑投料計算結果MEN-步驟-6FW 個數 控制組 總數 N(mmol)AA-m(mg) HOBt(mg) HBTu(mg) DIEA(mg)A Fmoc-Ala 311.3542562.088 650.0319.7752.3539.7C Fmoc-Cys(Trt) 585.76 06 0.216 126.533.1 77.8 55.8D Fmoc-Asp(OtBu)411.5250250.9370.4137.8324.3232.6E Fmoc-Glu(OtBu)425.5240240.864 367.6132.3311.3223.3F Fmoc-Phe 387.4270270.972 376.6148.8350.2251.2G Fmoc-Gly 297.3623652.448 727.8374.9882.0632.8H Fmoc-His(Trt) 619.78 08 0.288 178.544.1 103.874.4I Fmoc-Ile 353.4320321.152 407.1176.4415.1297.8K Fmoc-Lys(Boc) 468.5370371.332 624.0204.0479.9344.3L Fmoc-Leu 353.4673702.628 928.7402.4946.8679.3M Fmoc-Met 371.5120120.432 160.566.2 155.6111.7N Fmoc-Asn 354.4411421.548 548.6237.0557.7400.1P Fmoc-Pro 337.4370371.332 449.4204.0479.9344.3Q Fmoc-Gln 368.4370371.332 490.7204.0479.9344.3R Fmoc-Arg(Pbf) 648.8480481.728 1121.1 264.6622.6446.7S Fmoc-Ser(tBu) 383.4510511.836 703.9281.1661.5474.6T Fmoc-Thr(tBu) 397.5500501.8715.5275.6648.5465.3V Fmoc-Val 339.4392411.548 525.4237.0557.7400.1W Fmoc-Trp 426.5120120.432 184.266.2 155.6111.7Y Fmoc-Tyr(tBu) 459.6231240.9413.6137.8324.3232.6
表34合成上述1～692號「交叉重疊」多肽第七步時使用試劑投料計算結果MEN-步驟-7 FW 個數 控制組 總數 N(mmol) AA-m(mg) HOBt(mg)HBTu(mg)DIEA(mg)A Fmoc-Ala 311.3531541.98 616.4303.2 713.4 511.8C Fmoc-Cys(Trt)585.76 06 0.216 126.533.177.855.8D Fmoc-Asp(OtBu) 411.5250250.9 370.4137.8 324.3 232.6E Fmoc-Glu(OtBu) 425.5240240.864 367.6132.3 311.3 223.3F Fmoc-Phe 387.4281291.08 418.4165.4 389.1 279.2G Fmoc-Gly 297.3622642.376 706.4363.8 856.0 614.1H Fmoc-His(Trt)619.78 08 0.288 178.544.1103.8 74.4I Fmoc-Ile 353.4312331.26 445.3192.9 454.0 325.7K Fmoc-Lys(Boc)468.5370371.332 624.0204.0 479.9 344.3L Fmoc-Leu 353.4672692.556 903.3391.4 920.9 660.7M Fmoc-Met 371.5120120.432 160.566.2155.6 111.7N Fmoc-Asn 354.4431441.62 574.1248.1 583.7 418.7P Fmoc-Pro 337.4370371.332 449.4204.0 479.9 344.3Q Fmoc-Gln 368.4370371.332 490.7204.0 479.9 344.3R Fmoc-Arg(Pbf)648.8480481.728 1121.1 264.6 622.6 446.7S Fmoc-Ser(tBu)383.4510511.836 703.9281.1 661.5 474.6T Fmoc-Thr(tBu)397.5501511.872 744.1286.7 674.5 483.9V Fmoc-Val 339.4380381.368 464.3209.5 492.9 353.6W Fmoc-Trp 426.5121130.504 215.077.2181.6 130.3Y Fmoc-Tyr(tBu)459.6231240.9 413.6137.8 324.3 232.6
表35合成上述1～692號「交叉重疊」多肽第八步時使用試劑投料計算結果MEN-步驟-8 FW 個數控制組 總數 N(mmol)AA-m(mg) HOBt(mg) HBTu(mg) DIEA(mg)A Fmoc-Ala 311.353 1541.98 616.4303.2713.4511.8C Fmoc-Cys(Trt)585.76 06 0.216 126.533.1 77.8 55.8D Fmoc-Asp(OtBu) 411.527 0270.972 400.0148.8350.2251.2E Fmoc-Glu(OtBu) 425.524 1250.936 398.3143.3337.2241.9F Fmoc-Phe 387.428 1291.08 418.4165.4389.1279.2G Fmoc-Gly 297.361 0612.196 652.9336.3791.2567.6H Fmoc-His(Trt)619.78 08 0.288 178.544.1 103.874.4I Fmoc-Ile 353.431 1321.188 419.8181.9428.0307.1K Fmoc-Lys(Boc)468.537 0371.332 624.0204.0479.9344.3L Fmoc-Leu 353.467 4712.7954.2413.5972.8697.9M Fmoc-Met 371.512 0120.432 160.566.2 155.6111.7N Fmoc-Asn 354.442 0421.512 535.9231.5544.8390.8P Fmoc-Pro 337.437 0371.332 449.4204.0479.9344.3Q Fmoc-Gln 368.437 0371.332 490.7204.0479.9344.3R Fmoc-Arg(Pbf)648.848 1491.81167.8 275.6648.5465.3S Fmoc-Ser(tBu)383.451 0511.836 703.9281.1661.5474.6T Fmoc-Thr(tBu)397.550 2521.944 772.7297.7700.4502.5V Fmoc-Val 339.438 1391.44 488.7220.5518.8372.2W Fmoc-Trp 426.512 0120.432 184.266.2 155.6111.7Y Fmoc-Tyr(tBu)459.623 0230.828 380.5126.8298.3214.0
表36合成上述1～692號「交叉重疊」多肽第九步時使用試劑投料計算結果MEN-步驟-9 FW 個數 控制組 總數 N(mmol) AA-m(mg) HOBt(mg) HBTu(mg) DIEA(mg)A Fmoc-Ala 311.353 0531.908594.0 292.2 687.4 493.2C Fmoc-Cys(Trt)585.76 06 0.216126.5 33.1 77.8 55.8D Fmoc-Asp(OtBu) 411.526 0260.936385.2 143.3 337.2 241.9E Fmoc-Glu(OtBu) 425.523 0230.828352.3 126.8 298.3 214.0F Fmoc-Phe 387.428 3311.224474.2 187.4 441.0 316.4G Fmoc-Gly 297.361 2632.34 695.7 358.3 843.1 604.8H Fmoc-His(Trt)619.78 08 0.288178.5 44.1 103.8 74.4I Fmoc-Ile 353.431 0311.116394.4 170.9 402.1 288.5K Fmoc-Lys(Boc)468.537 0371.332624.0 204.0 479.9 344.3L Fmoc-Leu 353.467 2692.556903.3 391.4 920.9 660.7M Fmoc-Met 371.512 0120.432160.5 66.2 155.6 111.7N Fmoc-Asn 354.442 0421.512535.9 231.5 544.8 390.8P Fmoc-Pro 337.437 1381.404473.7 215.0 505.8 362.9Q Fmoc-Gln 368.437 0371.332490.7 204.0 479.9 344.3R Fmoc-Arg(Pbf)648.848 0481.7281121.1 264.6 622.6 446.7S Fmoc-Ser(tBu)383.452 0521.872717.7 286.7 674.5 483.9T Fmoc-Thr(tBu)397.550 0501.8 715.5 275.6 648.5 465.3V Fmoc-Val 339.438 3411.584537.6 242.6 570.7 409.4W Fmoc-Trp 426.512 0120.432184.2 66.2 155.6 111.7Y Fmoc-Tyr(tBu)459.624 1250.936430.2 143.3 337.2 241.9
表37合成上述1～692號「交叉重疊」多肽第十步時使用試劑投料計算結果MEN-步驟-10 FW 個數 控制組 總數 N(mmol)AA-m(mg) HOBt(mg) HBTu(mg) DIEA(mg)A Fmoc-Ala 311.3531541.98 616.4 303.2 713.4 511.8C Fmoc-Cys(Trt)585.76 06 0.216 126.5 33.1 77.8 55.8D Fmoc-Asp(OtBu) 411.5260260.936 385.2 143.3 337.2 241.9E Fmoc-Glu(OtBu) 425.5230230.828 352.3 126.8 298.3 214.0F Fmoc-Phe 387.4281291.08 418.4 165.4 389.1 279.2G Fmoc-Gly 297.3602622.304 685.0 352.8 830.1 595.5H Fmoc-His(Trt)619.78 08 0.288 178.5 44.1 103.8 74.4I Fmoc-Ile 353.4310311.116 394.4 170.9 402.1 288.5K Fmoc-Lys(Boc)468.5370371.332 624.0 204.0 479.9 344.3L Fmoc-Leu 353.4671682.484 877.8 380.4 895.0 642.1M Fmoc-Met 371.5150150.54 200.6 82.7 194.6 139.6N Fmoc-Asn 354.4412431.62 574.1 248.1 583.7 418.7P Fmoc-Pro 337.4370371.332 449.4 204.0 479.9 344.3Q Fmoc-Gln 368.4370371.332 490.7 204.0 479.9 344.3R Fmoc-Arg(Pbf)648.8481491.81167.8 275.6 648.5 465.3S Fmoc-Ser(tBu)383.4510511.836 703.9 281.1 661.5 474.6T Fmoc-Thr(tBu)397.5500501.8715.5 275.6 648.5 465.3V Fmoc-Val 339.4382401.512 513.2 231.5 544.8 390.8W Fmoc-Trp 426.5122140.576 245.7 88.2 207.5 148.9Y Fmoc-Tyr(tBu)459.6240240.864 397.1 132.3 311.3 223.3
上述1～692號「交叉重疊」多肽第一步時使用試劑投料合成操作步驟多肽脫Fmoc保護/氨基酶殘基的連接步驟1.均分樹脂採用IRORI均分樹脂裝置，在每一個Microkan中(共692個)加入15mg Rink Amide樹脂；2.均分IRORI射頻子採用IRORI均分射頻子裝置，在每一個Microkan中加入一個射頻子，並用IRORI加蓋器加蓋；3.脫Fmoc保護集中所有待反應的Microkan，置於一個2L錐形瓶中，加入20％哌啶/DMF振搖，15min×2次；4.洗滌樹脂DMF 3min×6，DCM 3min×3，晾乾；5.IRORI射頻子編碼(或解碼)採用IRORI Sorting程序及相應配件編碼。按照多肽化學庫的序列對射頻子編碼(或解碼)，每一特定編碼代表一個特定序列，並以此將Microkan分配至不同胺基酸反應瓶中；6.洗滌樹脂用重蒸DMF潤洗待反應的樹脂；7.Fmoc-保護胺基酸的連接準確稱取計算量的Fmoc保護胺基酸、HOBt、HBTU(或DIC或BOP)，並用重蒸DMF溶解。將Microkan加入特定胺基酸反應瓶中，振搖反應3h；8.茚三酮檢測取事先設定好的對照組序列，用茚三酮法檢測反應情況。如果呈陰性反應，表明反應完全，進行步驟9的操作；如果呈陽性反應，表明反應不完全，需繼續反應；如果延長時間或改用縮合能力更強的縮合劑後，仍不能完全反應，則採用步驟11的方法封閉後，進行步驟9的操作；9.洗滌樹脂DMF 3min×3，DCM 3min×1；10.脫Fmoc保護/連接根據序列要求，重複步驟3-9的操作，直至反應結束；11.封閉採用乙醯化多肽的氨基端，15％ Ac2O/CH2Cl2，30min；12.洗滌樹脂DCM 3min×2，晾乾。
多肽裂解步驟1.配製裂解液根據序列特點，從選取並配製適當的裂解液；2.裂解每一個Microkan使用1mL裂解液在室溫下裂解2h；3.洗滌再用1mL裂解液浸泡洗滌Microkan 5min；4.晾乾或離心乾燥合併兩次裂解液，離心濃縮至少量；5.沉澱將事先用冰水浴充分冷卻的甲基叔丁基醚/石油醚(v/v3/1)加入到殘餘物中，放入冰水浴中冷卻20min使析晶完全；6.離心離心沉澱多肽化合物(3000rpm以上)，10min後取出，小心傾去上清夜；7.洗滌多肽加入冷卻的甲基叔丁基醚/石油醚，充分洗滌後，離心，傾去上清夜；
8.重複步驟7的操作一次；9.晾乾將多肽殘餘物在室溫下揮幹，直至完全乾燥；10.檢測抽檢部分多肽樣品，用10％HOAc/H2O或30％CH3CN/H2O或60％CH3CN/H2O溶解，並用HPLC-MS檢測純度。
實施例4採用此法合成非「交叉重複」多肽化合物。
採用本發明的方法除了能合成如實施例1～3所述的具有10個胺基酸序列長度的胺基酸殘基序列，還能合成長度為2～50個胺基酸的胺基酸片段，應用此方法合成了具有如下序列的多肽。
合成多肽序列1.Ac-LYENQKQIANQFNKAISQIQ-CONH22.Ac-ESLTTTSTALGKLQDVVNQNAQ-CONH23.Ac-ALNTLVKQLSSNFGAISSVLND-CONH24.Ac-ILSRLDKVEAEVQIDRLITGRL-CONH25.Ac-QSLQTYVTQQLIRAAEIRASAN-CONH26.Ac-DISGINASVVNIQKEIDRLN-CONH27.Ac-EVAKNLNESLIDLQELGKYE-CONH28.NH2-NAQALNTLVKQLSSNFGAISSVLNDILSRLDKVE-CONH29.Ac-SSNFGAISSVLNDILSRLDKVE-CONH2以下本實施例多肽的製備步驟1.加樹脂及射頻子選取9個Microkan並分別裝入15毫克Rink樹脂和射頻子並蓋緊蓋子；2.合併Microkan，在一個適當的容器中用20％哌啶/DMF脫Fmoc保護基15min×2；3.洗滌樹脂DMF 3min×6，DCM 3min×3，重蒸DMF 3min×3；4.IRORI射頻子編碼(或解碼)採用IRORI Sorting程序，按照多肽編碼次序將Microkan分配至不同胺基酸反應瓶中；5.在每一個反應瓶中加入溶解於DMF中計算量的Fmoc-保護胺基酸、HOBt、HBTU，振搖反應3h；6.合併Microkan並洗滌樹脂DMF 3min×3次；7.重複步驟3～7，直至連接全部胺基酸；8.合併Microkan，重複3～4步；9.採用15％Ac2O/CH2Cl2乙醯化多肽的氨基端15min；10.用CH2Cl23min×3次後，室溫晾乾；11.採用IRORI Sorting解碼Microkan並分配到對應的15毫升裂解管中；
12.每一個Microkan用1mL在室溫下裂解液裂解2h；13.再用1mL裂解液浸泡洗滌Microkan 5min；14.合併裂解液和洗滌液；15.惰性氣體流吹乾濃縮至殘留少量裂解液；16.加入事先用冰水浴充分冷卻的甲基叔丁基醚/石油醚(v/v3/1)，放入冰水浴中冷卻20min；17.離心沉澱多肽化合物(3000rpm以上)，10min後小心傾去上清夜；18.再加入冷卻的甲基叔丁基醚/石油醚，充分洗滌、離心，傾去上清夜，共兩次；19.將多肽殘餘物置於室溫下，直至完全乾燥；20.將多肽化合物用10％HOAc/H2O或30％CH3CN/H2O或60％CH3CN/H2O溶解後，經HPLC-MS檢測純度和正確的分子量。
實施例5ELISA法篩選發現抗SARS-CoV陽性血清的抗原決定簇(B-細胞抗原決定簇)將實施例1、2和3獲得的分子多樣性化合物庫與SARS-CoV陽性血清反應，由於SARS-CoV陽性血清中含有SARS-CoV抗體，測定其與分子多樣性化合物庫中的具有10個胺基酸序列的短肽結合活性，由此測定多重最小多肽抗原決定簇，其具體步驟如下1、包被包被液為0.07M NaHCO3，0.03M Na2CO3(pH＝9.6)，包被濃度10μg/mL，100μL/孔，4℃過夜或37℃2小時。
2、清洗PBST(pH＝7.4，含0.05％的Tween 20)清洗5遍，拍幹。
3、封閉封閉液為1～3％的BSA，200μL/孔，4℃過夜或37℃2小時。
4、清洗PBST(pH＝7.4，含0.05％的Tween 20)清洗5遍，拍幹。
5、加入一抗病人血清用生理鹽水1∶100稀釋，100μL/孔，37℃孵育2小時。
6、清洗PBST(pH＝7.4，含0.05％的Tween 20)清洗5遍，拍幹。
7、加入二抗HRP-兔抗人IgG抗體用PBS1∶1000～1∶3000稀釋，100μL/孔，37℃孵育2小時。
8、清洗PBST(pH＝7.4，含0.05％的Tween 20)清洗5遍，拍幹。
9、加入酶反應底物酶反應底物為TMB，100μL/孔，37℃孵育15～30分鐘。
10、加終止液2MH2SO4，100μL/孔，37℃孵育5～10分鐘。
11、讀數在酶標儀中測量450nm和630nm的OD值。
12、中和實驗篩選得到的陽性多肽化合物與抗SARA-CoV陽性血清混合物在37℃孵育30分鐘後重複上述ELISA實驗步驟。
13、對照及數據處理每一個多肽樣品取三個抗SARA-CoV陽性血清反應復孔，和三個正常對照血清復孔進行對比實驗。三個正常對照血清復孔的OD平均值控制在0.1以下。三個抗SARA-CoV陽性血清反應復孔OD值的平均值減去三個正常對照血清復孔的OD平均值即為最終的結果。陽性多肽化合物經過中和實驗確認特異性結果。圖4是20個代表性多肽化合物的篩選結果。它們的序列結構如下11)TSGSDLDRCT12) SGSDLDRCTT13)GSDLDRCTTF14) SDLDRCTTFD15)DLDRCTTFDD16) LDRCTTFDDV17)DRCTTFDDVQ18) RCTTFDDVQA19)CTTFDDVQAP20) TTFDDVQAPN21)TFDDVQAPNY22) FDDVQAPNYT23)DDVQAPNYTQ24) DVQAPNYTQH25)VQAPNYTQHT26) QAPNYTQHTS27) APNYTQHTSS28) PNYTQHTSSM29) NYTQHTSSMR30) YTQHTSSMRG由圖4可以看出，在S蛋白中有101個能與SARS-CoV陽性血清結合的多肽序列，分別是1.MFIFLLFLTL(Compd 1)2.TSGSDLDRCT(Compd 11)3.SGSDLDRCTT(Compd 12)4.SDLDRCTTFD(compd 14)5.TTFDDVQAPN(Compd 20)6.FDDVQAPNYT(Compd 22)7.EIFRSDTLYL(Compd 45)8.FKDGIYFAAT(Compd 83)
9.NSTNVVIRAC(Compd 119)10.STNVVIRACN(Compd 120)11.VSKPMGTQTH(Compd 140)12.SKPMGTQTHT(Compd 141)13.PMGTQTHTMI(Compd 143)14.MIFDNAFNCT(Compd 151)15.DNAFNCTFEY(Compd 154)16.KSGNFKHLRE(Compd 175)17.KNKDGFLYVY(Compd 188)18.KDGFLYVYKG(Compd 190)19.KGYQPIDVVR(Compd 198)20.FKLPLGINIT(Compd 220)21.INITNFRAIL(Compd 226)22.VDCSQNPLAE(Compd 276)23.GIYQTSNFRV(Compd 298)24.YQTSNFRVVP(Compd 300)25.ISNCVADYSV(Compd 345)26.ADYSVLYNST(Compd 350)27.DYSVLYNSTF(Compd 351)28.YSVLYNSTFF(Compd 352)29.SVLYNSTFFS(Compd 353)30.NSTFFSTFKC(Compd 357)31.TFFSTFKCYG(Compd 359)32.GDDVRQIAPG(Compd 391)33.DDVRQIAPGQ(Compd 392)34.VRQIAPGQTG(Compd 394)35.FMGCVLAWNT(Compd 416)36.VLAWNTRNID(Compd 420)37.TRNIDATSTG(Compd 425)38.RNIDATSTGN(Compd 426)39.YNYKYRYLRH(Compd 436)40.PFERDISNVP(Compd 450)41.NCYWPLNDYG(Compd 463)42.WPLNDYGFYT(Compd 476)
43.YQPYRVVVLS(Compd 491)44.QPYRVVVLSF(Compd 492)45.YRVVVLSFEL(Compd 494)46.NQCVNFNFNG(Compd 522)47.QCVNFNFNGL(Compd 523)48.CVNFNFNGLT(Compd 524)49.NFNFNGLTGT(Compd 526)50.FNFNGLTGTG(Compd 527)51.NFNGLTGTGV(Compd 528)52.TDVSTAIHAD(Compd 604)53.DVSTAIHADQ(Compd 605)54.VSTAIHADQL(Compd 606)55.TPAWRIYSTG(Compd 616)56.IGAEHVDTSY(Compd 637)57.SIAYSNNTIA(Compd 686)58.IAYSNNTIAI(Compd 687)59.ITTEVMPVSM(Compd 704)60.AKTSVDCNMY(Compd 714)61.YGECLGDINA(Compd 819)62.AQKFNGLTVL(Compd 834)63.LTVLPPLLTD(Compd 840)64.TVLPPLLTDD(Compd 841)65.KQIANQFNKA(Compd 903)66.TALGKLQDVV(Compd 925)67.KQLSSNFGAI(Compd 946)68.NFGAISSVLN(Compd 951)69.FGAISSVLND(Compd 952)70.GAISSVLNDI(Compd 953)71.AISSVLNDIL(Compd 954)72.RLDKVEAEVQ(Compd 965)73.RLITGRLQSL(Compd 977)74.QLIRAAEIRA(Compd 993)75.SANLAATKMS(Compd 1003)76.VPSQERNFTT(Compd 1050)
77.REGVFVFNGT(Compd 1073)78.WFITQRNFFS(Compd 1084)79.FFSPQIITTD(Compd 1091)80.ANLAATKMSE(Compd 1104)81.NLAATKMSEC(Compd 1105)82.FKNHTSPDVD(Compd 1138)83.TSPDVDLGDI(Compd 1142)84.DVDLGDISGI(Compd 1145)85.VDLGDISGIN(Compd 1146)86.NASVVNIQKE(Compd 1155)87.ASVVNIQKEI(Compd 1156)88.SVVNIQKEID(Compd 1157)89.KEIDRLNEVA(Compd 1163)90.EIDRLNEVAK(Compd 1164)91.LNEVAKNLNE(Compd 1168)92.NLNESLIDLQ(Compd 1174)93.LQELGKYEQY(Compd 1182)94.VMVTILLCCM(Compd 1210)95.MVTILLCCMT(Compd 1211)96.VTILLCCMTS(Compd 1212)97.TILLCCMTSC(Compd 1213)98.CSCLKGACSC(Compd 1223)99.DDSEPVLKGV(Compd 1241)100.DSEPVLKGVK(Compd 1242)101.SEPVLKGVKL(Compd 1243)由圖5可以看出，在M蛋白中有2個能與SARS-CoV陽性血清結合的多肽序列，分別是篩選得到的M蛋白線性抗原決定簇1.A W I M L L Q F A Y(Compd 29)2.S N R N R F L Y I I(Compd 39)在E蛋白中有1個能與SARS-CoV陽性血清結合的多肽序列，分別是篩選得到的E蛋白線性抗原決定簇1.L T A L R L C A Y C(Compd 246)
在N蛋白中有8個能與SARS-CoV陽性血清結合的多肽序列，分別是篩選得到的N蛋白線性抗原決定簇1.G R N G A R P K Q R(Compd 311)2.K E E L R F P R G Q(Compd 441)3.S R G N S P A R M A(Compd 482)4.E A S K K P R Q K R(Compd 533)5.K K P R Q K R T A T(Compd 536)6.T E P K K D K K K K(Compd 646)7.A Q P L P Q R Q K K(Compd 659)8.Q P L P Q R Q K K Q(Compd 660)上述112個序列即為本發明所篩選的SARS-CoV的抗原決定簇。
序列表110中國醫學科學院藥物研究所120一種肽文庫、其合成方法及從該文庫中篩選的活性片段130
160116170PatentIn version 3.1210121110212PRT213SARS-CoV4001Met Phe Ile Phe Leu Leu Phe Leu Thr Leu1 5 10210221110212PRT213SARS-CoV4002Thr Ser Gly Ser Asp Leu Asp Arg Cys Thr1 5 10210321110212PRT213SARS-CoV4003Ser Gly Ser Asp Leu Asp Arg Cys Thr Thr1 5 10
210421110212PRT213SARS-CoV4004Ser Asp Leu Asp Arg Cys Thr Thr Phe Asp1 5 10210521110212PRT213SARS-CoV4005Thr Thr Phe Asp Asp Val Gln Ala Pro Asn1 5 10210621110212PRT213SARS-CoV4006Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr1 5 10210721110212PRT213SARS-CoV4007Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu1 5 10
210821110212PRT213SARS-CoV4008Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr1 5 10210921110212PRT213SARS-CoV4009Asn Ser Thr Asn Val Val Ile Arg Ala Cys1 5 102101021110212PRT213SARS-CoV40010Ser Thr Asn Val Val Ile Arg Ala Cys Asn1 5 102101121110212PRT213SARS-CoV40011Val Ser Lys Pro Met Gly Thr Gln Thr His1 5 10
2101221110212PRT213SARS-CoV40012Ser Lys Pro Met Gly Thr Gln Thr His Thr1 5 102101321110212PRT213SARS-CoV40013Pro Met Gly Thr Gln Thr His Thr Met Ile1 5 102101421110212PRT213SARS-CoV40014Met Ile Phe Asp Asn Ala Phe Asn Cys Thr1 5 102101521110212PRT213SARS-CoV40015Asp Asn Ala Phe Asn Cys Thr Phe Glu Tyr1 5 10
2101621110212PRT213SARS-CoV40016Lys Ser Gly Asn Phe Lys His Leu Arg Glu1 5 102101721110212PRT213SARS-CoV40017Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr1 5 102101821110212PRT213SARS-CoV40018Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly1 5 102101921110212PRT213SARS-CoV40019Lys Gly Tyr Gln Pro Ile Asp Val Val Arg1 5 10
2102021110212PRT213SARS-CoV40020Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr1 5 102102121110212PRT213SARS-CoV40021Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu1 5 102102221110212PRT213SARS-CoV40022Val Asp Cys Ser Gln Asn Pro Leu Ala Glu1 5 102102321110212PRT213SARS-CoV40023Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val1 5 10
2102421110212PRT213SARS-CoV40024Tyr Gln Thr Ser Asn Phe Arg Val Val Pro1 5 102102521110212PRT213SARS-CoV40025Ile Ser Asn Cys Val Ala Asp Tyr Ser Val1 5 102102621110212PRT213SARS-CoV40026Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr1 5 102102721110212PRT213SARS-CoV40027Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe1 5 10
2102821110212PRT213SARS-CoV40028Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe1 5 102102921110212PRT213SARS-CoV40029Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser1 5 102103021110212PRT213SARS-CoV40030Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys1 5 102103121110212PRT213SARS-CoV40031Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly1 5 10
2103221110212PRT213SARS-CoV40032Gly Asp Asp Val Arg Gln Ile Ala Pro Gly1 5 102103321110212PRT213SARS-CoV40033Asp Asp Val Arg Gln Ile Ala Pro Gly Gln1 5 102103421110212PRT213SARS-CoV40034Val Arg Gln Ile Ala Pro Gly Gln Thr Gly1 5 102103521110212PRT213SARS-CoV40035Phe Met Gly Cys Val Leu Ala Trp Asn Thr1 5 10
2103621110212PRT213SARS-CoV40036Val Leu Ala Trp Asn Thr Arg Asn Ile Asp1 5 102103721110212PRT213SARS-CoV40037Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly1 5 102103821110212PRT213SARS-CoV40038Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn1 5 102103921110212PRT213SARS-CoV40039Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His1 5 10
2104021110212PRT213SARS-CoV40040Pro Phe Glu Arg Asp Ile Ser Asn Val Pro1 5 102104121110212PRT213SARS-CoV40041Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly1 5 102104221110212PRT213SARS-CoV40042Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr1 5 102104321110212PRT213SARS-CoV40043Tyr Gln Pro Tyr Arg Val Val Val Leu Ser1 5 10
2104421110212PRT213SARS-CoV40044Tyr Arg Val Val Val Leu Ser Phe Glu Leu1 5 102104521110212PRT213SARS-CoV40045Asn Gln Cys Val Asn Phe Asn Phe Asn Gly1 5 102104621110212PRT213SARS-CoV40046Gln Cys Val Asn Phe Asn Phe Asn Gly Leu1 5 102104721110212PRT213SARS-CoV40047Cys Val Asn Phe Asn Phe Asn Gly Leu Thr1 5 10
2104821110212PRT213SARS-CoV40048Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr1 5 102104921110212PRT213SARS-CoV40049Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly1 5 102105021110212PRT213SARS-CoV40050Asn Phe Asn Gly Leu Thr Gly Thr Gly Val1 5 102105121110212PRT213SARS-CoV40051Thr Asp Val Ser Thr Ala Ile His Ala Asp1 5 10
2105221110212PRT213SARS-CoV40052Asp Val Ser Thr Ala Ile His Ala Asp Gln1 5 102105321110212PRT213SARS-CoV40053Val Ser Thr Ala Ile His Ala Asp Gln Leu1 5 102105421110212PRT213SARS-CoV40054Thr Pro Ala Trp Arg Ile Tyr Ser Thr Gly1 5 102105521110212PRT213SARS-CoV40055Ile Gly Ala Glu His Val Asp Thr Ser Tyr1 5 10
2105621110212PRT213SARS-CoV40056Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala1 5 102105721110212PRT213SARS-CoV40057Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile1 5 102105821110212PRT213SARS-CoV40058Ile Thr Thr Glu Val Met Pro Val Ser Met1 5 102105921110212PRT213SARS-CoV40059Ala Lys Thr Ser Val Asp Cys Asn Met Tyr1 5 10
2106021110212PRT213SARS-CoV40060Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala1 5 102106121110212PRT213SARS-CoV40061Ala Gln Lys Phe Asn Gly Leu Thr Val Leu1 5 102106221110212PRT213SARS-CoV40062Leu Thr Val Leu Pro Pro Leu Leu Thr Asp1 5 102106321110212PRT213SARS-CoV40063Thr Val Leu Pro Pro Leu Leu Thr Asp Asp1 5 10
2106421110212PRT213SARS-CoV40064Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala1 5 102106521110212PRT213SARS-CoV40065Thr Ala Leu Gly Lys Leu Gln Asp Val Val1 5 102106621110212PRT213SARS-CoV40066Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile1 5 102106721110212PRT213SARS-CoV40067Asn Phe Gly Ala Ile Ser Ser Val Leu Asn1 5 10
2106821110212PRT213SARS-CoV40068Phe Gly Ala Ile Ser Ser Val Leu Asn Asp1 5 102106921110212PRT213SARS-CoV40069Gly Ala Ile Ser Ser Val Leu Asn Asp Ile1 5 102107021110212PRT213SARS-CoV40070Ala Ile Ser Ser Val Leu Asn Asp Ile Leu1 5 102107121110212PRT213SARS-CoV40071Arg Leu Asp Lys Val Glu Ala Glu Val Gln1 5 10
2107221110212PRT213SARS-CoV40072Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu1 5 102107321110212PRT213SARS-CoV40073Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala1 5 102107421110212PRT213SARS-CoV40074Ser Ala Asn Leu Ala Ala Thr Lys Met Ser1 5 102107521110212PRT213SARS-CoV40075Val Pro Ser Gln Glu Arg Asn Phe Thr Thr1 5 10
2107621110212PRT213SARS-CoV40076Arg Glu Gly Val Phe Val Phe Asn Gly Thr1 5 102107721110212PRT213SARS-CoV40077Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser1 5 102107821110212PRT213SARS-CoV40078Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp1 5 102107921110212PRT213SARS-CoV40079Ala Asn Leu Ala Ala Thr Lys Met Ser Glu1 5 10
2108021110212PRT213SARS-CoV40080Asn Leu Ala Ala Thr Lys Met Ser Glu Cys1 5 102108121110212PRT213SARS-CoV40081Phe Lys Asn His Thr Ser Pro Asp Val Asp1 5 102108221110212PRT213SARS-CoV40082Thr Ser Pro Asp Val Asp Leu Gly Asp Ile1 5 102108321110212PRT213SARS-CoV40083Asp Val Asp Leu Gly Asp Ile Ser Gly Ile1 5 10
2108421110212PRT213SARS-CoV40084Val Asp Leu Gly Asp Ile Ser Gly Ile Asn1 5 102108521110212PRT213SARS-CoV40085Asn Ala Ser Val Val Asn Ile Gln Lys Glu1 5 102108621110212PRT213SARS-CoV40086Ala Ser Val Val Asn Ile Gln Lys Glu Ile1 5 102108721110212PRT213SARS-CoV40087Ser Val Val Asn Ile Gln Lys Glu Ile Asp1 5 10
2108821110212PRT213SARS-CoV40088Lys Glu Ile Asp Arg Leu Asn Glu Val Ala1 5 102108921110212PRT213SARS-CoV40089Glu Ile Asp Arg Leu Asn Glu Val Ala Lys1 5 102109021110212PRT213SARS-CoV40090Leu Asn Glu Val Ala Lys Asn Leu Asn Glu1 5 102109121110212PRT213SARS-CoV40091Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln1 5 10
2109221110212PRT213SARS-CoV40092Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr1 5 102109321110212PRT213SARS-CoV40093Val Met Val Thr Ile Leu Leu Cys Cys Met1 5 102109421110212PRT213SARS-CoV40094Met Val Thr Ile Leu Leu Cys Cys Met Thr1 5 102109521110212PRT213SARS-CoV40095Val Thr Ile Leu Leu Cys Cys Met Thr Ser1 5 10
2109621110212PRT213SARS-CoV40096Thr Ile Leu Leu Cys Cys Met Thr Ser Cys1 5 102109721110212PRT213SARS-CoV40097Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys1 5 102109821110212PRT213SARS-CoV40098Asp Asp Ser Glu Pro Val Leu Lys Gly Val1 5 102109921110212PRT213SARS-CoV40099Asp Ser Glu Pro Val Leu Lys Gly Val Lys1 5 10
21010021110212PRT213SARS-CoV400100Ser Glu Pro Val Leu Lys Gly Val Lys Leu1 5 1021010121110212PRT213SARS-CoV400101Ala Trp Ile Met Leu Leu Gln Phe Ala Tyr1 5 1021010221110212PRT213SARS-CoV400102Ser Asn Arg Asn Arg Phe Leu Tyr Ile Ile1 5 1021010321110212PRT213SARS-CoV400103Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys1 5 10
21010421110212PRT213SARS-CoV400104Gly Arg Asn Gly Ala Arg Pro Lys Gln Arg1 5 1021010521110212PRT213SARS-CoV400105Lys Glu Glu Leu Arg Phe Pro Arg Gly Gln1 5 1021010621110212PRT213SARS-CoV400106Ser Arg Gly Asn Ser Pro Ala Arg Met Ala1 5 1021010721110212PRT213SARS-CoV400107Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg1 5 10
21010821110212PRT213SARS-CoV400108Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr1 5 1021010921110212PRT213SARS-CoV400109Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys1 5 1021011021110212PRT213SARS-CoV400110Ala Gln Pro Leu Pro Gln Arg Gln Lys Lys1 5 1021011121110212PRT213SARS-CoV400111Gln Pro Leu Pro Gln Arg Gln Lys Lys Gln1 5 10
21011221110212PRT213SARS-CoV400112Gln Pro Tyr Arg Val Val Val Leu Ser Phe1 5 102101132111255212PRT213SARS-CoV400113Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu1 5 10 15Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln20 25 30His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg35 40 45Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser50 55 60Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Asp Asn Pro Val65 70 75 80Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn85 90 95Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln
100 105 110Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys115 120 125Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met130 135 140Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr145 150 155 160Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser165 170 175Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly180 185 190Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp195 200 205Leu Pro Ser Gly Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu210 215 220Gly Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu Thr Ala Phe Ser Pro225 230 235 240Ala Gln Asp Thr Trp Gly Thr Ser Ala Ala Ala Tyr Phe Val Gly Tyr245 250 255Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile260 265 270Thr Asp Ala Val Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys
275 280 285Ser Val Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr Gln Thr Ser Asn290 295 300Phe Arg Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr305 310 315 320Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser325 330 335Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val Ala Asp Tyr340 345 350Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly355 360 365Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn Val Tyr Ala370 375 380Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly385 390 395 400Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe405 410 415Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp Ala Thr Ser420 425 430Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His Gly Lys Leu435 440 445Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly
450 455 460Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp465 470 475 480Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val485 490 495Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly500 505 510Pro Lys Leu Ser Thr Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn515 520 525Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg530 535 540Phe Gln Pro Phe Gln Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp545 550 555 560Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys565 570 575Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser580 585 590Glu Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr595 600 605Ala Ile His Ala Asp Gln Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr610 615 620Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile Gly Ala Glu
625 630 635 640His Val Asp Thr Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile645 650 655Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys660 665 670Ser Ile Val Ala Tyr Thr Met Ser Leu Gly Ala Asp Ser Ser Ile Ala675 680 685Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn Phe Ser Ile Ser Ile690 695 700Thr Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser Val Asp Cys705 710 715 720Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ala Asn Leu Leu Leu725 730 735Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly Ile740 745 750Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val Lys755 760 765Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn Phe770 775 780Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile785 790 795 800Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Met
805 810 815Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu Ile820 825 830Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr835 840 845Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr Ala850 855 860Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe865 870 875 880Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn885 890 895Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala900 905 910Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly915 920 925Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu930 935 940Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn945 950 955 960Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp965 970 975Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln
980 985 990Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala995 10001005Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp101010151020Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala102510301035Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln104010451050Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys105510601065Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn Gly Thr Ser107010751080Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr108510901095Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly110011051110Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp111511201125Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser113011351140Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val
114511501155Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys116011651170Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr117511801185Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile119011951200Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu Cys Cys120512101215Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys Gly122012251230Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys123512401245Gly Val Lys Leu His Tyr Thr12501255210114211221212PRT213SARS-CoV400114Met Ala Asp Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Gln Leu Leu1 5 10 15Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Ala Trp Ile Met20 25 30
Leu Leu Gln Phe Ala Tyr Ser Asn Arg Asn Arg Phe Leu Tyr Ile Ile35 40 45Lys Leu Val Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys Phe50 55 60Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Val Thr Gly Gly Ile Ala65 70 75 80Ile Ala Met Ala Cys Ile Val Gly Leu Met Trp Leu Ser Tyr Phe Val85 90 95Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe Asn100 105 110Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu Arg Gly Thr Ile Val115 120 125Thr Arg Pro Leu Met Glu Ser Glu Leu Val Ile Gly Ala Val Ile Ile130 135 140Arg Gly His Leu Arg Met Ala Gly His Ser Leu Gly Arg Cys Asp Ile145 150 155 160Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser165 170 175Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Gly Thr Asp Ser Gly Phe180 185 190Ala Ala Tyr Asn Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp195 200 205
His Ala Gly Ser Asn Asp Asn Ile Ala Leu Leu Val Gln210 215 22021011521176212PRT213SARS-CoV400115Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser1 5 10 15Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala20 25 30Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn35 40 45Val Ser Leu Val Lys Pro Thr Val Tyr Val Tyr Ser Arg Val Lys Asn50 55 60Leu Asn Ser Ser Glu Gly Val Pro Asp Leu Leu Val65 70 75210116211422212PRT213SARS-CoV400116Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile1 5 10 15
Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly20 25 30Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn35 40 45Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu50 55 60Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Gly65 70 75 80Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg85 90 95Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr Phe Tyr100 105 110Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro Tyr Gly Ala Asn Lys115 120 125Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys130 135 140Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr Val Leu145 150 155 160Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly165 170 175Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg180 185 190
Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro195 200 205Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu210 215 220Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys Gly Gln225 230 235 240Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser245 250 255Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr260 265 270Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly275 280 285Asp Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln290 295 300Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg305 310 315 320Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly325 330 335Ala Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe LysAsp Asn Val Ile340 345 350Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu355 360 365
Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro370 375 380Gln Arg Gln Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp385 390 395 400Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser405 410 415Ala Asp Ser Thr Gln Ala420
權利要求
1.一種肽文庫，其中每一個肽的通式為Z-XnXn+1Xn+2Xn+3Xn+4Xn+5Xn+6Xn+7Xn+8Xn+9-Y其中X代表任何一種L-構型的天然胺基酸殘基，n是指第Xn胺基酸在蛋白質序列中的天然排列位置，n為整數，Y代表NH2或者OH，Z代表氫或者Ac，其中Ac為乙醯基。
2.根據權利要求1所述的肽文庫，其特徵在於所述的蛋白質序列為與人類相關的冠狀病毒SARS-CoV相關的S、M、E和N蛋白的多肽序列。
3.權利要求2所述的胺基酸序列，其特徵在於所述的與人類相關的冠狀病毒SARS-CoV為SARS-CoVBJ01號、SARS-CoVBJ02號、SARS-CoVBJ03號、SARS-CoVBJ04號、SARS-CoVCUHK-W1、SARS-CoVGZ01、SARS-CoVHKU-39849、SARS-CoVTaiwan、SARS-CoVTor2、SARS-CoVUrbani、SARS-CoVVietnam。
4.權利要求2或3所述的胺基酸序列，其特徵在於所述的與人類相關的冠狀病毒SARS-CoV為SARS-CoVBJ01號。
5.一種選自如SEQ ID NO1至SEQ ID NO101所示的胺基酸序列的任意一種或其結合的胺基酸序列。
6.一種如SEQ ID NO102至103所示的胺基酸序列的任意一種或其結合的胺基酸序列。
7.一種如SEQ ID NO104所示的胺基酸序列。
8.一種如SEQ ID NO105至112所示的胺基酸序列的任意一種或其結合的胺基酸序列。
9.具有抗SARS-CoV陽性血清抗原決定簇(B-細胞抗原決定簇)、T-細胞抗原決定簇、以及由此發現B-細胞抗原決定簇和T-細胞抗原決定簇作用的、含有SEQ ID NO1至SEQ ID NO112任意一種或其結合的共價或者非共價化合物。
10.含有如SEQ ID NO1至112所示的胺基酸序列的任意一種或其結合的胺基酸序列在用於製備治療SARS-CoV引起的疾病的藥物中的應用。
11.通過封閉SEQ ID NO1至SEQ ID NO112所示的胺基酸序列從而阻止SARS-CoV病毒進入宿主的阻斷劑。
12.一種採用「混-分」物理編碼法合成含有至少一個具有2～50個胺基酸殘基的肽文庫的方法，其包括以下步驟1)選擇合適的射頻子編碼確定胺基酸序列的多肽片段；2)混合併對多肽母鏈氨基端進行脫Fmoc保護；3)混合併進行充分的洗滌；4)解碼並按所要連接的特定胺基酸進行分組；5)將特定胺基酸以3～10倍摩爾過量的形式加入到已經分組的反應瓶中，進而連接到步驟2)的多肽母鏈；以及6)重複步驟2)和5)直至達到所需長度的片段；7)最後，進行解碼並將多肽化合物從樹脂上裂解下來。
13.根據權利要求9所述的方法，其特徵在於所述的胺基酸殘基長度為10-34。
14.根據權利要求9所述的方法，其特徵在於所述的保護為Boc/Bzl保護。
全文摘要
本發明涉及一種肽文庫，其中每一個肽的通式為Z－X
文檔編號A61P11/00GK1609119SQ20031010189
公開日2005年4月27日 申請日期2003年10月23日 優先權日2003年10月23日
發明者劉剛, 何維, 李莉, 崔蓮仙, 寇賓賓, 胡洪波, 王佔國, 郝志勇, 張亮, 李旭琴, 倪安平, 劉勇, 孫曼霽, 李前 申請人:中國醫學科學院藥物研究所