一种肽文库、其合成方法及从该文库中筛选的活性片段的制作方法

专利名称：一种肽文库、其合成方法及从该文库中筛选的活性片段的制作方法
背景技术：
与人类相关的冠状病毒(Severe Acute Respiratory SyndromeCoronavirus(SARS-CoV))已经被确认为新的一类病毒，人类以迄今最快的速度测定了其基因序列，主要由RNA-dependent RNApolymerase、Spike(S)、Membrane(M)、Envelope(E)、Nucleocapside(N)、Polymerase(P)等蛋白组成(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)蛋白组成了病毒外壳，是病毒识别、结合并进入宿主细胞的蛋白质。尤其是刺突S蛋白是关键性蛋白(图1)。虽然文献研究结果表明HCoV-229E刺突S蛋白的417-546序列是宿主受体结合的部位，但同时也指出不同的冠状病毒通过使用不同的受体进入宿主细胞。这很可能与刺突S蛋白的变异有关(1、Marra MA etal.，The genome sequence of the SARS-associated Coronavirus，Sciencexpress，published online atwww.sciencexpress.org，1 May，2003)。Nucleocapsid(N)蛋白属于胞浆内蛋白，处于病毒颗粒的核心部分，和Genomic RNA以结合的形式存在。病毒RNA在细胞质中复制完成后会和N蛋白结合，结合产物可以被M蛋白识别并被包装到病毒颗粒中。所以N和M蛋白与病毒在宿主细胞中的复制具有明显的关系。N蛋白是宿主T-细胞识别的主要位点之一(见图1)。因而，发展合成病毒蛋白的多肽化合物化学库，对于寻找病毒表面蛋白的多重最小多肽抗原决定簇(包括B-细胞和T-细胞抗原决定簇)，进而寻求和发展合成多肽疫苗、临床诊断试剂和血清治疗方案有极其重要的意义。同时，该化学库还可用于筛选并发现病毒结合受体的多重配基，进而阻断冠状病毒进入宿主细胞，发展治疗药物(特别是对于耐药病毒的药物治疗)有极大的参考价值。
目前，寻找抗原决定簇最为常用的方法是利用各种计算机软件，通过已经发表的序列采用亲水性、疏水性、转角结构、HPLC滞留系数、螺旋结构、保守性等参数进行预测。我们的实验结果表明，这种预测方法与实际测得的序列仅有30％～50％的重复性(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.)。另一种方法是采用组合化学技术对蛋白抗原决定簇谱的研究(“交叉重叠”多肽化合物，见表1)。
表1 HR2区的“交叉重叠”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组合化学是用于新药合成和筛选的一种全新的方法，始于90年代初，被誉为是对传统药物化学合成和筛选的一场“革命性突破”。它打破了逐一合成、逐一筛选的模式，以合成和筛选化合物库的形式寻找和优化先导化合物，从而大大加快了发现药物先导化合物的速度。
组合化学把化学合成、电脑设计、计算机技术结为一体，能同时产生许多种结构相关但有序变化的化合物，然后用高度灵敏的生物学方法对这些化合物同时进行筛选，从中确定具有生物活性的物质，再经结构测定，以期找到全新的先导化合物。组合化学以其高效、微量、高度自动化的特点而很快受到世人的瞩目并被许多大的制药公司采纳，成为近年来用以发现新药先导化合物的一种重要手段。该法的特点是以一定数目的氨基酸残基肽(比如1到50个氨基酸残基)为合成“交叉重叠”片段，将蛋白序列通过逐个错位(或者间隔错位，包括从2到合成肽片段的氨基酸的总位数)的方式全部合成出来，然后进行抗原-抗体反应(或者其它生物目的的筛选反应等，如筛选发现T-细胞免疫抗原决定簇，以及SARS-CoV的受体配基等)，一次便可以得到所有最短的抗原决定簇，进而绘成抗原决定簇谱，或者其它最小有效活性片段的多肽化合物以及它们的图谱。
目前，通过组合化学技术在化合物合成领域已经成功用于小分子物质的合成，但却没有关于合成大分子物质如肽的报道。

发明内容
本发明人通过应用物理编码法成功合成了一种“交叉重叠”多肽化学库，并使该多肽化学库与SARS-CoV阳性血清反应，成功筛选得到了BJ01号冠状病毒变种的抗原决定簇，从而完成了本发明。
本发明一方面涉及一种“交叉重叠”肽库，其中每一个肽的通式为Z-XnXn+1Xn+2Xn+3Xn+4Xn+5Xn+6Xn+7Xn+8Xn+9-Y其中X代表任何一种L-构型的天然氨基酸残基，n是指第Xn氨基酸在蛋白质序列中的天然排列位置，n为整数，例如第1，2，3，4....n；Y代表NH2或者OH，Z代表氢或者Ac，其中Ac为乙酰基。
所述的蛋白质序列的多肽优选为与人类相关的冠状病毒SARS-CoV相关的S、M、E和N蛋白的多肽序列。
所述的与人类相关的冠状病毒SARS-CoV优选为SARS-CoVBJ01号、SARS-CoVBJ02号、SARS-CoVBJ03号、SARS-CoVBJ04号、SARS-CoVCUHK-W1、SARS-CoVGZ01、SARS-CoVHKU-39849、SARS-CoVTaiwan、SARS-CoVTor2、SARS-CoVUrbani、SARS-CoVVietnam。所述的与人类相关的冠状病毒SARS-CoV最优选为SARS-CoVBJ01号。
本发明另一方面涉及一种选自如SEQ ID NO1至SEQ ID NO101所示的氨基酸序列的任意一种或其结合的氨基酸序列。
本发明再一方面涉及一种如SEQ ID NO102至103所示的氨基酸序列的任意一种或其结合的氨基酸序列。
本发明还涉及一种如SEQ ID NO104所示的氨基酸序列。
本发明还涉及一种如SEQ ID NO105至112所示的氨基酸序列的任意一种或其结合的氨基酸序列。
本发明还涉及具有抗SARS-CoV阳性血清抗原决定簇(B-细胞抗原决定簇)、T-细胞抗原决定簇、以及由此发现B-细胞抗原决定簇和T-细胞抗原决定簇作用的、含有SEQ ID NO1至SEQ ID NO112任意一种或其结合的共价或者非共价化合物。
本发明还涉及含有如SEQ ID NO1至112所示的氨基酸序列的任意一种或其结合的氨基酸序列在用于制备治疗SARS-CoV引起的疾病的药物中的应用。
本发明进一步涉及通过封闭SEQ ID NO1至SEQ ID NO112所示的氨基酸序列从而阻止SARS-CoV病毒进入宿主的阻断剂。
本发明还涉及一种采用“混-分”物理编码法合成含有至少一个具有2～50个氨基酸残基的肽文库的方法，其包括以下步骤1)选择合适的射频子编码确定氨基酸序列的多肽片段；2)混合并对多肽母链氨基端进行脱Fmoc保护；3)混合并进行充分的洗涤；4)解码并按所要连接的特定氨基酸进行分组；5)将特定氨基酸以3～10倍摩尔过量的形式加入到已经分组的反应瓶中，进而连接到步骤2)的多肽母链；以及6)重复步骤2)和5)直至达到所需长度的片段；7)最后，进行解码并将多肽化合物从树脂上裂解下来。
所述的多肽为具有2～50个、优选3-34个氨基酸残基的多肽。
该方法所述的保护策略包括Fmoc/t-Bu或者Boc/Bz1保护，优选为Fmoc/tBu。
本发明的优点在于采用固相合成技术、射频编码技术在短时间内合成了大量的“交叉重叠”多肽化合物，并首次合成了与SARS-CoV相关的S、M、E和N蛋白的全部“交叉重叠”多肽。发明中，首次使用SARS-CoV感染的人的阳性血清对化学库进行了筛选，并绘制了B-细胞抗原决定簇谱。


图1描述了Spike蛋白及其结构。
图2为合成多肽随机抽样LC-MS分析结果。
图3显示合成NH2-NAQALNTLVKQLSSNFGAISSVLNDILSRLDKVE-CONH2HPLC-MS的分析结果。
图4显示S蛋白筛选结果。
图5显示M、E和N蛋白筛选结果。
11～30号多肽ELISA筛选抗SARS-CoV阳性血清结果，其中纵坐标代表OD值，横坐标代表合成多肽序列编号。
具体实施例方式
组合化学研究分三个阶段分子多样性化合物库的合成；群集筛选(进行活性检测)；活性分子的结构测定。
1、肽库的建立要建立肽文库必须事先考虑许多方面如何设计模板分子；选择合适的构建单元；确定何种合成方案；如何完成半自动或者自动合成等。
本发明中的主要目的是寻找针对SARS-CoV病毒的人的B-细胞线型抗原决定簇，并绘制抗原决定簇谱。选择逐位错位的合成方式可以直接找到最小的抗原决定簇。因而，线型10肽被选择为摸板分子。蛋白质以天然L-构型氨基酸组成，因而，本化学库的构建单元自然选择为20种天然L-构型氨基酸。高通量地合成方法一般采用固相合成技术，特点是中间体的纯化仅采用简单的洗涤和过滤操作即可完成。同时，反应中可以使用大大过量的构建单元来保证反应进行完全，并易半自动化或者自动化。因而，我们选择了固相合成技术，在树脂上完成了全部多肽化合物的合成。使用20种天然L-构型氨基酸为构建单元来合成数千个多肽化合物时，相同的反应条件可以在多肽编码的情况下同步进行，如相同的脱保护步骤、不同的多肽序列与相同的保护氨基酸进行反应步骤等。这样就可以完成大规模、快速地合成。其中编码方式有多种，如化学编码、物理编码等。物理编码的特点是无“化学污染”，无须多余的化学解码步骤等。因而，我们选择了IRORI编码-解码的分类技术。一次可操作数千个化合物的合成，本实验中我们选择了一次合成600～700个多肽化合物的规模。
2、群集筛选总的来讲，筛选可分为随机筛选和定向筛选。但无论是随机筛选还是定向筛选，都要考虑选定筛选模式为固相筛选或用何法(细胞功能筛选、受体筛选、抗体筛选等)进行筛选，用何种指示剂(同位素标记、荧光标记、染料染色)等。具体的筛选方法有三种固相筛选、液相筛选和两者的结合。本发明中使用的ELISA筛选方法属于定向液相筛选，生物反应是抗原-抗体结合实验。人抗SARS-CoV阳性血清中含有人抗SARS-CoV多克隆抗体(包括IgG和IgM)。当包被在96孔反应板中的多肽化合物与人抗SARS-CoV多克隆抗体结合后，经过仔细的洗涤即可洗去为结合多余的、甚至是非特异的结合抗体和血清。此时，加入用辣根过氧化酶标记的抗IgG或者抗IgM抗体就会完成二次结合，并将辣根过氧化酶保留在体系内。该辣根过氧化酶在一定的条件下可以催化水解对应的底物，并可以在特定的波长下测得吸收值。此吸收值的大小与人抗SARS-CoV多克隆抗体结合的强弱和多少相关。
3、确认活性分子结构编码技术已应用于解析组合库中高活性化合物的结构。最早提出此技术的是Brenner和Lemer，Nicolaou等提出了射频编码合成仪突破了以往的编码形式，它是建立在射频信号及应用多功能微反应仪的半导体记忆装置基础上的。
本发明中使用的射频编码技术可以直接将裂解到96孔板中多肽化合物编码对应孔号。因而，筛选得到的活性孔经过与编码子序号对比即可给出多肽的序列。
本实施例中以筛选抗SARS-CoV阳性血清，寻找并绘制B-细胞抗原决定簇谱为目标，筛选了本化学库。筛选并绘制T-细胞抗原决定簇谱和SARS-CoV融合阻断剂正在进行中。本实施例中选择了SARS-CoV的BJ01号序列，其组成成分的氨基酸序列如下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。
实施例1设计合成Spike(S)蛋白从1至600个氨基酸序列中每相邻的10个氨基酸序列的“交叉重叠”序列，如下表所示。从右至左为合成次序，代表合成步骤号码为阿拉伯数字1到10。
表2 所合成的600个以10个氨基酸为长度的序列编号 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)蛋白从1至600个氨基酸序列中每相邻的10个氨基酸序列的合成操作步骤如下A.多肽脱Fmoc保护/氨基酸残基的连接步骤1.均分树脂采用IRORI均分树脂装置，在每一个Microkan中(共600个)加入15mg Rink Amide树脂；
2.均分IRORI射频子采用IRORI均分射频子装置，在每一个Microkan中加入一个射频子，并用IRORI加盖器加盖；3.脱Fmoc保护集中所有待反应的Microkan，置于一个2L锥形瓶中，加入20％哌啶/DMF振摇，15min×2次；4.洗涤树脂DMF 3min×6，DCM 3min×3，晾干；5.IRORI射频子编码(或解码)采用IRORI Sorting程序及相应配件编码。按照多肽化学库的序列对射频子编码(或解码)，每一特定编码代表一个特定序列，并以此将Microkan分配至不同氨基酸反应瓶中；6.洗涤树脂用重蒸DMF润洗待反应的树脂；7.Fmoc-保护氨基酸的连接准确称取计算量的Fmoc保护氨基酸、HOBt、HBTU(或DIC或BOP)，并用重蒸DMF溶解。将Microkan加入特定氨基酸反应瓶中，振摇反应3h；8.茚三酮检测取事先设定好的对照组序列，用茚三酮法检测反应情况。如果呈阴性反应，表明反应完全，进行步骤9的操作；如果呈阳性反应，表明反应不完全，需继续反应；如果延长时间或改用缩合能力更强的缩合剂后，仍不能完全反应，则采用步骤11的方法封闭后，进行步骤9的操作；9.洗涤树脂DMF 3min×3，DCM 3min×1；10.脱Fmoc保护/连接根据序列要求，重复步骤3-9的操作，直至反应结束；11.封闭采用乙酰化多肽的氨基端，15％Ac2O/CH2Cl2，30min；12.洗涤树脂DCM 3min×2，晾干。
B.多肽裂解步骤1.配制裂解液根据序列特点，从选取并配制适当的裂解液；2.裂解每一个Microkan使用1mL裂解液在室温下裂解2h；3.洗涤再用1mL裂解液浸泡洗涤Microkan 5min；4.晾干或离心干燥合并两次裂解液，离心浓缩至少量；
5.沉淀将事先用冰水浴充分冷却的甲基叔丁基醚/石油醚(v/v3/1)加入到残余物中，放入冰水浴中冷却20min使析晶完全；6.离心离心沉淀多肽化合物，10min后取出，小心倾去上清夜；7.洗涤多肽加入冷却的甲基叔丁基醚/石油醚，充分洗涤后，离心，倾去上清夜；8.重复步骤7的操作一次；9.晾干将多肽残余物在室温下挥干，直至完全干燥；10.检测抽检部分多肽样品，用10％HOAc/H2O或30％CH3CN/H2O或60％CH3CN/H2O溶解，并用HPLC-MS检测纯度。
C.以下表3-表12为第1步至第10步的投料D.从肽库中随机抽取的多肽化合物的纯度以及正确的分子量见表13
表3合成1～600号“交叉重叠”多肽第一步投料计算结果S-1-步骤-1 FW 总数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号“交叉重叠”多肽第二步使用试剂投料计算结果S-1-步骤-2 FW 个数控制组总数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号“交叉重叠”多肽第三步投料计算结果S-1-步骤3FW 个数 控制组 总数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号“交叉重叠”多肽第四步投料计算结果S-1-步骤-4 FW 个数 控制组 总数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号“交叉重叠”多肽第五步投料计算结果S-1-步骤-5 FW 个数控制组 总数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号“交叉重叠”多肽第六步投料计算结果S-1-步骤-6 FW 个数 控制组 总数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号“交叉重叠”多肽第七步投料计算结果S-1-步骤-7FW 个数 控制组 总数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号“交叉重叠”多肽第八步投料计算结果S-1-步骤-8 FW 个数 控制组 总数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号“交叉重叠”多肽第九步投料计算结果S-1-步骤-9 FW 个数 控制组 总数 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号“交叉重叠”多肽第十步投料计算结果S-1-步骤-10 FW 个数 控制组 总数 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合成肽库中随机抽取的多肽化合物的纯度以及正确的分子量

实施例2设计合成Spike(S)蛋白601至1246位氨基酸的每10位序列的“交叉重叠”序列如下。从右至左为合成次序，代表合成步骤号码为阿拉伯数字1到10。
表14 Spike(S)蛋白中所合成的646个以10个氨基酸为长度的序列编号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其合成方法同实施例1，所不同的是从第1步至第10步的投料。以下表15至表24为第1步至第10步的投料
表15合成Spike(S)蛋白601～1246号“交叉重叠”多肽第一步时使用试剂投料计算结果S-2-步骤-1FW 个数 控制组 总数 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号“交叉重叠”多肽第二步时使用试剂投料计算结果S-2-步骤-2 FW 个数 控制组 总数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号“交叉重叠”多肽第三步时使用试剂投料计算结果S-2-步骤-3 FW 个数 控制组 总数 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号“交叉重叠”多肽第四步时使用试剂投料计算结果S-2-步骤-4FW 个数 控制组 总数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号“交叉重叠”多肽第五步时使用试剂投料计算结果S-2-步骤-5 FW 个数 控制组 总数 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号“交叉重叠”多肽第六步时使用试剂投料计算结果S-2-步骤-6 FW 个数 控制组 总数 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号“交叉重叠”多肽第七步时使用试剂投料计算结果S-2-步骤-7 FW 个数 控制组总数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号“交叉重叠”多肽第八步时使用试剂投料计算结果S-2-步骤-8 FW 个数 控制组 总数 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号“交叉重叠”多肽第九步时使用试剂投料计算结果S-2-步骤-9 FW 个数 控制组 总数 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号“交叉重叠”多肽第十步时使用试剂投料计算结果S-2-步骤-10 FW 个数 控制组 总数 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号合成操作步骤多肽脱Fmoc保护/氨基酸残基的连接步骤1.均分树脂采用IRORI均分树脂装置，在每一个Microkan中(共646个)加入15mg Rink Amide树脂；2.均分IRORI射频子采用IRORI均分射频子装置，在每一个Microkan中加入一个射频子，并用IRORI加盖器加盖；3.脱Fmoc保护集中所有待反应的Microkan，置于一个2L锥形瓶中，加入20％哌啶/DMF振摇，15min×2次；4.洗涤树脂DMF 3min×6，DCM 3min×3，晾干；5.IRORI射频子编码(或解码)采用IRORI Sorting程序及相应配件编码。按照多肽化学库的序列对射频子编码(或解码)，每一特定编码代表一个特定序列，并以此将Microkan分配至不同氨基酸反应瓶中；6.洗涤树脂用重蒸DMF润洗待反应的树脂；7.Fmoc-保护氨基酸的连接准确称取计算量的Fmoc保护氨基酸、HOBt、HBTU(或DIC或BOP)，并用重蒸DMF溶解。将Microkan加入特定氨基酸反应瓶中，振摇反应3h；8.茚三酮检测取事先设定好的对照组序列，用茚三酮法检测反应情况。如果呈阴性反应，表明反应完全，进行步骤9的操作；如果呈阳性反应，表明反应不完全，需继续反应；如果延长时间或改用缩合能力更强的缩合剂后，仍不能完全反应，则采用步骤11的方法封闭后，进行步骤9的操作；9.洗涤树脂DMF 3min×3，DCM 3min×1；10.脱Fmoc保护/连接根据序列要求，重复步骤3-9的操作，直至反应结束；11.封闭采用乙酰化多肽的氨基端，15％Ac2O/CH2Cl2，30min；12.洗涤树脂DCM 3min×2，晾干。
多肽裂解步骤1.配制裂解液根据序列特点，从选取并配制适当的裂解液；2.裂解每一个Microkan使用1mL裂解液在室温下裂解2h；3.洗涤再用1mL裂解液浸泡洗涤Microkan 5min；4.晾干或离心干燥合并两次裂解液，离心浓缩至少量；
5.沉淀将事先用冰水浴充分冷却的甲基叔丁基醚/石油醚(v/v3/1)加入到残余物中，放入冰水浴中冷却20min使析晶完全；6.离心离心沉淀多肽化合物(3000rpm以上)，10min后取出，小心倾去上清夜；7.洗涤多肽加入冷却的甲基叔丁基醚/石油醚，充分洗涤后，离心，倾去上清夜；8.重复步骤7的操作一次；9.晾干将多肽残余物在室温下挥干，直至完全干燥；10.检测抽检部分多肽样品，用10％HOAc/H2O或30％CH3CN/H2O或60％CH3CN/H2O溶解，并用HPLC-MS检测纯度。
实施例3设计合成Membrane(M)蛋白、Envelop(E)蛋白和Nucleocapside(N)蛋白全部氨基酸序列的每10位氨基酸“交叉重叠”序列如下。从右至左为合成次序，代表合成步骤号码为阿拉伯数字1到10。
M-蛋白表25 所合成的M-蛋白212个以10个氨基酸为长度的序列编号 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个以10个氨基酸为长度的序列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个以10个氨基酸为长度的序列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为上述1～692号“交叉重叠”多肽第1-10步使用的试剂投料
表28合成上述1～692号“交叉重叠”多肽第一步时使用试剂投料计算结果MEN-步骤-1 FW 个数 控制组 总数 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号“交叉重叠”多肽第二步时使用试剂投料计算结果MEN-步骤-2 FW 个数 控制组 总数 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号“交叉重叠”多肽第三步时使用试剂投料计算结果MEN-步骤-3 FW 个数 控制组 总数 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号“交叉重叠”多肽第四步时使用试剂投料计算结果MEN-步骤-4 FW 个数 控制组 总数 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号“交叉重叠”多肽第五步时使用试剂投料计算结果MEN-步骤-5 FW 个数 控制组 总数 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号“交叉重叠”多肽第六步时使用试剂投料计算结果MEN-步骤-6FW 个数 控制组 总数 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号“交叉重叠”多肽第七步时使用试剂投料计算结果MEN-步骤-7 FW 个数 控制组 总数 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号“交叉重叠”多肽第八步时使用试剂投料计算结果MEN-步骤-8 FW 个数控制组 总数 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号“交叉重叠”多肽第九步时使用试剂投料计算结果MEN-步骤-9 FW 个数 控制组 总数 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号“交叉重叠”多肽第十步时使用试剂投料计算结果MEN-步骤-10 FW 个数 控制组 总数 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号“交叉重叠”多肽第一步时使用试剂投料合成操作步骤多肽脱Fmoc保护/氨基酶残基的连接步骤1.均分树脂采用IRORI均分树脂装置，在每一个Microkan中(共692个)加入15mg Rink Amide树脂；2.均分IRORI射频子采用IRORI均分射频子装置，在每一个Microkan中加入一个射频子，并用IRORI加盖器加盖；3.脱Fmoc保护集中所有待反应的Microkan，置于一个2L锥形瓶中，加入20％哌啶/DMF振摇，15min×2次；4.洗涤树脂DMF 3min×6，DCM 3min×3，晾干；5.IRORI射频子编码(或解码)采用IRORI Sorting程序及相应配件编码。按照多肽化学库的序列对射频子编码(或解码)，每一特定编码代表一个特定序列，并以此将Microkan分配至不同氨基酸反应瓶中；6.洗涤树脂用重蒸DMF润洗待反应的树脂；7.Fmoc-保护氨基酸的连接准确称取计算量的Fmoc保护氨基酸、HOBt、HBTU(或DIC或BOP)，并用重蒸DMF溶解。将Microkan加入特定氨基酸反应瓶中，振摇反应3h；8.茚三酮检测取事先设定好的对照组序列，用茚三酮法检测反应情况。如果呈阴性反应，表明反应完全，进行步骤9的操作；如果呈阳性反应，表明反应不完全，需继续反应；如果延长时间或改用缩合能力更强的缩合剂后，仍不能完全反应，则采用步骤11的方法封闭后，进行步骤9的操作；9.洗涤树脂DMF 3min×3，DCM 3min×1；10.脱Fmoc保护/连接根据序列要求，重复步骤3-9的操作，直至反应结束；11.封闭采用乙酰化多肽的氨基端，15％ Ac2O/CH2Cl2，30min；12.洗涤树脂DCM 3min×2，晾干。
多肽裂解步骤1.配制裂解液根据序列特点，从选取并配制适当的裂解液；2.裂解每一个Microkan使用1mL裂解液在室温下裂解2h；3.洗涤再用1mL裂解液浸泡洗涤Microkan 5min；4.晾干或离心干燥合并两次裂解液，离心浓缩至少量；5.沉淀将事先用冰水浴充分冷却的甲基叔丁基醚/石油醚(v/v3/1)加入到残余物中，放入冰水浴中冷却20min使析晶完全；6.离心离心沉淀多肽化合物(3000rpm以上)，10min后取出，小心倾去上清夜；7.洗涤多肽加入冷却的甲基叔丁基醚/石油醚，充分洗涤后，离心，倾去上清夜；
8.重复步骤7的操作一次；9.晾干将多肽残余物在室温下挥干，直至完全干燥；10.检测抽检部分多肽样品，用10％HOAc/H2O或30％CH3CN/H2O或60％CH3CN/H2O溶解，并用HPLC-MS检测纯度。
实施例4采用此法合成非“交叉重复”多肽化合物。
采用本发明的方法除了能合成如实施例1～3所述的具有10个氨基酸序列长度的氨基酸残基序列，还能合成长度为2～50个氨基酸的氨基酸片段，应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以下本实施例多肽的制备步骤1.加树脂及射频子选取9个Microkan并分别装入15毫克Rink树脂和射频子并盖紧盖子；2.合并Microkan，在一个适当的容器中用20％哌啶/DMF脱Fmoc保护基15min×2；3.洗涤树脂DMF 3min×6，DCM 3min×3，重蒸DMF 3min×3；4.IRORI射频子编码(或解码)采用IRORI Sorting程序，按照多肽编码次序将Microkan分配至不同氨基酸反应瓶中；5.在每一个反应瓶中加入溶解于DMF中计算量的Fmoc-保护氨基酸、HOBt、HBTU，振摇反应3h；6.合并Microkan并洗涤树脂DMF 3min×3次；7.重复步骤3～7，直至连接全部氨基酸；8.合并Microkan，重复3～4步；9.采用15％Ac2O/CH2Cl2乙酰化多肽的氨基端15min；10.用CH2Cl23min×3次后，室温晾干；11.采用IRORI Sorting解码Microkan并分配到对应的15毫升裂解管中；
12.每一个Microkan用1mL在室温下裂解液裂解2h；13.再用1mL裂解液浸泡洗涤Microkan 5min；14.合并裂解液和洗涤液；15.惰性气体流吹干浓缩至残留少量裂解液；16.加入事先用冰水浴充分冷却的甲基叔丁基醚/石油醚(v/v3/1)，放入冰水浴中冷却20min；17.离心沉淀多肽化合物(3000rpm以上)，10min后小心倾去上清夜；18.再加入冷却的甲基叔丁基醚/石油醚，充分洗涤、离心，倾去上清夜，共两次；19.将多肽残余物置于室温下，直至完全干燥；20.将多肽化合物用10％HOAc/H2O或30％CH3CN/H2O或60％CH3CN/H2O溶解后，经HPLC-MS检测纯度和正确的分子量。
实施例5ELISA法筛选发现抗SARS-CoV阳性血清的抗原决定簇(B-细胞抗原决定簇)将实施例1、2和3获得的分子多样性化合物库与SARS-CoV阳性血清反应，由于SARS-CoV阳性血清中含有SARS-CoV抗体，测定其与分子多样性化合物库中的具有10个氨基酸序列的短肽结合活性，由此测定多重最小多肽抗原决定簇，其具体步骤如下1、包被包被液为0.07M NaHCO3，0.03M Na2CO3(pH＝9.6)，包被浓度10μg/mL，100μL/孔，4℃过夜或37℃2小时。
2、清洗PBST(pH＝7.4，含0.05％的Tween 20)清洗5遍，拍干。
3、封闭封闭液为1～3％的BSA，200μL/孔，4℃过夜或37℃2小时。
4、清洗PBST(pH＝7.4，含0.05％的Tween 20)清洗5遍，拍干。
5、加入一抗病人血清用生理盐水1∶100稀释，100μL/孔，37℃孵育2小时。
6、清洗PBST(pH＝7.4，含0.05％的Tween 20)清洗5遍，拍干。
7、加入二抗HRP-兔抗人IgG抗体用PBS1∶1000～1∶3000稀释，100μL/孔，37℃孵育2小时。
8、清洗PBST(pH＝7.4，含0.05％的Tween 20)清洗5遍，拍干。
9、加入酶反应底物酶反应底物为TMB，100μL/孔，37℃孵育15～30分钟。
10、加终止液2MH2SO4，100μL/孔，37℃孵育5～10分钟。
11、读数在酶标仪中测量450nm和630nm的OD值。
12、中和实验筛选得到的阳性多肽化合物与抗SARA-CoV阳性血清混合物在37℃孵育30分钟后重复上述ELISA实验步骤。
13、对照及数据处理每一个多肽样品取三个抗SARA-CoV阳性血清反应复孔，和三个正常对照血清复孔进行对比实验。三个正常对照血清复孔的OD平均值控制在0.1以下。三个抗SARA-CoV阳性血清反应复孔OD值的平均值减去三个正常对照血清复孔的OD平均值即为最终的结果。阳性多肽化合物经过中和实验确认特异性结果。图4是20个代表性多肽化合物的筛选结果。它们的序列结构如下11)TSGSDLDRCT12) SGSDLDRCTT13)GSDLDRCTTF14) SDLDRCTTFD15)DLDRCTTFDD16) LDRCTTFDDV17)DRCTTFDDVQ18) RCTTFDDVQA19)CTTFDDVQAP20) TTFDDVQAPN21)TFDDVQAPNY22) FDDVQAPNYT23)DDVQAPNYTQ24) DVQAPNYTQH25)VQAPNYTQHT26) QAPNYTQHTS27) APNYTQHTSS28) PNYTQHTSSM29) NYTQHTSSMR30) YTQHTSSMRG由图4可以看出，在S蛋白中有101个能与SARS-CoV阳性血清结合的多肽序列，分别是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)由图5可以看出，在M蛋白中有2个能与SARS-CoV阳性血清结合的多肽序列，分别是筛选得到的M蛋白线性抗原决定簇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个能与SARS-CoV阳性血清结合的多肽序列，分别是筛选得到的E蛋白线性抗原决定簇1.L T A L R L C A Y C(Compd 246)
在N蛋白中有8个能与SARS-CoV阳性血清结合的多肽序列，分别是筛选得到的N蛋白线性抗原决定簇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个序列即为本发明所筛选的SARS-CoV的抗原决定簇。
序列表<110>中国医学科学院药物研究所<120>一种肽文库、其合成方法及从该文库中筛选的活性片段<130>
<160>116<170>PatentIn version 3.1<210>1<211>10<212>PRT<213>SARS-CoV<400>1Met Phe Ile Phe Leu Leu Phe Leu Thr Leu1 5 10<210>2<211>10<212>PRT<213>SARS-CoV<400>2Thr Ser Gly Ser Asp Leu Asp Arg Cys Thr1 5 10<210>3<211>10<212>PRT<213>SARS-CoV<400>3Ser Gly Ser Asp Leu Asp Arg Cys Thr Thr1 5 10
<210>4<211>10<212>PRT<213>SARS-CoV<400>4Ser Asp Leu Asp Arg Cys Thr Thr Phe Asp1 5 10<210>5<211>10<212>PRT<213>SARS-CoV<400>5Thr Thr Phe Asp Asp Val Gln Ala Pro Asn1 5 10<210>6<211>10<212>PRT<213>SARS-CoV<400>6Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr1 5 10<210>7<211>10<212>PRT<213>SARS-CoV<400>7Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu1 5 10
<210>8<211>10<212>PRT<213>SARS-CoV<400>8Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr1 5 10<210>9<211>10<212>PRT<213>SARS-CoV<400>9Asn Ser Thr Asn Val Val Ile Arg Ala Cys1 5 10<210>10<211>10<212>PRT<213>SARS-CoV<400>10Ser Thr Asn Val Val Ile Arg Ala Cys Asn1 5 10<210>11<211>10<212>PRT<213>SARS-CoV<400>11Val Ser Lys Pro Met Gly Thr Gln Thr His1 5 10
<210>12<211>10<212>PRT<213>SARS-CoV<400>12Ser Lys Pro Met Gly Thr Gln Thr His Thr1 5 10<210>13<211>10<212>PRT<213>SARS-CoV<400>13Pro Met Gly Thr Gln Thr His Thr Met Ile1 5 10<210>14<211>10<212>PRT<213>SARS-CoV<400>14Met Ile Phe Asp Asn Ala Phe Asn Cys Thr1 5 10<210>15<211>10<212>PRT<213>SARS-CoV<400>15Asp Asn Ala Phe Asn Cys Thr Phe Glu Tyr1 5 10
<210>16<211>10<212>PRT<213>SARS-CoV<400>16Lys Ser Gly Asn Phe Lys His Leu Arg Glu1 5 10<210>17<211>10<212>PRT<213>SARS-CoV<400>17Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr1 5 10<210>18<211>10<212>PRT<213>SARS-CoV<400>18Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly1 5 10<210>19<211>10<212>PRT<213>SARS-CoV<400>19Lys Gly Tyr Gln Pro Ile Asp Val Val Arg1 5 10
<210>20<211>10<212>PRT<213>SARS-CoV<400>20Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr1 5 10<210>21<211>10<212>PRT<213>SARS-CoV<400>21Ile Asn Ile Thr Asn Phe Arg Ala Ile Leu1 5 10<210>22<211>10<212>PRT<213>SARS-CoV<400>22Val Asp Cys Ser Gln Asn Pro Leu Ala Glu1 5 10<210>23<211>10<212>PRT<213>SARS-CoV<400>23Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val1 5 10
<210>24<211>10<212>PRT<213>SARS-CoV<400>24Tyr Gln Thr Ser Asn Phe Arg Val Val Pro1 5 10<210>25<211>10<212>PRT<213>SARS-CoV<400>25Ile Ser Asn Cys Val Ala Asp Tyr Ser Val1 5 10<210>26<211>10<212>PRT<213>SARS-CoV<400>26Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr1 5 10<210>27<211>10<212>PRT<213>SARS-CoV<400>27Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe1 5 10
<210>28<211>10<212>PRT<213>SARS-CoV<400>28Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe1 5 10<210>29<211>10<212>PRT<213>SARS-CoV<400>29Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser1 5 10<210>30<211>10<212>PRT<213>SARS-CoV<400>30Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys1 5 10<210>31<211>10<212>PRT<213>SARS-CoV<400>31Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly1 5 10
<210>32<211>10<212>PRT<213>SARS-CoV<400>32Gly Asp Asp Val Arg Gln Ile Ala Pro Gly1 5 10<210>33<211>10<212>PRT<213>SARS-CoV<400>33Asp Asp Val Arg Gln Ile Ala Pro Gly Gln1 5 10<210>34<211>10<212>PRT<213>SARS-CoV<400>34Val Arg Gln Ile Ala Pro Gly Gln Thr Gly1 5 10<210>35<211>10<212>PRT<213>SARS-CoV<400>35Phe Met Gly Cys Val Leu Ala Trp Asn Thr1 5 10
<210>36<211>10<212>PRT<213>SARS-CoV<400>36Val Leu Ala Trp Asn Thr Arg Asn Ile Asp1 5 10<210>37<211>10<212>PRT<213>SARS-CoV<400>37Thr Arg Asn Ile Asp Ala Thr Ser Thr Gly1 5 10<210>38<211>10<212>PRT<213>SARS-CoV<400>38Arg Asn Ile Asp Ala Thr Ser Thr Gly Asn1 5 10<210>39<211>10<212>PRT<213>SARS-CoV<400>39Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His1 5 10
<210>40<211>10<212>PRT<213>SARS-CoV<400>40Pro Phe Glu Arg Asp Ile Ser Asn Val Pro1 5 10<210>41<211>10<212>PRT<213>SARS-CoV<400>41Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly1 5 10<210>42<211>10<212>PRT<213>SARS-CoV<400>42Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr1 5 10<210>43<211>10<212>PRT<213>SARS-CoV<400>43Tyr Gln Pro Tyr Arg Val Val Val Leu Ser1 5 10
<210>44<211>10<212>PRT<213>SARS-CoV<400>44Tyr Arg Val Val Val Leu Ser Phe Glu Leu1 5 10<210>45<211>10<212>PRT<213>SARS-CoV<400>45Asn Gln Cys Val Asn Phe Asn Phe Asn Gly1 5 10<210>46<211>10<212>PRT<213>SARS-CoV<400>46Gln Cys Val Asn Phe Asn Phe Asn Gly Leu1 5 10<210>47<211>10<212>PRT<213>SARS-CoV<400>47Cys Val Asn Phe Asn Phe Asn Gly Leu Thr1 5 10
<210>48<211>10<212>PRT<213>SARS-CoV<400>48Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr1 5 10<210>49<211>10<212>PRT<213>SARS-CoV<400>49Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly1 5 10<210>50<211>10<212>PRT<213>SARS-CoV<400>50Asn Phe Asn Gly Leu Thr Gly Thr Gly Val1 5 10<210>51<211>10<212>PRT<213>SARS-CoV<400>51Thr Asp Val Ser Thr Ala Ile His Ala Asp1 5 10
<210>52<211>10<212>PRT<213>SARS-CoV<400>52Asp Val Ser Thr Ala Ile His Ala Asp Gln1 5 10<210>53<211>10<212>PRT<213>SARS-CoV<400>53Val Ser Thr Ala Ile His Ala Asp Gln Leu1 5 10<210>54<211>10<212>PRT<213>SARS-CoV<400>54Thr Pro Ala Trp Arg Ile Tyr Ser Thr Gly1 5 10<210>55<211>10<212>PRT<213>SARS-CoV<400>55Ile Gly Ala Glu His Val Asp Thr Ser Tyr1 5 10
<210>56<211>10<212>PRT<213>SARS-CoV<400>56Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala1 5 10<210>57<211>10<212>PRT<213>SARS-CoV<400>57Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile1 5 10<210>58<211>10<212>PRT<213>SARS-CoV<400>58Ile Thr Thr Glu Val Met Pro Val Ser Met1 5 10<210>59<211>10<212>PRT<213>SARS-CoV<400>59Ala Lys Thr Ser Val Asp Cys Asn Met Tyr1 5 10
<210>60<211>10<212>PRT<213>SARS-CoV<400>60Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala1 5 10<210>61<211>10<212>PRT<213>SARS-CoV<400>61Ala Gln Lys Phe Asn Gly Leu Thr Val Leu1 5 10<210>62<211>10<212>PRT<213>SARS-CoV<400>62Leu Thr Val Leu Pro Pro Leu Leu Thr Asp1 5 10<210>63<211>10<212>PRT<213>SARS-CoV<400>63Thr Val Leu Pro Pro Leu Leu Thr Asp Asp1 5 10
<210>64<211>10<212>PRT<213>SARS-CoV<400>64Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala1 5 10<210>65<211>10<212>PRT<213>SARS-CoV<400>65Thr Ala Leu Gly Lys Leu Gln Asp Val Val1 5 10<210>66<211>10<212>PRT<213>SARS-CoV<400>66Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile1 5 10<210>67<211>10<212>PRT<213>SARS-CoV<400>67Asn Phe Gly Ala Ile Ser Ser Val Leu Asn1 5 10
<210>68<211>10<212>PRT<213>SARS-CoV<400>68Phe Gly Ala Ile Ser Ser Val Leu Asn Asp1 5 10<210>69<211>10<212>PRT<213>SARS-CoV<400>69Gly Ala Ile Ser Ser Val Leu Asn Asp Ile1 5 10<210>70<211>10<212>PRT<213>SARS-CoV<400>70Ala Ile Ser Ser Val Leu Asn Asp Ile Leu1 5 10<210>71<211>10<212>PRT<213>SARS-CoV<400>71Arg Leu Asp Lys Val Glu Ala Glu Val Gln1 5 10
<210>72<211>10<212>PRT<213>SARS-CoV<400>72Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu1 5 10<210>73<211>10<212>PRT<213>SARS-CoV<400>73Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala1 5 10<210>74<211>10<212>PRT<213>SARS-CoV<400>74Ser Ala Asn Leu Ala Ala Thr Lys Met Ser1 5 10<210>75<211>10<212>PRT<213>SARS-CoV<400>75Val Pro Ser Gln Glu Arg Asn Phe Thr Thr1 5 10
<210>76<211>10<212>PRT<213>SARS-CoV<400>76Arg Glu Gly Val Phe Val Phe Asn Gly Thr1 5 10<210>77<211>10<212>PRT<213>SARS-CoV<400>77Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser1 5 10<210>78<211>10<212>PRT<213>SARS-CoV<400>78Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp1 5 10<210>79<211>10<212>PRT<213>SARS-CoV<400>79Ala Asn Leu Ala Ala Thr Lys Met Ser Glu1 5 10
<210>80<211>10<212>PRT<213>SARS-CoV<400>80Asn Leu Ala Ala Thr Lys Met Ser Glu Cys1 5 10<210>81<211>10<212>PRT<213>SARS-CoV<400>81Phe Lys Asn His Thr Ser Pro Asp Val Asp1 5 10<210>82<211>10<212>PRT<213>SARS-CoV<400>82Thr Ser Pro Asp Val Asp Leu Gly Asp Ile1 5 10<210>83<211>10<212>PRT<213>SARS-CoV<400>83Asp Val Asp Leu Gly Asp Ile Ser Gly Ile1 5 10
<210>84<211>10<212>PRT<213>SARS-CoV<400>84Val Asp Leu Gly Asp Ile Ser Gly Ile Asn1 5 10<210>85<211>10<212>PRT<213>SARS-CoV<400>85Asn Ala Ser Val Val Asn Ile Gln Lys Glu1 5 10<210>86<211>10<212>PRT<213>SARS-CoV<400>86Ala Ser Val Val Asn Ile Gln Lys Glu Ile1 5 10<210>87<211>10<212>PRT<213>SARS-CoV<400>87Ser Val Val Asn Ile Gln Lys Glu Ile Asp1 5 10
<210>88<211>10<212>PRT<213>SARS-CoV<400>88Lys Glu Ile Asp Arg Leu Asn Glu Val Ala1 5 10<210>89<211>10<212>PRT<213>SARS-CoV<400>89Glu Ile Asp Arg Leu Asn Glu Val Ala Lys1 5 10<210>90<211>10<212>PRT<213>SARS-CoV<400>90Leu Asn Glu Val Ala Lys Asn Leu Asn Glu1 5 10<210>91<211>10<212>PRT<213>SARS-CoV<400>91Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln1 5 10
<210>92<211>10<212>PRT<213>SARS-CoV<400>92Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr1 5 10<210>93<211>10<212>PRT<213>SARS-CoV<400>93Val Met Val Thr Ile Leu Leu Cys Cys Met1 5 10<210>94<211>10<212>PRT<213>SARS-CoV<400>94Met Val Thr Ile Leu Leu Cys Cys Met Thr1 5 10<210>95<211>10<212>PRT<213>SARS-CoV<400>95Val Thr Ile Leu Leu Cys Cys Met Thr Ser1 5 10
<210>96<211>10<212>PRT<213>SARS-CoV<400>96Thr Ile Leu Leu Cys Cys Met Thr Ser Cys1 5 10<210>97<211>10<212>PRT<213>SARS-CoV<400>97Cys Ser Cys Leu Lys Gly Ala Cys Ser Cys1 5 10<210>98<211>10<212>PRT<213>SARS-CoV<400>98Asp Asp Ser Glu Pro Val Leu Lys Gly Val1 5 10<210>99<211>10<212>PRT<213>SARS-CoV<400>99Asp Ser Glu Pro Val Leu Lys Gly Val Lys1 5 10
<210>100<211>10<212>PRT<213>SARS-CoV<400>100Ser Glu Pro Val Leu Lys Gly Val Lys Leu1 5 10<210>101<211>10<212>PRT<213>SARS-CoV<400>101Ala Trp Ile Met Leu Leu Gln Phe Ala Tyr1 5 10<210>102<211>10<212>PRT<213>SARS-CoV<400>102Ser Asn Arg Asn Arg Phe Leu Tyr Ile Ile1 5 10<210>103<211>10<212>PRT<213>SARS-CoV<400>103Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys1 5 10
<210>104<211>10<212>PRT<213>SARS-CoV<400>104Gly Arg Asn Gly Ala Arg Pro Lys Gln Arg1 5 10<210>105<211>10<212>PRT<213>SARS-CoV<400>105Lys Glu Glu Leu Arg Phe Pro Arg Gly Gln1 5 10<210>106<211>10<212>PRT<213>SARS-CoV<400>106Ser Arg Gly Asn Ser Pro Ala Arg Met Ala1 5 10<210>107<211>10<212>PRT<213>SARS-CoV<400>107Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg1 5 10
<210>108<211>10<212>PRT<213>SARS-CoV<400>108Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr1 5 10<210>109<211>10<212>PRT<213>SARS-CoV<400>109Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys1 5 10<210>110<211>10<212>PRT<213>SARS-CoV<400>110Ala Gln Pro Leu Pro Gln Arg Gln Lys Lys1 5 10<210>111<211>10<212>PRT<213>SARS-CoV<400>111Gln Pro Leu Pro Gln Arg Gln Lys Lys Gln1 5 10
<210>112<211>10<212>PRT<213>SARS-CoV<400>112Gln Pro Tyr Arg Val Val Val Leu Ser Phe1 5 10<210>113<211>1255<212>PRT<213>SARS-CoV<400>113Met 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 Thr12501255<210>114<211>221<212>PRT<213>SARS-CoV<400>114Met 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 215 220<210>115<211>76<212>PRT<213>SARS-CoV<400>115Met 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 75<210>116<211>422<212>PRT<213>SARS-CoV<400>116Met 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
权利要求
1.一种肽文库，其中每一个肽的通式为Z-XnXn+1Xn+2Xn+3Xn+4Xn+5Xn+6Xn+7Xn+8Xn+9-Y其中X代表任何一种L-构型的天然氨基酸残基，n是指第Xn氨基酸在蛋白质序列中的天然排列位置，n为整数，Y代表NH2或者OH，Z代表氢或者Ac，其中Ac为乙酰基。
2.根据权利要求1所述的肽文库，其特征在于所述的蛋白质序列为与人类相关的冠状病毒SARS-CoV相关的S、M、E和N蛋白的多肽序列。
3.权利要求2所述的氨基酸序列，其特征在于所述的与人类相关的冠状病毒SARS-CoV为SARS-CoVBJ01号、SARS-CoVBJ02号、SARS-CoVBJ03号、SARS-CoVBJ04号、SARS-CoVCUHK-W1、SARS-CoVGZ01、SARS-CoVHKU-39849、SARS-CoVTaiwan、SARS-CoVTor2、SARS-CoVUrbani、SARS-CoVVietnam。
4.权利要求2或3所述的氨基酸序列，其特征在于所述的与人类相关的冠状病毒SARS-CoV为SARS-CoVBJ01号。
5.一种选自如SEQ ID NO1至SEQ ID NO101所示的氨基酸序列的任意一种或其结合的氨基酸序列。
6.一种如SEQ ID NO102至103所示的氨基酸序列的任意一种或其结合的氨基酸序列。
7.一种如SEQ ID NO104所示的氨基酸序列。
8.一种如SEQ ID NO105至112所示的氨基酸序列的任意一种或其结合的氨基酸序列。
9.具有抗SARS-CoV阳性血清抗原决定簇(B-细胞抗原决定簇)、T-细胞抗原决定簇、以及由此发现B-细胞抗原决定簇和T-细胞抗原决定簇作用的、含有SEQ ID NO1至SEQ ID NO112任意一种或其结合的共价或者非共价化合物。
10.含有如SEQ ID NO1至112所示的氨基酸序列的任意一种或其结合的氨基酸序列在用于制备治疗SARS-CoV引起的疾病的药物中的应用。
11.通过封闭SEQ ID NO1至SEQ ID NO112所示的氨基酸序列从而阻止SARS-CoV病毒进入宿主的阻断剂。
12.一种采用“混-分”物理编码法合成含有至少一个具有2～50个氨基酸残基的肽文库的方法，其包括以下步骤1)选择合适的射频子编码确定氨基酸序列的多肽片段；2)混合并对多肽母链氨基端进行脱Fmoc保护；3)混合并进行充分的洗涤；4)解码并按所要连接的特定氨基酸进行分组；5)将特定氨基酸以3～10倍摩尔过量的形式加入到已经分组的反应瓶中，进而连接到步骤2)的多肽母链；以及6)重复步骤2)和5)直至达到所需长度的片段；7)最后，进行解码并将多肽化合物从树脂上裂解下来。
13.根据权利要求9所述的方法，其特征在于所述的氨基酸残基长度为10-34。
14.根据权利要求9所述的方法，其特征在于所述的保护为Boc/Bzl保护。
全文摘要
本发明涉及一种肽文库，其中每一个肽的通式为Z－X
文档编号A61P11/00GK1609119SQ20031010189
公开日2005年4月27日 申请日期2003年10月23日 优先权日2003年10月23日
发明者刘刚, 何维, 李莉, 崔莲仙, 寇宾宾, 胡洪波, 王占国, 郝志勇, 张亮, 李旭琴, 倪安平, 刘勇, 孙曼霁, 李前 申请人:中国医学科学院药物研究所