CN111533790A

CN111533790A - 基于马赛克策略的冠状病毒抗原的构建方法及其应用

Info

Publication number: CN111533790A
Application number: CN202010387460.XA
Authority: CN
Inventors: 孙彩军; 杜向军; 舒跃龙; 李敏超; 曾金锋
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2020-08-14

Abstract

本发明提供了基于马赛克策略的冠状病毒抗原的构建方法及其应用，所述方法包括：(1)对人冠状病毒的天然结构蛋白进行分析，获取天然结构蛋白上存在的细胞毒性T淋巴细胞表位；(2)采用马赛克策略对人冠状病毒的结构蛋白进行分析，去除在天然结构蛋白中出现次数低的罕见表位后，获得由短肽组装形成的冠状病毒抗原。本发明基于人工智能算法对所有已知的CoV基因组序列进行大数据分析，基于马赛克策略设计新型抗原序列；新型抗原覆盖了所有已知的7种HCoVs上的天然S、M、N、E蛋白的大部分CTL表位，并与天然蛋白具有很好的结构相似性，在制备冠状病毒通用疫苗方面具有潜在的应用前景和价值。

Description

基于马赛克策略的冠状病毒抗原的构建方法及其应用

技术领域

本发明属于生物医药技术领域，涉及基于马赛克策略的冠状病毒抗原的构建方法及其应用，尤其涉及基于马赛克策略的冠状病毒抗原的构建方法、由此构建的涵盖大量保守T细胞表位的冠状病毒抗原及其在制备冠状病毒通用疫苗中的应用。

背景技术

目前，研究人冠状病毒(human coronavirus，HCoV)疫苗的方法主要包括灭活疫苗、减毒活疫苗、亚单位疫苗、DNA疫苗、mRNA疫苗、重组载体疫苗和联合疫苗等。但是考虑到安全性和有效性等因素，尚没有针对HCoV的疫苗产品。

在以往的冠状病毒疫苗研制过程中，往往忽略了T细胞免疫应答的重要性。T细胞免疫应答是抵抗病毒感染的关键因素，对控制流感病毒、艾滋病毒具有重要作用。在SARS、MERS 和COVID-19患者的临床病理解剖中发现，多个免疫组织器官中的T淋巴细胞数量大幅降低，而且CTL反应强度与疾病的严重程度呈负相关，这提示抗原特异性的T细胞免疫应答对于识别和清除冠状病毒感染细胞非常重要。此外，有研究显示，抗原特异性的T细胞反应在 SARS幸存者中可以长达11年之久。已有研究证明针对免疫优势表位的特异性记忆CD8+T细胞反应可以有效保护小鼠免受SARS-CoV感染。另一项研究表明，CD4+T细胞耗竭可以导致肺部炎症病理的增强和SARS-CoV从肺内清除的延迟等，因此CD4+T细胞在控制疾病发生和SARS-CoV复制中也起到重要作用。相比于抗体应答，诱导T细胞免疫应答的抗原表位更保守，因此T细胞免疫应答的交叉反应能力更强。

除了S蛋白，E蛋白和M蛋白在病毒装配中起着重要作用，M蛋白是SARS病毒中含量最丰富的蛋白，包含大量的B细胞和CTL抗原表位，N蛋白是病毒RNA合成所必需的，序列保守性强，免疫原性强，可以诱发强烈的特异性抗体和T细胞反应。研究发现，S蛋白、 N蛋白、E蛋白、M蛋白和3a蛋白都包含大量的CTL抗原表位，例如免疫SARS-CoV N蛋白后，试验动物的呼吸道中诱导产生了与其他冠状病毒N抗原表位有交叉反应的记忆性T细胞，在遇到同源或异源病毒攻击时可以快速发挥保护作用。因此，针对S蛋白、N蛋白、M 蛋白和E蛋白等抗原的T细胞免疫应答有望成为研发冠状病毒疫苗的重要考虑方向。

发明内容

针对现有技术的不足和实际需求，本发明提供了基于马赛克策略的冠状病毒抗原的构建方法及其应用，所述方法通过对现有已知的所有CoV基因组序列进行大数据分析，利用马赛克策略(Mosaic Strategy)设计得到能最大程度覆盖保守的T细胞表位的新型抗原序列。

为达此目的，本发明采用以下技术方案：

第一方面，本发明提供了基于马赛克策略的冠状病毒抗原的构建方法，所述方法包括：

(1)对人冠状病毒的结构蛋白进行分析，获取结构蛋白上存在的细胞毒性T淋巴细胞表位；

(2)采用马赛克策略对人冠状病毒的结构蛋白进行分析，去除在天然结构蛋白中出现次数低的罕见表位后，获得由短肽组装形成的冠状病毒抗原。

疫苗的免疫效果受到抗原设计、递送系统、免疫途径和佐剂选择等多种因素的综合影响，其中抗原设计至关重要；本发明从多个角度对抗原靶标进行选择、设计、改造和优化，基于人工智能算法对所有已知的CoV基因组序列进行大数据分析，利用马赛克策略设计出能最大程度覆盖保守的T细胞表位的新型抗原序列。

优选地，所述人冠状病毒包括HCoV-NL63、HCoV-229e、HCoV-OC43、HCoV-HKU1、SARS-CoV、MERS-CoV或SARS-CoV-2中的任意一种或至少两种的组合，优选为HCoV-NL63、HCoV-229e、HCoV-OC43、HCoV-HKU1、SARS-CoV、MERS-CoV和SARS-CoV-2的组合。

优选地，所述结构蛋白包括S蛋白(spike-surface glycoprotein)、M蛋白(matrixprotein)、 N蛋白(nucleocapsid protein)或E蛋白(small envelope protein)中的任意一种或至少两种的组合，优选为S蛋白、M蛋白、N蛋白和E蛋白的组合。

优选地，所述S蛋白上包括221～228个细胞毒性T淋巴细胞表位。

优选地，所述M蛋白上包括51～61个细胞毒性T淋巴细胞表位。

优选地，所述N蛋白上包括54～65个细胞毒性T淋巴细胞表位。

优选地，所述E蛋白上包括14～21个细胞毒性T淋巴细胞表位。

本发明中，冠状病毒的四组结构蛋白上均分布有细胞毒性T淋巴细胞表位(CTL)，说明T细胞免疫应答具有抵抗冠状病毒感染的作用。

优选地，所述马赛克策略包括Mosaic Vaccine Designer程序。

根据本发明，采用马赛克策略设计抗原，可以最大程度覆盖存在于天然抗原上的T细胞表位，获得的抗原由来源于天然序列的短肽组装形成。

优选地，所述罕见表位在结构蛋白中出现的次数小于3。

本发明中，在天然结构蛋白的CTL表位中出现次数低于3的罕见表位由于可能削弱表位的免疫应答，故在设计新型抗原时需要去除。

优选地，所述短肽的长度为8～10aa，例如可以是8aa、9aa或10aa，优选为9aa。

根据本发明，由于常规的CTL表位的长度为9个氨基酸(aa)，因此筛选长度为8～10aa、优选为9aa的短肽组装新型抗原。

优选地，所述冠状病毒抗原包括S蛋白马赛克抗原、M蛋白马赛克抗原、N蛋白马赛克抗原或E蛋白马赛克抗原中的任意一种或至少两种的组合，优选为S蛋白马赛克抗原、M蛋白马赛克抗原、N蛋白马赛克抗原和E蛋白马赛克抗原的组合。

第二方面，本发明提供了冠状病毒抗原，所述冠状病毒抗原采用第一方面所述的方法构建得到。

优选地，所述S蛋白马赛克抗原中含有的天然细胞毒性T淋巴细胞表位的百分比为89.01％。

优选地，所述M蛋白马赛克抗原中含有的天然细胞毒性T淋巴细胞表位的百分比为87.77％。

优选地，所述N蛋白马赛克抗原中含有的天然细胞毒性T淋巴细胞表位的百分比为91.96％。

优选地，所述E蛋白马赛克抗原中含有的天然细胞毒性T淋巴细胞表位的百分比为92.86％。

本发明中，设计的冠状病毒抗原覆盖了所有已知的7种HCoVs上的天然S、M、N、E 蛋白的大部分CTL表位，并与天然蛋白具有很好的结构相似性，有望用于制备冠状病毒通用疫苗。

优选地，所述S蛋白马赛克抗原包括SEQ ID NO:1～4所示的氨基酸序列；

SEQ ID NO:1：

MFLILLISLPTAFAVIGDLNCPLDPRLKGSFNNRDTGPPSISTDTVDVTNGLGTYYVLDR VYLNTTLFLNGYYPTSGSTYRNMALKGTDKLSTLWFKPPFLSDFINGIFAKVKNTKVFKDG VMYSEFPAITIGSTFVNTSYSVVVQPRTINSTQDGVNKLQGLLEVSVCQYNMCEYPHTICHP KLGNHFKELWHLDTGVVSCLYKRNFTYDVNATYLYFHFYQEGGTFYAYFTDTGFVTKFLF NVYLGMALSHYYVMPLTCISRRDIGFTLEYWVTPLTPRQYLLAFNQDGIIFNAVDCMSDFM SEIKCKTQSIAPPTGVYELNGYTVQPIADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKT FSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNRRKVDLQLGNLGYLQ SSNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNKRFGFIEDSVFVPQPTGVFTNHSVVYA QHCFKAPKNFCPCSSCPGKNNGIGTCPAGTNYLTCDNLCTLDPITFKAPDTYKCPQTKSLVG IGEHCSGLAVKSDYCGNNSCTCQPQAFLGWSADSCLQGDKCNIFANFILHDVNNGLTCSTD LQKANTEIELGVCVNYDLYGISGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYITNRTF MIHSCYSGRVSAAYHANSSEPALLFRNIKCNYVFNNSLTRQLQPINYSFDSYLGCVVNAYNS TAISVQTCDLTVGSGYCVDYSKNRRSRRAITTGYRFTNFEPFTVNSVNDSLEPVGGLYEIQIP SEFTIGNMEEFIQTSSPKVTIDCAAFVCGDYAACKLQLVEYGSFCDNINAILTEVNELLDTTQ LQVANSLMNGVTLSTKLKDGVNFNVDDINFSPVLGCLGSECSKASSRSAIEDLLFDKVKLS DVGFVEAYNNCTGGAEIRDLICVQSYKGIKVLPPLLSENQISGYTLAATSASLFPPWTAAAG VPFYLNVQYRINGLGVTMDVLSQNQKLIANAFNNALHAIQQGFDATNSALVKIQAVVNAN AEALNNLLQQLSNRFGAISASLQEILSRLDALEAEAQIDRLINGRLTALNAYVSQQLSDSTLV KFSAAQAMEKVNECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFNYVPTKYVTAKVSP GLCIAGNRGIAPKSGYFVNVNNTWMYTGSGYYYPEPITENNVVVMSTCAVNYTKAPYVM LNTSIPNLPDFKEELDQWFKNQTSVAPDLSLDYINVTFLDLQVEMNRLQEAIKVLNHSYINL KDIGTYEYYVKWPWYVWLLICLAGVAMLVLLFFICCCTGCGTSCFKKCGGCCDDYTGYQ ELVIKTSHDD；

SEQ ID NO:2：

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVN NATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEG KQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLAL HRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS FTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTG VLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASY QTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDC TMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPP LLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIA NQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDK VEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGY HLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQR NFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVT IMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT；

SEQ ID NO:3：

MKLFLILLVLPLASCFFTCNSNANLSMLQLGVPDNSSTIVTGLLPTHWICANQSTSVYS ANGFFYIDVGNHRSAFALHTGYYDVNQYYIYVTNEIGLNASVTLKICKFGINTTFDFLSNSS SSFDCIVNLLFTEQLGAPLGITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKCYFNYSCVFS VVNATVTVNVTTHNGRVVNYTVCDDCNGYTDNIFSVQQDGRIPNGFPFNNWFLLTNGSTL VDGVSRLYQPLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGYQHNSVADVMRYNLNFSA NSVDNLKSGVIVFKTLQYDVLFYCSNSSSGVLDTTIPFGPSSQPYYCFINSTINTTHVSTFVGVLPPTVREIVVARTGQFYINGFKYFDLGFIEAVNFNVTTASATDFWTVAFATFVDVLVNVSA TNIQNLLYCDSPFEKLQCEHLQFGLQDGFYSANFLDDNVLPETYVALPIYYQHTDINFTATA SFGGSCYVCKPHQVNISLNGNTSVCVRTSHFSIRYIYNRVKSGSPGDSSWHIYLKSGTCPFSF SKLNNFQKFKTICFSTVAVPGSCNFPLEATWHYTSYTIVGALYVTWSEGNSITGVPYPVSGI REFSNLVLNNCTKYNIYDYVGTGIIRSSNQSLAGGITYVSNSGNLLGFKNVSTGNIFIVTPCN QPDQVAVYQQSIIGAMTAVNESRYGLQNLLQLPNFYYVSNGGNNCTTAVMTYSNFGICAD GSLIPVRPRNSSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNL LKQYTSACKTIEDALRLSAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIHSS RIAGRSALEDLLFSKVVTSGLGTVDVDYKSCTKGLSIADLACAQYYNGIMVLPGVADAER MAMYTGSLIGGMVLGGLTSAAAIPFSLALQARLNYVALQTDVLQENQKILAASFNKAINNI VASFSSVNDAITQTAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQAISNSIQAIYDR LDSIQADQQVDRLITGRLAALNAFVSQVLNKYTEVRSSRRLAQQKINECVKSQSNRYGFC GNGTHIFSIVNSAPDGLLFLHTVLLPTDYKNVKAWSGICVDGIYGYVLRQPNLVLYSDNGV FRVTSRVMFQPRLPVLSDFVQIYNCNVTFVNISRVELHTVIPDYVDVNKTLQEFAQNLPKY VKPNFDLTPFNLTYLNLSSELKQLEAKTASLFQTTVELQGLIDQINSTYVDLKLLNRFENYIK WPWWVWLIISVVFVVLLSLLVFCCLSTGCCGCCNCLTSSMRGCCDCGSTKLPYYEFEKVH VQ；

SEQ ID NO:4：

MIHSVFLLMFLLTPTESYVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGIIYPQ GRTYSNITITYQGLFPYQGDHGDMYVYSAGHATGTTPQKLFVANYSQDVKQFANGFVVRI GAAANSTGTVIISPSTSATIRKIYPAFMLGSSVGNFSDGKMGRFFNHTLVLLPDGCGTLLRA FYCILEPRSGNHCPAGNSYTSFATYHTPATDCSDGNYNRNASLNSFKEYFNLRNCTFMYTY NITEDEILEWFGITQTAQGVHLFSSRYVDLYGGNMFQFATLPVYDTIKYYSIIPHSIRSIQSDR KAWAAFYVYKLQPLTFLLDFSVDGYIRRAIDCGFNDLSQLHCSYESFDVESGVYSVSSFEAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISP AAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKP LKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLV ASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEYSLYGVSGRG VFQNCTAVGVRQQRFVYDAYQNLVGYYSDDGNYYCLRACVSVPVSVIYDKETKTHATLF GSVACEHISSTMSQYSRSTRSMLKRRDSTYGPLQTPVGCVLGLVNSSLFVEDCKLPLGQSL CALPDTPSTLTPRSVRSVPGEMRLASIAFNHPIQVDQLNSSYFKLSIPTNFSFGVTQEYIQTTI QKVTVDCKQYVCNGFQKCEQLLREYGQFCSKINQALHGANLRQDDSVRNLFASVKSSQS SPIIPGFGGDFNLTLLEPVSISTGSRSARSAIEDLLFDKVTIADPGYMQGYDDCMQQGPASAR DLICAQYVAGYKVLPPLMDVNMEAAYTSSLLGSIAGVGWTAGLSSFAAIPFAQSIFYRLNG VGITQQVLSENQKLIANKFNQALGAMQTGFTTTNEAFRKVQDAVNNNAQALSKLASELSNTFGAISASIGDIIQRLDVLEQDAQIDRLINGRLTTLNAFVAQQLVRSESAALSAQLAKDKVNE CVKAQSKRSGFCGQGTHIVSFVVNAPNGLYFMHVGYYPSNHIEVVSAYGLCDAANPTNCI APVNGYFIKTNNTRIVDEWSYTGSSFYAPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTG IDFQDELDEFFKNVSTSIPNFGSLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKELGNYT YYNKWPWYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYDLEPHK VHVH。

优选地，所述M蛋白马赛克抗原包括SEQ ID NO:5～8所示的氨基酸序列；

SEQ ID NO:5：

MSNMTQLTEAQIIAIIKDWNFAWSLIFLLITIVLQYGYPSRSMTVYVFKMFVLWLLWPS SMALSIFSAIYPIDLASQIISGIVAAVSAMMWISYFVQSIRLFMRTGSWWSFNPETNILLNVPL HGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVA GDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ；

SEQ ID NO:6：

MSNSSVPLSEVYVHLRNWNFSWNLILTLFIVVLQYGHYKYSRLLYGLKMSVLWCLWP LVLALSIFDCFVNFNVDWVFFGFSILMSIITLCLWVMYFVNSFRLWRRVKTFWAFNPETNAI ISLQVYGHNYYLPVMAAPTGVTLTLLSGVLLVDGHKIATRVQVGQLPKYVIVATPSTTIVCD RVGRSVNETSQTGWAFYVRAKHGDFSGVASQEGVLSEREKLLHLI；

SEQ ID NO:7：

MSSKTTPAPVYIWTADEAIKFLKEWNFSLGIILLFITIILQFGYTSRSMFVYVIKMIILWL MWPITIILTTFNCVYALNNVYLGLSIVFTIVAIIMWIVYFVNSIRLFIRTGSFWSFNPETNCLLN VPIGGTTVVRPLVEDSTSVTAVVTNGHLKMAGMHFGACDYDRLPNEVTVAKPNVLIALKM VKRQSYGTNSGVAIYHRYKAGNYRSPPITADIELALLRA；

SEQ ID NO:8：

MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPV TLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNNLMC IDMKGTMYVRPIIEDYHTLTVTIIRGHLYIQGIKLGIGYSLADLPAYMTVAKVTHLCTYKRG FLDRISDTSGFAVYVKSKVGNYRLPSTQKGSGMDTALLRNNI。

优选地，所述N蛋白马赛克抗原包括SEQ ID NO:9～12示的氨基酸序列；

SEQ ID NO:9：

MASPAAPRAVSFADNNDITNTNLSRGRGRNPKPRAAPNNTVSWYTGLTQHGKVPLTF PPGQGVPLNANSTPAQNAGYWRRQDRKINTGNGIKQLAPRWYFYYTGTGPEAALPFRAVK DGIVWVHEDGATDAPSTFGTRNPNNDSAIVTQFAPGTKLPKNFHIEGTGGNSQSSSRASSVS RNSSRSSSQGSRSGNSTRGTSPGPSGIGAVGGDLLYLDLLNRLQALESGKVKQSQPKVITKK DAAAAKNKMRHKRTSTKSFNMVQAFGLRGPGDLQGNFGDLQLNKLGTEDPRWPQIAELA PTASAFMGMSQFKLTHQNNDDHGNPVYFLRYSGAIKLDPKNPNYNKWLELLEQNIDAYKT FPKKEKKQKAPKEESTDQMSEPPKEQRVQGSITQRTRTRPSVQPGPMIDVNTD；

SEQ ID NO:10：

MASVNWADDRAARKKFPPPSFYMPLLVSSDKAPYRVIPRNLVPIGKGNKDEQIGYWN VQERWRMRRGQRVDLPPKVHFYYLGTGPHAKDQYGTDINGVYWVASNQADVNTPADIV DRDPSSDEAIPTRFPPGTVLPQGYYIEGSGRSAPNSRSTSRTSSRASSAGSRSRANSGNRTPT SGVTPDMADQIASLVLAKLGKDATKPQQVTKHTAKEVRQKILNKPRQKRSPNKQCTVQQC FGKRGPNQNFGGGEMLKLGTSDPQFPILAELAPTAGAFFFGSKLELAKVQNLSGNPDEPQK DVYELRYNGAIRFDSTLSGFETIMKVLSENLNAYQQQDGMMNMSPKPQRQRGHKNGQGE NDNISVAVPKSRVQQNKSIELTAEDISLLKKMDEPFTEDTSEI；

SEQ ID NO:11：

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALT QHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGP EAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRG GSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKG QQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTD YKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHID AYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQ A；

SEQ ID NO:12：

MSFTPGKQSSSRASSGNRSGNGILKWADQSDQFRNVQTRGRRAQPKQTATSQQPSGG NVVPYYSWFSGITQFQKGKEFEFAEGQGVPIAPGVPATEAKGYWYRHNRRSFKTADGNQR QLLPRWYFYYLGTGPHKDLKFRQRSDGVVWVAKEGAKTVNTSLGNRKRNQKPLEPKFSI ALPPELSVVEFEDRSNNSSRASSRSSTRNNSRDSSRSTSRQQSRTRSDSNQSSSDLVAAVTLA LKNLGFDNQSKSPSSSGTSTPKKPNKPLSQPRADKPSQLKKPRWKRVPTREENVIQCFGPR DFNHNMGDSDLVQNGVDAKGFPQLAELIPNQAALFFDSEVSTDEVGDNVQITYTYKMLVA KDNKNLPKFIEQISAFTKPSSIKEMQSQSSHVVQNTVLNASIPESKPLADDDSAIIEIVNEVL H。

优选地，所述E蛋白马赛克抗原包括SEQ ID NO:13～16所示的氨基酸序列；

SEQ ID NO:13：

MFMADAYLADTVWYVGQIIFIVAICLLVTIVVVAFLATFKLCIQLCGMCNTLVLSPSIYVFNRGRQFYEFYNDIKPPVLDVDDVIQTL；

SEQ ID NO:14：

MLPFVQERIGLFIVNFFIFTVVCAITLLVCMAFLTATRLCVQCMTGFNTLLVQPALYLYNTGRSVYVKFQDSKPPLPPDEWV；

SEQ ID NO:15：

MFLRLIDDNGIVLNSILWLLVMIFFFVLAMTFIKLIQLCFTCHYFFSRTLYQPVYKIFLAYQDYMQIAPVPAEVLNV；

SEQ ID NO:16：

MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV。

第三方面，本发明提供了一种第二方面所述的冠状病毒抗原在制备冠状病毒通用疫苗中的应用。

第四方面，本发明提供了一种携带第二方面所述的冠状病毒抗原的冠状病毒通用疫苗，所述疫苗以pVAX1为DNA疫苗载体，或以删除E1和E3的复制缺陷性2型和5型腺病毒为疫苗载体。

与现有技术相比，本发明具有如下有益效果：

(1)本发明通过对现有已知的所有CoV基因组序列进行大数据分析，利用马赛克策略 (Mosaic Strategy)设计得到能最大程度覆盖保守的T细胞表位的冠状病毒抗原序列；

(2)本发明设计的冠状病毒抗原由来源于天然序列的短肽组装形成，覆盖了所有已知的7种HCoVs上的天然S、M、N、E蛋白的大部分CTL表位，并与天然蛋白具有很好的结构相似性；

(3)本发明的冠状病毒抗原构建方法及由此构建的冠状病毒抗原在制备冠状病毒通用疫苗方面具有潜在的应用前景和价值。

附图说明

图1为四种马赛克抗原上的CTL表位分析；

图2(A)为S protein mosaic cocktail与天然S蛋白的CTL表位匹配度分析，图2(B) 为M protein mosaic cocktail与天然M蛋白的CTL表位匹配度分析，图2(C)为Nprotein mosaic cocktail与天然N蛋白的CTL表位匹配度分析，图2(D)为E proteinmosaic cocktail与天然 E蛋白的CTL表位匹配度分析；

图3为四种马赛克抗原和天然蛋白之间的关系；

图4(A)为天然S蛋白和S protein mosaic cocktail其中一条序列的三维结构，图4(B) 为天然N蛋白和N protein mosaic cocktail其中一条序列的三维结构，图4(C)为天然M蛋白和M protein mosaic cocktail其中一条序列的三维结构，图4(D)为天然E蛋白和Eprotein mosaic cocktail其中一条序列的三维结构。

具体实施方式

为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。

实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。

实施例1 HCoVs结构蛋白上的保守CTL表位分析

为研究所有已知的HCoVs上存在的保守T细胞表位，本实施例首先从NCBI、VIPR和GISAID上下载所有地区、所有时间、所有宿主的冠状病毒基因组序列共3132条，其中包括928条HCoVs和2204条其他CoVs，并重点分析HCoVs基因组编码的四种结构蛋白：S蛋白(spike-surface glycoprotein)、E蛋白(small envelope protein)、M蛋白(matrixprotein) 和N蛋白(nucleocapsid protein)；

利用计算机算法去除重复序列和测序质量较差的序列后，得到534条S蛋白序列、485 条M蛋白序列、410条N蛋白序列和478条E蛋白序列，利用NetMHCpan-4.0工具预测这些蛋白序列上存在的潜在的细胞毒性T淋巴细胞(CTL)表位；

根据表型频率≥6％，共筛选得到15种在中国人群中频率最高的HLA-I类等位基因： HLA-A*02:01、HLA-A*24:02、HLA-A*11:01、HLA-A*33:03、HLA-A*30:01、HLA-B*40:01、HLA-B*46:01、HLA-B*13:02、HLA-C*04:01、HLA-C*03:03、HLA-C*08:01、HLA-C*03:04、 HLA-C*06:02、HLA-C*01:02和HLA-C*07:02；统计15种HLA-I类等位基因的表位能够很好的代表7种天然HCoVs与所生成的mosaic cocktail在中国人群中的表位数量。

基于NCBI数据库中七种HCoVs(HCoV-NL63、HCoV-229e、HCoV-OC43、HCoV-HKU1、SARS-CoV、MERS-CoV和SARS-CoV-2)的参考序列，分析潜在的CTL表位在S、M、N、 E四种结构蛋白上的分布。

分析发现，在7种HCoVs中，全长S蛋白上包括221～228个CTL表位，每100个氨基酸中包括16.33～19.76个CTL表位；M蛋白上包括51～61个CTL表位，每100个氨基酸中包括22.87～27.60个CTL表位；N蛋白上包括54～65个CTL表位，每100个氨基酸中包括 13.08～16.98个CTL表位；E蛋白上包括14～21个CTL表位，每100个氨基酸中包括17.07～25.00 个CTL表位；说明S蛋白的CTL表位总数量最高，但是E蛋白的每100个氨基酸中的表位数量最高。

实施例2马赛克抗原的设计和CTL表位分析

为了获得最大程度涵盖保守CTL表位的人工抗原，本实施例利用Mosaic VaccineDesigner程序进一步分析了S、M、N和E蛋白序列，去除了罕见的、在天然表位中出现次数低(出现次数小于3)的表位后，获得了一系列由短肽(由9个氨基酸组成)组装形成的马赛克抗原S protein mosaic cocktail(SEQ ID NO:1～4)、M protein mosaic cocktail(SEQ IDNO: 5～8)、N protein mosaic cocktail(SEQ ID NO:9～12)和E protein mosaiccocktail(SEQ ID NO: 13～16)。

对四种马赛克抗原上的CTL表位进行分析发现，天然S、M、N、E蛋白上的大部分CTL表位均存在于马赛克抗原上。如图1所示，在马赛克抗原上90％的CTL表位的9个氨基酸与天然S、M、N、E蛋白上的CTL表位的9个氨基酸完全匹配(9/9匹配)，95％的CTL表位的8个以上氨基酸与天然S、M、N、E蛋白上的CTL表位的9个氨基酸完全匹配(8/9匹配)，另有一些CTL表位的7个氨基酸与410条天然N蛋白的CTL表位的9个氨基酸完全匹配(7/9匹配)。例如，如表1所示，在N protein mosaic cocktail中，91.96％CTL表位与 410条天然N蛋白的CTL表位完全匹配(Exact)，95.53％CTL表位的8个以上氨基酸与410 条天然N蛋白的CTL表位的9个氨基酸完全匹配(Off-by-1)。

表1

马赛克抗原(n＝4)	天然序列条数	Exact	Off-by-1
				S protein mosaic cocktail	534	0.8901	0.9339
M protein mosaic cocktail	485	0.8777	0.9505
				N protein mosaic cocktail	410	0.9196	0.9553
E protein mosaic cocktail	478	0.9286	0.9447

为进一步评价这些表位的使用频率，对马赛克抗原和天然蛋白上的表位分布进行分析。结果如图2(A)、图2(B)、图2(C)和图2(D)所示，马赛克抗原上的CTL表位与天然蛋白的CTL表位匹配度高，说明四种马赛克抗原中的CTL表位覆盖了大部分天然蛋白中的 CTL表位。

实施例3马赛克抗原的系统发育分析

本实施例探究马赛克抗原和天然蛋白之间的关系。如图3所示，7种HCoVs形成4个进化枝，其中，A为HCoV-229E和HCoV-NL63的进化枝，B为SARS-CoV和SARS-CoV-2的进化枝，C为HCoV-HKU1和HCoV-OC43的进化枝，D为MERS-CoV的进化枝；四种马赛克抗原(星型)均存在于各个进化枝上，说明设计的马赛克抗原广泛覆盖了所有已知的HCoVs。

实施例4马赛克抗原的构象预测

本实施例通过构建马赛克抗原的三维结构模型，评估马赛克抗原的天然免疫功能。如图 4(A)、图4(B)、图4(C)和图4(D)分别为S蛋白和S protein mosaic cocktail、N蛋白和N protein mosaic cocktail、M蛋白和M protein mosaic cocktail、E蛋白和Eprotein mosaic cocktail的三维结构，a1、b1、c1、d1为天然蛋白和马赛克抗原的合并图像，a2、b2、c2、 d2为天然蛋白，a3、b3、c3、d3为相应的马赛克抗原；四种马赛克抗原与其天然蛋白的 Qualitative Model Energy Analysis(QMEA)评分分别为-0.97(S proteinmosaic cocktail)、0.8 (N protein mosaic cocktail)、-4.32(M protein mosaiccocktail)和-2.82(E protein mosaic cocktail)，说明马赛克抗原与其天然蛋白具有很好的结构相似性。

实施例5马赛克抗原的基因优化与合成

本实施例对设计得到的四种马赛克抗原S protein mosaic cocktail、M proteinmosaic cocktail、N protein mosaic cocktail和E protein mosaic cocktail的编码基因进行优化，根据哺乳动物细胞的密码子偏好性进行基因改造，得到优化的基因序列optMosaicCoV-S、 optMosaicCoV-N、optMosaicCoV-M和optMosaicCoV-E。

实施例6表达载体构建

本实施例以pVAX1作为DNA载体，以删除E1和E3的复制缺陷性2型和5型腺病毒(Ad2和Ad5)作为病毒载体，利用重组技术将优化的基因序列optMosaicCoV-S、optMosaicCoV-N、optMosaicCoV-M和optMosaicCoV-E分别克隆至DNA载体和腺病毒载体中，得到重组质粒和重组腺病毒。

重组质粒经无内毒素的质粒大提试剂盒进行抽提；重组腺病毒在293细胞中进行大量扩增后，通过CsCl密度梯度离心纯化病毒，并检测其物理颗粒浓度和TCID₅₀滴度。

综上所述，本发明基于人工智能算法对所有已知的CoV基因组序列进行大数据分析，设计出能最大程度覆盖广谱交叉抗原表位、尤其是保守的T细胞表位的新型抗原序列；对新型抗原序列进行优化得到广谱保护性免疫原，在冠状病毒新型疫苗的研发领域具有潜在应用前景。

申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

SEQUENCE LISTING

<110> 中山大学

<120> 基于马赛克策略的冠状病毒抗原的构建方法及其应用

<130> 20200414

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 1358

<212> PRT

<213> 人工

<400> 1

Met Phe Leu Ile Leu Leu Ile Ser Leu Pro Thr Ala Phe Ala Val Ile

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Gly Asp Leu Asn Cys Pro Leu Asp Pro Arg Leu Lys Gly Ser Phe Asn

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Asn Arg Asp Thr Gly Pro Pro Ser Ile Ser Thr Asp Thr Val Asp Val

35 40 45

Thr Asn Gly Leu Gly Thr Tyr Tyr Val Leu Asp Arg Val Tyr Leu Asn

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Thr Thr Leu Phe Leu Asn Gly Tyr Tyr Pro Thr Ser Gly Ser Thr Tyr

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Arg Asn Met Ala Leu Lys Gly Thr Asp Lys Leu Ser Thr Leu Trp Phe

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Lys Pro Pro Phe Leu Ser Asp Phe Ile Asn Gly Ile Phe Ala Lys Val

100 105 110

Lys Asn Thr Lys Val Phe Lys Asp Gly Val Met Tyr Ser Glu Phe Pro

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Ala Ile Thr Ile Gly Ser Thr Phe Val Asn Thr Ser Tyr Ser Val Val

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Val Gln Pro Arg Thr Ile Asn Ser Thr Gln Asp Gly Val Asn Lys Leu

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Gln Gly Leu Leu Glu Val Ser Val Cys Gln Tyr Asn Met Cys Glu Tyr

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Pro His Thr Ile Cys His Pro Lys Leu Gly Asn His Phe Lys Glu Leu

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Trp His Leu Asp Thr Gly Val Val Ser Cys Leu Tyr Lys Arg Asn Phe

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Thr Tyr Asp Val Asn Ala Thr Tyr Leu Tyr Phe His Phe Tyr Gln Glu

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Gly Gly Thr Phe Tyr Ala Tyr Phe Thr Asp Thr Gly Phe Val Thr Lys

225 230 235 240

Phe Leu Phe Asn Val Tyr Leu Gly Met Ala Leu Ser His Tyr Tyr Val

245 250 255

Met Pro Leu Thr Cys Ile Ser Arg Arg Asp Ile Gly Phe Thr Leu Glu

260 265 270

Tyr Trp Val Thr Pro Leu Thr Pro Arg Gln Tyr Leu Leu Ala Phe Asn

275 280 285

Gln Asp Gly Ile Ile Phe Asn Ala Val Asp Cys Met Ser Asp Phe Met

290 295 300

Ser Glu Ile Lys Cys Lys Thr Gln Ser Ile Ala Pro Pro Thr Gly Val

305 310 315 320

Tyr Glu Leu Asn Gly Tyr Thr Val Gln Pro Ile Ala Asp Val Tyr Arg

325 330 335

Arg Lys Pro Asp Leu Pro Asn Cys Asn Ile Glu Ala Trp Leu Asn Asp

340 345 350

Lys Ser Val Pro Ser Pro Leu Asn Trp Glu Arg Lys Thr Phe Ser Asn

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Cys Asn Phe Asn Met Ser Ser Leu Met Ser Phe Ile Gln Ala Asp Ser

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Phe Thr Cys Asn Asn Ile Asp Ala Ala Lys Ile Tyr Gly Met Cys Phe

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Ser Ser Ile Thr Ile Asp Lys Phe Ala Ile Pro Asn Arg Arg Lys Val

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Asp Leu Gln Leu Gly Asn Leu Gly Tyr Leu Gln Ser Ser Asn Tyr Arg

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Ile Asp Thr Thr Ala Thr Ser Cys Gln Leu Tyr Tyr Asn Leu Pro Ala

435 440 445

Ala Asn Val Ser Val Ser Arg Phe Asn Pro Ser Thr Trp Asn Lys Arg

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Phe Gly Phe Ile Glu Asp Ser Val Phe Val Pro Gln Pro Thr Gly Val

465 470 475 480

Phe Thr Asn His Ser Val Val Tyr Ala Gln His Cys Phe Lys Ala Pro

485 490 495

Lys Asn Phe Cys Pro Cys Ser Ser Cys Pro Gly Lys Asn Asn Gly Ile

500 505 510

Gly Thr Cys Pro Ala Gly Thr Asn Tyr Leu Thr Cys Asp Asn Leu Cys

515 520 525

Thr Leu Asp Pro Ile Thr Phe Lys Ala Pro Asp Thr Tyr Lys Cys Pro

530 535 540

Gln Thr Lys Ser Leu Val Gly Ile Gly Glu His Cys Ser Gly Leu Ala

545 550 555 560

Val Lys Ser Asp Tyr Cys Gly Asn Asn Ser Cys Thr Cys Gln Pro Gln

565 570 575

Ala Phe Leu Gly Trp Ser Ala Asp Ser Cys Leu Gln Gly Asp Lys Cys

580 585 590

Asn Ile Phe Ala Asn Phe Ile Leu His Asp Val Asn Asn Gly Leu Thr

595 600 605

Cys Ser Thr Asp Leu Gln Lys Ala Asn Thr Glu Ile Glu Leu Gly Val

610 615 620

Cys Val Asn Tyr Asp Leu Tyr Gly Ile Ser Gly Gln Gly Ile Phe Val

625 630 635 640

Glu Val Asn Ala Thr Tyr Tyr Asn Ser Trp Gln Asn Leu Leu Tyr Asp

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Ser Asn Gly Asn Leu Tyr Gly Phe Arg Asp Tyr Ile Thr Asn Arg Thr

660 665 670

Phe Met Ile His Ser Cys Tyr Ser Gly Arg Val Ser Ala Ala Tyr His

675 680 685

Ala Asn Ser Ser Glu Pro Ala Leu Leu Phe Arg Asn Ile Lys Cys Asn

690 695 700

Tyr Val Phe Asn Asn Ser Leu Thr Arg Gln Leu Gln Pro Ile Asn Tyr

705 710 715 720

Ser Phe Asp Ser Tyr Leu Gly Cys Val Val Asn Ala Tyr Asn Ser Thr

725 730 735

Ala Ile Ser Val Gln Thr Cys Asp Leu Thr Val Gly Ser Gly Tyr Cys

740 745 750

Val Asp Tyr Ser Lys Asn Arg Arg Ser Arg Arg Ala Ile Thr Thr Gly

755 760 765

Tyr Arg Phe Thr Asn Phe Glu Pro Phe Thr Val Asn Ser Val Asn Asp

770 775 780

Ser Leu Glu Pro Val Gly Gly Leu Tyr Glu Ile Gln Ile Pro Ser Glu

785 790 795 800

Phe Thr Ile Gly Asn Met Glu Glu Phe Ile Gln Thr Ser Ser Pro Lys

805 810 815

Val Thr Ile Asp Cys Ala Ala Phe Val Cys Gly Asp Tyr Ala Ala Cys

820 825 830

Lys Leu Gln Leu Val Glu Tyr Gly Ser Phe Cys Asp Asn Ile Asn Ala

835 840 845

Ile Leu Thr Glu Val Asn Glu Leu Leu Asp Thr Thr Gln Leu Gln Val

850 855 860

Ala Asn Ser Leu Met Asn Gly Val Thr Leu Ser Thr Lys Leu Lys Asp

865 870 875 880

Gly Val Asn Phe Asn Val Asp Asp Ile Asn Phe Ser Pro Val Leu Gly

885 890 895

Cys Leu Gly Ser Glu Cys Ser Lys Ala Ser Ser Arg Ser Ala Ile Glu

900 905 910

Asp Leu Leu Phe Asp Lys Val Lys Leu Ser Asp Val Gly Phe Val Glu

915 920 925

Ala Tyr Asn Asn Cys Thr Gly Gly Ala Glu Ile Arg Asp Leu Ile Cys

930 935 940

Val Gln Ser Tyr Lys Gly Ile Lys Val Leu Pro Pro Leu Leu Ser Glu

945 950 955 960

Asn Gln Ile Ser Gly Tyr Thr Leu Ala Ala Thr Ser Ala Ser Leu Phe

965 970 975

Pro Pro Trp Thr Ala Ala Ala Gly Val Pro Phe Tyr Leu Asn Val Gln

980 985 990

Tyr Arg Ile Asn Gly Leu Gly Val Thr Met Asp Val Leu Ser Gln Asn

995 1000 1005

Gln Lys Leu Ile Ala Asn Ala Phe Asn Asn Ala Leu His Ala Ile

1010 1015 1020

Gln Gln Gly Phe Asp Ala Thr Asn Ser Ala Leu Val Lys Ile Gln

1025 1030 1035

Ala Val Val Asn Ala Asn Ala Glu Ala Leu Asn Asn Leu Leu Gln

1040 1045 1050

Gln Leu Ser Asn Arg Phe Gly Ala Ile Ser Ala Ser Leu Gln Glu

1055 1060 1065

Ile Leu Ser Arg Leu Asp Ala Leu Glu Ala Glu Ala Gln Ile Asp

1070 1075 1080

Arg Leu Ile Asn Gly Arg Leu Thr Ala Leu Asn Ala Tyr Val Ser

1085 1090 1095

Gln Gln Leu Ser Asp Ser Thr Leu Val Lys Phe Ser Ala Ala Gln

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Ala Met Glu Lys Val Asn Glu Cys Val Lys Ser Gln Ser Ser Arg

1115 1120 1125

Ile Asn Phe Cys Gly Asn Gly Asn His Ile Ile Ser Leu Val Gln

1130 1135 1140

Asn Ala Pro Tyr Gly Leu Tyr Phe Ile His Phe Asn Tyr Val Pro

1145 1150 1155

Thr Lys Tyr Val Thr Ala Lys Val Ser Pro Gly Leu Cys Ile Ala

1160 1165 1170

Gly Asn Arg Gly Ile Ala Pro Lys Ser Gly Tyr Phe Val Asn Val

1175 1180 1185

Asn Asn Thr Trp Met Tyr Thr Gly Ser Gly Tyr Tyr Tyr Pro Glu

1190 1195 1200

Pro Ile Thr Glu Asn Asn Val Val Val Met Ser Thr Cys Ala Val

1205 1210 1215

Asn Tyr Thr Lys Ala Pro Tyr Val Met Leu Asn Thr Ser Ile Pro

1220 1225 1230

Asn Leu Pro Asp Phe Lys Glu Glu Leu Asp Gln Trp Phe Lys Asn

1235 1240 1245

Gln Thr Ser Val Ala Pro Asp Leu Ser Leu Asp Tyr Ile Asn Val

1250 1255 1260

Thr Phe Leu Asp Leu Gln Val Glu Met Asn Arg Leu Gln Glu Ala

1265 1270 1275

Ile Lys Val Leu Asn His Ser Tyr Ile Asn Leu Lys Asp Ile Gly

1280 1285 1290

Thr Tyr Glu Tyr Tyr Val Lys Trp Pro Trp Tyr Val Trp Leu Leu

1295 1300 1305

Ile Cys Leu Ala Gly Val Ala Met Leu Val Leu Leu Phe Phe Ile

1310 1315 1320

Cys Cys Cys Thr Gly Cys Gly Thr Ser Cys Phe Lys Lys Cys Gly

1325 1330 1335

Gly Cys Cys Asp Asp Tyr Thr Gly Tyr Gln Glu Leu Val Ile Lys

1340 1345 1350

Thr Ser His Asp Asp

1355

<210> 2

<211> 1273

<212> PRT

<213> 人工

<400> 2

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu

1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 3

<211> 1356

<212> PRT

<213> 人工

<400> 3

Met Lys Leu Phe Leu Ile Leu Leu Val Leu Pro Leu Ala Ser Cys Phe

1 5 10 15

Phe Thr Cys Asn Ser Asn Ala Asn Leu Ser Met Leu Gln Leu Gly Val

20 25 30

Pro Asp Asn Ser Ser Thr Ile Val Thr Gly Leu Leu Pro Thr His Trp

35 40 45

Ile Cys Ala Asn Gln Ser Thr Ser Val Tyr Ser Ala Asn Gly Phe Phe

50 55 60

Tyr Ile Asp Val Gly Asn His Arg Ser Ala Phe Ala Leu His Thr Gly

65 70 75 80

Tyr Tyr Asp Val Asn Gln Tyr Tyr Ile Tyr Val Thr Asn Glu Ile Gly

85 90 95

Leu Asn Ala Ser Val Thr Leu Lys Ile Cys Lys Phe Gly Ile Asn Thr

100 105 110

Thr Phe Asp Phe Leu Ser Asn Ser Ser Ser Ser Phe Asp Cys Ile Val

115 120 125

Asn Leu Leu Phe Thr Glu Gln Leu Gly Ala Pro Leu Gly Ile Thr Ile

130 135 140

Ser Gly Glu Thr Val Arg Leu His Leu Tyr Asn Val Thr Arg Thr Phe

145 150 155 160

Tyr Val Pro Ala Ala Tyr Lys Leu Thr Lys Leu Ser Val Lys Cys Tyr

165 170 175

Phe Asn Tyr Ser Cys Val Phe Ser Val Val Asn Ala Thr Val Thr Val

180 185 190

Asn Val Thr Thr His Asn Gly Arg Val Val Asn Tyr Thr Val Cys Asp

195 200 205

Asp Cys Asn Gly Tyr Thr Asp Asn Ile Phe Ser Val Gln Gln Asp Gly

210 215 220

Arg Ile Pro Asn Gly Phe Pro Phe Asn Asn Trp Phe Leu Leu Thr Asn

225 230 235 240

Gly Ser Thr Leu Val Asp Gly Val Ser Arg Leu Tyr Gln Pro Leu Arg

245 250 255

Leu Thr Cys Leu Trp Pro Val Pro Gly Leu Lys Ser Ser Thr Gly Phe

260 265 270

Val Tyr Phe Asn Ala Thr Gly Ser Asp Val Asn Cys Asn Gly Tyr Gln

275 280 285

His Asn Ser Val Ala Asp Val Met Arg Tyr Asn Leu Asn Phe Ser Ala

290 295 300

Asn Ser Val Asp Asn Leu Lys Ser Gly Val Ile Val Phe Lys Thr Leu

305 310 315 320

Gln Tyr Asp Val Leu Phe Tyr Cys Ser Asn Ser Ser Ser Gly Val Leu

325 330 335

Asp Thr Thr Ile Pro Phe Gly Pro Ser Ser Gln Pro Tyr Tyr Cys Phe

340 345 350

Ile Asn Ser Thr Ile Asn Thr Thr His Val Ser Thr Phe Val Gly Val

355 360 365

Leu Pro Pro Thr Val Arg Glu Ile Val Val Ala Arg Thr Gly Gln Phe

370 375 380

Tyr Ile Asn Gly Phe Lys Tyr Phe Asp Leu Gly Phe Ile Glu Ala Val

385 390 395 400

Asn Phe Asn Val Thr Thr Ala Ser Ala Thr Asp Phe Trp Thr Val Ala

405 410 415

Phe Ala Thr Phe Val Asp Val Leu Val Asn Val Ser Ala Thr Asn Ile

420 425 430

Gln Asn Leu Leu Tyr Cys Asp Ser Pro Phe Glu Lys Leu Gln Cys Glu

435 440 445

His Leu Gln Phe Gly Leu Gln Asp Gly Phe Tyr Ser Ala Asn Phe Leu

450 455 460

Asp Asp Asn Val Leu Pro Glu Thr Tyr Val Ala Leu Pro Ile Tyr Tyr

465 470 475 480

Gln His Thr Asp Ile Asn Phe Thr Ala Thr Ala Ser Phe Gly Gly Ser

485 490 495

Cys Tyr Val Cys Lys Pro His Gln Val Asn Ile Ser Leu Asn Gly Asn

500 505 510

Thr Ser Val Cys Val Arg Thr Ser His Phe Ser Ile Arg Tyr Ile Tyr

515 520 525

Asn Arg Val Lys Ser Gly Ser Pro Gly Asp Ser Ser Trp His Ile Tyr

530 535 540

Leu Lys Ser Gly Thr Cys Pro Phe Ser Phe Ser Lys Leu Asn Asn Phe

545 550 555 560

Gln Lys Phe Lys Thr Ile Cys Phe Ser Thr Val Ala Val Pro Gly Ser

565 570 575

Cys Asn Phe Pro Leu Glu Ala Thr Trp His Tyr Thr Ser Tyr Thr Ile

580 585 590

Val Gly Ala Leu Tyr Val Thr Trp Ser Glu Gly Asn Ser Ile Thr Gly

595 600 605

Val Pro Tyr Pro Val Ser Gly Ile Arg Glu Phe Ser Asn Leu Val Leu

610 615 620

Asn Asn Cys Thr Lys Tyr Asn Ile Tyr Asp Tyr Val Gly Thr Gly Ile

625 630 635 640

Ile Arg Ser Ser Asn Gln Ser Leu Ala Gly Gly Ile Thr Tyr Val Ser

645 650 655

Asn Ser Gly Asn Leu Leu Gly Phe Lys Asn Val Ser Thr Gly Asn Ile

660 665 670

Phe Ile Val Thr Pro Cys Asn Gln Pro Asp Gln Val Ala Val Tyr Gln

675 680 685

Gln Ser Ile Ile Gly Ala Met Thr Ala Val Asn Glu Ser Arg Tyr Gly

690 695 700

Leu Gln Asn Leu Leu Gln Leu Pro Asn Phe Tyr Tyr Val Ser Asn Gly

705 710 715 720

Gly Asn Asn Cys Thr Thr Ala Val Met Thr Tyr Ser Asn Phe Gly Ile

725 730 735

Cys Ala Asp Gly Ser Leu Ile Pro Val Arg Pro Arg Asn Ser Ser Asp

740 745 750

Asn Gly Ile Ser Ala Ile Ile Thr Ala Asn Leu Ser Ile Pro Ser Asn

755 760 765

Trp Thr Thr Ser Val Gln Val Glu Tyr Leu Gln Ile Thr Ser Thr Pro

770 775 780

Ile Val Val Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn Pro Arg Cys

785 790 795 800

Lys Asn Leu Leu Lys Gln Tyr Thr Ser Ala Cys Lys Thr Ile Glu Asp

805 810 815

Ala Leu Arg Leu Ser Ala His Leu Glu Thr Asn Asp Val Ser Ser Met

820 825 830

Leu Thr Phe Asp Ser Asn Ala Phe Ser Leu Ala Asn Val Thr Ser Phe

835 840 845

Gly Asp Tyr Asn Leu Ser Ser Val Leu Pro Gln Arg Asn Ile His Ser

850 855 860

Ser Arg Ile Ala Gly Arg Ser Ala Leu Glu Asp Leu Leu Phe Ser Lys

865 870 875 880

Val Val Thr Ser Gly Leu Gly Thr Val Asp Val Asp Tyr Lys Ser Cys

885 890 895

Thr Lys Gly Leu Ser Ile Ala Asp Leu Ala Cys Ala Gln Tyr Tyr Asn

900 905 910

Gly Ile Met Val Leu Pro Gly Val Ala Asp Ala Glu Arg Met Ala Met

915 920 925

Tyr Thr Gly Ser Leu Ile Gly Gly Met Val Leu Gly Gly Leu Thr Ser

930 935 940

Ala Ala Ala Ile Pro Phe Ser Leu Ala Leu Gln Ala Arg Leu Asn Tyr

945 950 955 960

Val Ala Leu Gln Thr Asp Val Leu Gln Glu Asn Gln Lys Ile Leu Ala

965 970 975

Ala Ser Phe Asn Lys Ala Ile Asn Asn Ile Val Ala Ser Phe Ser Ser

980 985 990

Val Asn Asp Ala Ile Thr Gln Thr Ala Glu Ala Ile His Thr Val Thr

995 1000 1005

Ile Ala Leu Asn Lys Ile Gln Asp Val Val Asn Gln Gln Gly Ser

1010 1015 1020

Ala Leu Asn His Leu Thr Ser Gln Leu Arg His Asn Phe Gln Ala

1025 1030 1035

Ile Ser Asn Ser Ile Gln Ala Ile Tyr Asp Arg Leu Asp Ser Ile

1040 1045 1050

Gln Ala Asp Gln Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ala

1055 1060 1065

Ala Leu Asn Ala Phe Val Ser Gln Val Leu Asn Lys Tyr Thr Glu

1070 1075 1080

Val Arg Ser Ser Arg Arg Leu Ala Gln Gln Lys Ile Asn Glu Cys

1085 1090 1095

Val Lys Ser Gln Ser Asn Arg Tyr Gly Phe Cys Gly Asn Gly Thr

1100 1105 1110

His Ile Phe Ser Ile Val Asn Ser Ala Pro Asp Gly Leu Leu Phe

1115 1120 1125

Leu His Thr Val Leu Leu Pro Thr Asp Tyr Lys Asn Val Lys Ala

1130 1135 1140

Trp Ser Gly Ile Cys Val Asp Gly Ile Tyr Gly Tyr Val Leu Arg

1145 1150 1155

Gln Pro Asn Leu Val Leu Tyr Ser Asp Asn Gly Val Phe Arg Val

1160 1165 1170

Thr Ser Arg Val Met Phe Gln Pro Arg Leu Pro Val Leu Ser Asp

1175 1180 1185

Phe Val Gln Ile Tyr Asn Cys Asn Val Thr Phe Val Asn Ile Ser

1190 1195 1200

Arg Val Glu Leu His Thr Val Ile Pro Asp Tyr Val Asp Val Asn

1205 1210 1215

Lys Thr Leu Gln Glu Phe Ala Gln Asn Leu Pro Lys Tyr Val Lys

1220 1225 1230

Pro Asn Phe Asp Leu Thr Pro Phe Asn Leu Thr Tyr Leu Asn Leu

1235 1240 1245

Ser Ser Glu Leu Lys Gln Leu Glu Ala Lys Thr Ala Ser Leu Phe

1250 1255 1260

Gln Thr Thr Val Glu Leu Gln Gly Leu Ile Asp Gln Ile Asn Ser

1265 1270 1275

Thr Tyr Val Asp Leu Lys Leu Leu Asn Arg Phe Glu Asn Tyr Ile

1280 1285 1290

Lys Trp Pro Trp Trp Val Trp Leu Ile Ile Ser Val Val Phe Val

1295 1300 1305

Val Leu Leu Ser Leu Leu Val Phe Cys Cys Leu Ser Thr Gly Cys

1310 1315 1320

Cys Gly Cys Cys Asn Cys Leu Thr Ser Ser Met Arg Gly Cys Cys

1325 1330 1335

Asp Cys Gly Ser Thr Lys Leu Pro Tyr Tyr Glu Phe Glu Lys Val

1340 1345 1350

His Val Gln

1355

<210> 4

<211> 1353

<212> PRT

<213> 人工

<400> 4

Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu

1 5 10 15

Ser Tyr Val Asp Val Gly Pro Asp Ser Val Lys Ser Ala Cys Ile Glu

20 25 30

Val Asp Ile Gln Gln Thr Phe Phe Asp Lys Thr Trp Pro Arg Pro Ile

35 40 45

Asp Val Ser Lys Ala Asp Gly Ile Ile Tyr Pro Gln Gly Arg Thr Tyr

50 55 60

Ser Asn Ile Thr Ile Thr Tyr Gln Gly Leu Phe Pro Tyr Gln Gly Asp

65 70 75 80

His Gly Asp Met Tyr Val Tyr Ser Ala Gly His Ala Thr Gly Thr Thr

85 90 95

Pro Gln Lys Leu Phe Val Ala Asn Tyr Ser Gln Asp Val Lys Gln Phe

100 105 110

Ala Asn Gly Phe Val Val Arg Ile Gly Ala Ala Ala Asn Ser Thr Gly

115 120 125

Thr Val Ile Ile Ser Pro Ser Thr Ser Ala Thr Ile Arg Lys Ile Tyr

130 135 140

Pro Ala Phe Met Leu Gly Ser Ser Val Gly Asn Phe Ser Asp Gly Lys

145 150 155 160

Met Gly Arg Phe Phe Asn His Thr Leu Val Leu Leu Pro Asp Gly Cys

165 170 175

Gly Thr Leu Leu Arg Ala Phe Tyr Cys Ile Leu Glu Pro Arg Ser Gly

180 185 190

Asn His Cys Pro Ala Gly Asn Ser Tyr Thr Ser Phe Ala Thr Tyr His

195 200 205

Thr Pro Ala Thr Asp Cys Ser Asp Gly Asn Tyr Asn Arg Asn Ala Ser

210 215 220

Leu Asn Ser Phe Lys Glu Tyr Phe Asn Leu Arg Asn Cys Thr Phe Met

225 230 235 240

Tyr Thr Tyr Asn Ile Thr Glu Asp Glu Ile Leu Glu Trp Phe Gly Ile

245 250 255

Thr Gln Thr Ala Gln Gly Val His Leu Phe Ser Ser Arg Tyr Val Asp

260 265 270

Leu Tyr Gly Gly Asn Met Phe Gln Phe Ala Thr Leu Pro Val Tyr Asp

275 280 285

Thr Ile Lys Tyr Tyr Ser Ile Ile Pro His Ser Ile Arg Ser Ile Gln

290 295 300

Ser Asp Arg Lys Ala Trp Ala Ala Phe Tyr Val Tyr Lys Leu Gln Pro

305 310 315 320

Leu Thr Phe Leu Leu Asp Phe Ser Val Asp Gly Tyr Ile Arg Arg Ala

325 330 335

Ile Asp Cys Gly Phe Asn Asp Leu Ser Gln Leu His Cys Ser Tyr Glu

340 345 350

Ser Phe Asp Val Glu Ser Gly Val Tyr Ser Val Ser Ser Phe Glu Ala

355 360 365

Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp

370 375 380

Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe Lys

385 390 395 400

Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu Ser

405 410 415

Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala Ala

420 425 430

Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser Tyr

435 440 445

Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile

450 455 460

Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile

465 470 475 480

Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys

485 490 495

Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg Thr

500 505 510

Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys Val Ser

515 520 525

Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys Gln

530 535 540

Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly Ser Thr

545 550 555 560

Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln

565 570 575

Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe Ala Asn

580 585 590

Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr Ser Leu

595 600 605

Tyr Gly Val Ser Gly Arg Gly Val Phe Gln Asn Cys Thr Ala Val Gly

610 615 620

Val Arg Gln Gln Arg Phe Val Tyr Asp Ala Tyr Gln Asn Leu Val Gly

625 630 635 640

Tyr Tyr Ser Asp Asp Gly Asn Tyr Tyr Cys Leu Arg Ala Cys Val Ser

645 650 655

Val Pro Val Ser Val Ile Tyr Asp Lys Glu Thr Lys Thr His Ala Thr

660 665 670

Leu Phe Gly Ser Val Ala Cys Glu His Ile Ser Ser Thr Met Ser Gln

675 680 685

Tyr Ser Arg Ser Thr Arg Ser Met Leu Lys Arg Arg Asp Ser Thr Tyr

690 695 700

Gly Pro Leu Gln Thr Pro Val Gly Cys Val Leu Gly Leu Val Asn Ser

705 710 715 720

Ser Leu Phe Val Glu Asp Cys Lys Leu Pro Leu Gly Gln Ser Leu Cys

725 730 735

Ala Leu Pro Asp Thr Pro Ser Thr Leu Thr Pro Arg Ser Val Arg Ser

740 745 750

Val Pro Gly Glu Met Arg Leu Ala Ser Ile Ala Phe Asn His Pro Ile

755 760 765

Gln Val Asp Gln Leu Asn Ser Ser Tyr Phe Lys Leu Ser Ile Pro Thr

770 775 780

Asn Phe Ser Phe Gly Val Thr Gln Glu Tyr Ile Gln Thr Thr Ile Gln

785 790 795 800

Lys Val Thr Val Asp Cys Lys Gln Tyr Val Cys Asn Gly Phe Gln Lys

805 810 815

Cys Glu Gln Leu Leu Arg Glu Tyr Gly Gln Phe Cys Ser Lys Ile Asn

820 825 830

Gln Ala Leu His Gly Ala Asn Leu Arg Gln Asp Asp Ser Val Arg Asn

835 840 845

Leu Phe Ala Ser Val Lys Ser Ser Gln Ser Ser Pro Ile Ile Pro Gly

850 855 860

Phe Gly Gly Asp Phe Asn Leu Thr Leu Leu Glu Pro Val Ser Ile Ser

865 870 875 880

Thr Gly Ser Arg Ser Ala Arg Ser Ala Ile Glu Asp Leu Leu Phe Asp

885 890 895

Lys Val Thr Ile Ala Asp Pro Gly Tyr Met Gln Gly Tyr Asp Asp Cys

900 905 910

Met Gln Gln Gly Pro Ala Ser Ala Arg Asp Leu Ile Cys Ala Gln Tyr

915 920 925

Val Ala Gly Tyr Lys Val Leu Pro Pro Leu Met Asp Val Asn Met Glu

930 935 940

Ala Ala Tyr Thr Ser Ser Leu Leu Gly Ser Ile Ala Gly Val Gly Trp

945 950 955 960

Thr Ala Gly Leu Ser Ser Phe Ala Ala Ile Pro Phe Ala Gln Ser Ile

965 970 975

Phe Tyr Arg Leu Asn Gly Val Gly Ile Thr Gln Gln Val Leu Ser Glu

980 985 990

Asn Gln Lys Leu Ile Ala Asn Lys Phe Asn Gln Ala Leu Gly Ala Met

995 1000 1005

Gln Thr Gly Phe Thr Thr Thr Asn Glu Ala Phe Arg Lys Val Gln

1010 1015 1020

Asp Ala Val Asn Asn Asn Ala Gln Ala Leu Ser Lys Leu Ala Ser

1025 1030 1035

Glu Leu Ser Asn Thr Phe Gly Ala Ile Ser Ala Ser Ile Gly Asp

1040 1045 1050

Ile Ile Gln Arg Leu Asp Val Leu Glu Gln Asp Ala Gln Ile Asp

1055 1060 1065

Arg Leu Ile Asn Gly Arg Leu Thr Thr Leu Asn Ala Phe Val Ala

1070 1075 1080

Gln Gln Leu Val Arg Ser Glu Ser Ala Ala Leu Ser Ala Gln Leu

1085 1090 1095

Ala Lys Asp Lys Val Asn Glu Cys Val Lys Ala Gln Ser Lys Arg

1100 1105 1110

Ser Gly Phe Cys Gly Gln Gly Thr His Ile Val Ser Phe Val Val

1115 1120 1125

Asn Ala Pro Asn Gly Leu Tyr Phe Met His Val Gly Tyr Tyr Pro

1130 1135 1140

Ser Asn His Ile Glu Val Val Ser Ala Tyr Gly Leu Cys Asp Ala

1145 1150 1155

Ala Asn Pro Thr Asn Cys Ile Ala Pro Val Asn Gly Tyr Phe Ile

1160 1165 1170

Lys Thr Asn Asn Thr Arg Ile Val Asp Glu Trp Ser Tyr Thr Gly

1175 1180 1185

Ser Ser Phe Tyr Ala Pro Glu Pro Ile Thr Ser Leu Asn Thr Lys

1190 1195 1200

Tyr Val Ala Pro Gln Val Thr Tyr Gln Asn Ile Ser Thr Asn Leu

1205 1210 1215

Pro Pro Pro Leu Leu Gly Asn Ser Thr Gly Ile Asp Phe Gln Asp

1220 1225 1230

Glu Leu Asp Glu Phe Phe Lys Asn Val Ser Thr Ser Ile Pro Asn

1235 1240 1245

Phe Gly Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp Leu Thr

1250 1255 1260

Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Ala Leu Asn Glu

1265 1270 1275

Ser Tyr Ile Asp Leu Lys Glu Leu Gly Asn Tyr Thr Tyr Tyr Asn

1280 1285 1290

Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Val

1295 1300 1305

Ala Leu Ala Leu Cys Val Phe Phe Ile Leu Cys Cys Thr Gly Cys

1310 1315 1320

Gly Thr Asn Cys Met Gly Lys Leu Lys Cys Asn Arg Cys Cys Asp

1325 1330 1335

Arg Tyr Glu Glu Tyr Asp Leu Glu Pro His Lys Val His Val His

1340 1345 1350

<210> 5

<211> 221

<212> PRT

<213> 人工

<400> 5

Met Ser Asn Met Thr Gln Leu Thr Glu Ala Gln Ile Ile Ala Ile Ile

1 5 10 15

Lys Asp Trp Asn Phe Ala Trp Ser Leu Ile Phe Leu Leu Ile Thr Ile

20 25 30

Val Leu Gln Tyr Gly Tyr Pro Ser Arg Ser Met Thr Val Tyr Val Phe

35 40 45

Lys Met Phe Val Leu Trp Leu Leu Trp Pro Ser Ser Met Ala Leu Ser

50 55 60

Ile Phe Ser Ala Ile Tyr Pro Ile Asp Leu Ala Ser Gln Ile Ile Ser

65 70 75 80

Gly Ile Val Ala Ala Val Ser Ala Met Met Trp Ile Ser Tyr Phe Val

85 90 95

Gln Ser Ile Arg Leu Phe Met Arg Thr Gly Ser Trp Trp Ser Phe Asn

100 105 110

Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile Leu

115 120 125

Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile Leu

130 135 140

Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp Ile

145 150 155 160

Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser

165 170 175

Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly Phe

180 185 190

Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp

195 200 205

His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln

210 215 220

<210> 6

<211> 226

<212> PRT

<213> 人工

<400> 6

Met Ser Asn Ser Ser Val Pro Leu Ser Glu Val Tyr Val His Leu Arg

1 5 10 15

Asn Trp Asn Phe Ser Trp Asn Leu Ile Leu Thr Leu Phe Ile Val Val

20 25 30

Leu Gln Tyr Gly His Tyr Lys Tyr Ser Arg Leu Leu Tyr Gly Leu Lys

35 40 45

Met Ser Val Leu Trp Cys Leu Trp Pro Leu Val Leu Ala Leu Ser Ile

50 55 60

Phe Asp Cys Phe Val Asn Phe Asn Val Asp Trp Val Phe Phe Gly Phe

65 70 75 80

Ser Ile Leu Met Ser Ile Ile Thr Leu Cys Leu Trp Val Met Tyr Phe

85 90 95

Val Asn Ser Phe Arg Leu Trp Arg Arg Val Lys Thr Phe Trp Ala Phe

100 105 110

Asn Pro Glu Thr Asn Ala Ile Ile Ser Leu Gln Val Tyr Gly His Asn

115 120 125

Tyr Tyr Leu Pro Val Met Ala Ala Pro Thr Gly Val Thr Leu Thr Leu

130 135 140

Leu Ser Gly Val Leu Leu Val Asp Gly His Lys Ile Ala Thr Arg Val

145 150 155 160

Gln Val Gly Gln Leu Pro Lys Tyr Val Ile Val Ala Thr Pro Ser Thr

165 170 175

Thr Ile Val Cys Asp Arg Val Gly Arg Ser Val Asn Glu Thr Ser Gln

180 185 190

Thr Gly Trp Ala Phe Tyr Val Arg Ala Lys His Gly Asp Phe Ser Gly

195 200 205

Val Ala Ser Gln Glu Gly Val Leu Ser Glu Arg Glu Lys Leu Leu His

210 215 220

Leu Ile

225

<210> 7

<211> 225

<212> PRT

<213> 人工

<400> 7

Met Ser Ser Lys Thr Thr Pro Ala Pro Val Tyr Ile Trp Thr Ala Asp

1 5 10 15

Glu Ala Ile Lys Phe Leu Lys Glu Trp Asn Phe Ser Leu Gly Ile Ile

20 25 30

Leu Leu Phe Ile Thr Ile Ile Leu Gln Phe Gly Tyr Thr Ser Arg Ser

35 40 45

Met Phe Val Tyr Val Ile Lys Met Ile Ile Leu Trp Leu Met Trp Pro

50 55 60

Ile Thr Ile Ile Leu Thr Thr Phe Asn Cys Val Tyr Ala Leu Asn Asn

65 70 75 80

Val Tyr Leu Gly Leu Ser Ile Val Phe Thr Ile Val Ala Ile Ile Met

85 90 95

Trp Ile Val Tyr Phe Val Asn Ser Ile Arg Leu Phe Ile Arg Thr Gly

100 105 110

Ser Phe Trp Ser Phe Asn Pro Glu Thr Asn Cys Leu Leu Asn Val Pro

115 120 125

Ile Gly Gly Thr Thr Val Val Arg Pro Leu Val Glu Asp Ser Thr Ser

130 135 140

Val Thr Ala Val Val Thr Asn Gly His Leu Lys Met Ala Gly Met His

145 150 155 160

Phe Gly Ala Cys Asp Tyr Asp Arg Leu Pro Asn Glu Val Thr Val Ala

165 170 175

Lys Pro Asn Val Leu Ile Ala Leu Lys Met Val Lys Arg Gln Ser Tyr

180 185 190

Gly Thr Asn Ser Gly Val Ala Ile Tyr His Arg Tyr Lys Ala Gly Asn

195 200 205

Tyr Arg Ser Pro Pro Ile Thr Ala Asp Ile Glu Leu Ala Leu Leu Arg

210 215 220

Ala

225

<210> 8

<211> 225

<212> PRT

<213> 人工

<400> 8

Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu

1 5 10 15

Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile

20 25 30

Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile

35 40 45

Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys

50 55 60

Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile

65 70 75 80

Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe

85 90 95

Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe

100 105 110

Asn Pro Glu Thr Asn Asn Leu Met Cys Ile Asp Met Lys Gly Thr Met

115 120 125

Tyr Val Arg Pro Ile Ile Glu Asp Tyr His Thr Leu Thr Val Thr Ile

130 135 140

Ile Arg Gly His Leu Tyr Ile Gln Gly Ile Lys Leu Gly Ile Gly Tyr

145 150 155 160

Ser Leu Ala Asp Leu Pro Ala Tyr Met Thr Val Ala Lys Val Thr His

165 170 175

Leu Cys Thr Tyr Lys Arg Gly Phe Leu Asp Arg Ile Ser Asp Thr Ser

180 185 190

Gly Phe Ala Val Tyr Val Lys Ser Lys Val Gly Asn Tyr Arg Leu Pro

195 200 205

Ser Thr Gln Lys Gly Ser Gly Met Asp Thr Ala Leu Leu Arg Asn Asn

210 215 220

Ile

225

<210> 9

<211> 413

<212> PRT

<213> 人工

<400> 9

Met Ala Ser Pro Ala Ala Pro Arg Ala Val Ser Phe Ala Asp Asn Asn

1 5 10 15

Asp Ile Thr Asn Thr Asn Leu Ser Arg Gly Arg Gly Arg Asn Pro Lys

20 25 30

Pro Arg Ala Ala Pro Asn Asn Thr Val Ser Trp Tyr Thr Gly Leu Thr

35 40 45

Gln His Gly Lys Val Pro Leu Thr Phe Pro Pro Gly Gln Gly Val Pro

50 55 60

Leu Asn Ala Asn Ser Thr Pro Ala Gln Asn Ala Gly Tyr Trp Arg Arg

65 70 75 80

Gln Asp Arg Lys Ile Asn Thr Gly Asn Gly Ile Lys Gln Leu Ala Pro

85 90 95

Arg Trp Tyr Phe Tyr Tyr Thr Gly Thr Gly Pro Glu Ala Ala Leu Pro

100 105 110

Phe Arg Ala Val Lys Asp Gly Ile Val Trp Val His Glu Asp Gly Ala

115 120 125

Thr Asp Ala Pro Ser Thr Phe Gly Thr Arg Asn Pro Asn Asn Asp Ser

130 135 140

Ala Ile Val Thr Gln Phe Ala Pro Gly Thr Lys Leu Pro Lys Asn Phe

145 150 155 160

His Ile Glu Gly Thr Gly Gly Asn Ser Gln Ser Ser Ser Arg Ala Ser

165 170 175

Ser Val Ser Arg Asn Ser Ser Arg Ser Ser Ser Gln Gly Ser Arg Ser

180 185 190

Gly Asn Ser Thr Arg Gly Thr Ser Pro Gly Pro Ser Gly Ile Gly Ala

195 200 205

Val Gly Gly Asp Leu Leu Tyr Leu Asp Leu Leu Asn Arg Leu Gln Ala

210 215 220

Leu Glu Ser Gly Lys Val Lys Gln Ser Gln Pro Lys Val Ile Thr Lys

225 230 235 240

Lys Asp Ala Ala Ala Ala Lys Asn Lys Met Arg His Lys Arg Thr Ser

245 250 255

Thr Lys Ser Phe Asn Met Val Gln Ala Phe Gly Leu Arg Gly Pro Gly

260 265 270

Asp Leu Gln Gly Asn Phe Gly Asp Leu Gln Leu Asn Lys Leu Gly Thr

275 280 285

Glu Asp Pro Arg Trp Pro Gln Ile Ala Glu Leu Ala Pro Thr Ala Ser

290 295 300

Ala Phe Met Gly Met Ser Gln Phe Lys Leu Thr His Gln Asn Asn Asp

305 310 315 320

Asp His Gly Asn Pro Val Tyr Phe Leu Arg Tyr Ser Gly Ala Ile Lys

325 330 335

Leu Asp Pro Lys Asn Pro Asn Tyr Asn Lys Trp Leu Glu Leu Leu Glu

340 345 350

Gln Asn Ile Asp Ala Tyr Lys Thr Phe Pro Lys Lys Glu Lys Lys Gln

355 360 365

Lys Ala Pro Lys Glu Glu Ser Thr Asp Gln Met Ser Glu Pro Pro Lys

370 375 380

Glu Gln Arg Val Gln Gly Ser Ile Thr Gln Arg Thr Arg Thr Arg Pro

385 390 395 400

Ser Val Gln Pro Gly Pro Met Ile Asp Val Asn Thr Asp

405 410

<210> 10

<211> 401

<212> PRT

<213> 人工

<400> 10

Met Ala Ser Val Asn Trp Ala Asp Asp Arg Ala Ala Arg Lys Lys Phe

1 5 10 15

Pro Pro Pro Ser Phe Tyr Met Pro Leu Leu Val Ser Ser Asp Lys Ala

20 25 30

Pro Tyr Arg Val Ile Pro Arg Asn Leu Val Pro Ile Gly Lys Gly Asn

35 40 45

Lys Asp Glu Gln Ile Gly Tyr Trp Asn Val Gln Glu Arg Trp Arg Met

50 55 60

Arg Arg Gly Gln Arg Val Asp Leu Pro Pro Lys Val His Phe Tyr Tyr

65 70 75 80

Leu Gly Thr Gly Pro His Ala Lys Asp Gln Tyr Gly Thr Asp Ile Asn

85 90 95

Gly Val Tyr Trp Val Ala Ser Asn Gln Ala Asp Val Asn Thr Pro Ala

100 105 110

Asp Ile Val Asp Arg Asp Pro Ser Ser Asp Glu Ala Ile Pro Thr Arg

115 120 125

Phe Pro Pro Gly Thr Val Leu Pro Gln Gly Tyr Tyr Ile Glu Gly Ser

130 135 140

Gly Arg Ser Ala Pro Asn Ser Arg Ser Thr Ser Arg Thr Ser Ser Arg

145 150 155 160

Ala Ser Ser Ala Gly Ser Arg Ser Arg Ala Asn Ser Gly Asn Arg Thr

165 170 175

Pro Thr Ser Gly Val Thr Pro Asp Met Ala Asp Gln Ile Ala Ser Leu

180 185 190

Val Leu Ala Lys Leu Gly Lys Asp Ala Thr Lys Pro Gln Gln Val Thr

195 200 205

Lys His Thr Ala Lys Glu Val Arg Gln Lys Ile Leu Asn Lys Pro Arg

210 215 220

Gln Lys Arg Ser Pro Asn Lys Gln Cys Thr Val Gln Gln Cys Phe Gly

225 230 235 240

Lys Arg Gly Pro Asn Gln Asn Phe Gly Gly Gly Glu Met Leu Lys Leu

245 250 255

Gly Thr Ser Asp Pro Gln Phe Pro Ile Leu Ala Glu Leu Ala Pro Thr

260 265 270

Ala Gly Ala Phe Phe Phe Gly Ser Lys Leu Glu Leu Ala Lys Val Gln

275 280 285

Asn Leu Ser Gly Asn Pro Asp Glu Pro Gln Lys Asp Val Tyr Glu Leu

290 295 300

Arg Tyr Asn Gly Ala Ile Arg Phe Asp Ser Thr Leu Ser Gly Phe Glu

305 310 315 320

Thr Ile Met Lys Val Leu Ser Glu Asn Leu Asn Ala Tyr Gln Gln Gln

325 330 335

Asp Gly Met Met Asn Met Ser Pro Lys Pro Gln Arg Gln Arg Gly His

340 345 350

Lys Asn Gly Gln Gly Glu Asn Asp Asn Ile Ser Val Ala Val Pro Lys

355 360 365

Ser Arg Val Gln Gln Asn Lys Ser Ile Glu Leu Thr Ala Glu Asp Ile

370 375 380

Ser Leu Leu Lys Lys Met Asp Glu Pro Phe Thr Glu Asp Thr Ser Glu

385 390 395 400

Ile

<210> 11

<211> 419

<212> PRT

<213> 人工

<400> 11

Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr

1 5 10 15

Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg

20 25 30

Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

35 40 45

Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

50 55 60

Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

65 70 75 80

Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

85 90 95

Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

100 105 110

Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

115 120 125

Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

130 135 140

His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

145 150 155 160

Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

165 170 175

Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

180 185 190

Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

195 200 205

Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

210 215 220

Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

225 230 235 240

Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

245 250 255

Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

275 280 285

Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

290 295 300

Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

305 310 315 320

Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala

325 330 335

Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

340 345 350

Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

370 375 380

Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

385 390 395 400

Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser

405 410 415

Thr Gln Ala

<210> 12

<211> 424

<212> PRT

<213> 人工

<400> 12

Met Ser Phe Thr Pro Gly Lys Gln Ser Ser Ser Arg Ala Ser Ser Gly

1 5 10 15

Asn Arg Ser Gly Asn Gly Ile Leu Lys Trp Ala Asp Gln Ser Asp Gln

20 25 30

Phe Arg Asn Val Gln Thr Arg Gly Arg Arg Ala Gln Pro Lys Gln Thr

35 40 45

Ala Thr Ser Gln Gln Pro Ser Gly Gly Asn Val Val Pro Tyr Tyr Ser

50 55 60

Trp Phe Ser Gly Ile Thr Gln Phe Gln Lys Gly Lys Glu Phe Glu Phe

65 70 75 80

Ala Glu Gly Gln Gly Val Pro Ile Ala Pro Gly Val Pro Ala Thr Glu

85 90 95

Ala Lys Gly Tyr Trp Tyr Arg His Asn Arg Arg Ser Phe Lys Thr Ala

100 105 110

Asp Gly Asn Gln Arg Gln Leu Leu Pro Arg Trp Tyr Phe Tyr Tyr Leu

115 120 125

Gly Thr Gly Pro His Lys Asp Leu Lys Phe Arg Gln Arg Ser Asp Gly

130 135 140

Val Val Trp Val Ala Lys Glu Gly Ala Lys Thr Val Asn Thr Ser Leu

145 150 155 160

Gly Asn Arg Lys Arg Asn Gln Lys Pro Leu Glu Pro Lys Phe Ser Ile

165 170 175

Ala Leu Pro Pro Glu Leu Ser Val Val Glu Phe Glu Asp Arg Ser Asn

180 185 190

Asn Ser Ser Arg Ala Ser Ser Arg Ser Ser Thr Arg Asn Asn Ser Arg

195 200 205

Asp Ser Ser Arg Ser Thr Ser Arg Gln Gln Ser Arg Thr Arg Ser Asp

210 215 220

Ser Asn Gln Ser Ser Ser Asp Leu Val Ala Ala Val Thr Leu Ala Leu

225 230 235 240

Lys Asn Leu Gly Phe Asp Asn Gln Ser Lys Ser Pro Ser Ser Ser Gly

245 250 255

Thr Ser Thr Pro Lys Lys Pro Asn Lys Pro Leu Ser Gln Pro Arg Ala

260 265 270

Asp Lys Pro Ser Gln Leu Lys Lys Pro Arg Trp Lys Arg Val Pro Thr

275 280 285

Arg Glu Glu Asn Val Ile Gln Cys Phe Gly Pro Arg Asp Phe Asn His

290 295 300

Asn Met Gly Asp Ser Asp Leu Val Gln Asn Gly Val Asp Ala Lys Gly

305 310 315 320

Phe Pro Gln Leu Ala Glu Leu Ile Pro Asn Gln Ala Ala Leu Phe Phe

325 330 335

Asp Ser Glu Val Ser Thr Asp Glu Val Gly Asp Asn Val Gln Ile Thr

340 345 350

Tyr Thr Tyr Lys Met Leu Val Ala Lys Asp Asn Lys Asn Leu Pro Lys

355 360 365

Phe Ile Glu Gln Ile Ser Ala Phe Thr Lys Pro Ser Ser Ile Lys Glu

370 375 380

Met Gln Ser Gln Ser Ser His Val Val Gln Asn Thr Val Leu Asn Ala

385 390 395 400

Ser Ile Pro Glu Ser Lys Pro Leu Ala Asp Asp Asp Ser Ala Ile Ile

405 410 415

Glu Ile Val Asn Glu Val Leu His

420

<210> 13

<211> 88

<212> PRT

<213> 人工

<400> 13

Met Phe Met Ala Asp Ala Tyr Leu Ala Asp Thr Val Trp Tyr Val Gly

1 5 10 15

Gln Ile Ile Phe Ile Val Ala Ile Cys Leu Leu Val Thr Ile Val Val

20 25 30

Val Ala Phe Leu Ala Thr Phe Lys Leu Cys Ile Gln Leu Cys Gly Met

35 40 45

Cys Asn Thr Leu Val Leu Ser Pro Ser Ile Tyr Val Phe Asn Arg Gly

50 55 60

Arg Gln Phe Tyr Glu Phe Tyr Asn Asp Ile Lys Pro Pro Val Leu Asp

65 70 75 80

Val Asp Asp Val Ile Gln Thr Leu

85

<210> 14

<211> 82

<212> PRT

<213> 人工

<400> 14

Met Leu Pro Phe Val Gln Glu Arg Ile Gly Leu Phe Ile Val Asn Phe

1 5 10 15

Phe Ile Phe Thr Val Val Cys Ala Ile Thr Leu Leu Val Cys Met Ala

20 25 30

Phe Leu Thr Ala Thr Arg Leu Cys Val Gln Cys Met Thr Gly Phe Asn

35 40 45

Thr Leu Leu Val Gln Pro Ala Leu Tyr Leu Tyr Asn Thr Gly Arg Ser

50 55 60

Val Tyr Val Lys Phe Gln Asp Ser Lys Pro Pro Leu Pro Pro Asp Glu

65 70 75 80

Trp Val

<210> 15

<211> 77

<212> PRT

<213> 人工

<400> 15

Met Phe Leu Arg Leu Ile Asp Asp Asn Gly Ile Val Leu Asn Ser Ile

1 5 10 15

Leu Trp Leu Leu Val Met Ile Phe Phe Phe Val Leu Ala Met Thr Phe

20 25 30

Ile Lys Leu Ile Gln Leu Cys Phe Thr Cys His Tyr Phe Phe Ser Arg

35 40 45

Thr Leu Tyr Gln Pro Val Tyr Lys Ile Phe Leu Ala Tyr Gln Asp Tyr

50 55 60

Met Gln Ile Ala Pro Val Pro Ala Glu Val Leu Asn Val

65 70 75

<210> 16

<211> 75

<212> PRT

<213> 人工

<400> 16

Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser

1 5 10 15

Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala

20 25 30

Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

35 40 45

Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

50 55 60

Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val

65 70 75

Claims

1.基于马赛克策略的冠状病毒抗原的构建方法，其特征在于，所述方法包括：

(2)采用马赛克策略对人冠状病毒的结构蛋白进行分析，去除在结构蛋白中出现次数低的罕见表位后，获得由短肽组装形成的冠状病毒抗原。

2.根据权利要求1所述的方法，其特征在于，所述人冠状病毒包括HCoV-NL63、HCoV-229e、HCoV-OC43、HCoV-HKU1、SARS-CoV、MERS-CoV或SARS-CoV-2中的任意一种或至少两种的组合。

3.根据权利要求1或2所述的方法，其特征在于，所述结构蛋白包括S蛋白、M蛋白、N蛋白或E蛋白中的任意一种或至少两种的组合；

优选地，所述S蛋白上包括221～228个细胞毒性T淋巴细胞表位；

优选地，所述M蛋白上包括51～61个细胞毒性T淋巴细胞表位；

优选地，所述N蛋白上包括54～65个细胞毒性T淋巴细胞表位；

优选地，所述E蛋白上包括14～21个细胞毒性T淋巴细胞表位。

4.根据权利要求1-3任一项所述的方法，其特征在于，所述马赛克策略包括MosaicVaccine Designer程序；

优选地，所述罕见表位在结构蛋白中出现的次数小于3；

优选地，所述短肽的长度为8～10aa。

5.根据权利要求1-4任一项所述的方法，其特征在于，所述冠状病毒抗原包括S蛋白马赛克抗原、M蛋白马赛克抗原、N蛋白马赛克抗原或E蛋白马赛克抗原中的任意一种或至少两种的组合。

6.冠状病毒抗原，其特征在于，所述冠状病毒抗原采用权利要求1-5任一项所述的方法构建得到。

7.根据权利要求6所述的冠状病毒抗原，其特征在于，所述冠状病毒抗原包括S蛋白马赛克抗原、M蛋白马赛克抗原、N蛋白马赛克抗原或E蛋白马赛克抗原中的任意一种或至少两种的组合；

优选地，所述S蛋白马赛克抗原中含有的天然细胞毒性T淋巴细胞表位的百分比为89.01％；

优选地，所述M蛋白马赛克抗原中含有的天然细胞毒性T淋巴细胞表位的百分比为87.77％；

优选地，所述N蛋白马赛克抗原中含有的天然细胞毒性T淋巴细胞表位的百分比为91.96％；

8.根据权利要求6或7所述的冠状病毒抗原，其特征在于，所述S蛋白马赛克抗原包括SEQ ID NO:1～4所示的氨基酸序列；

优选地，所述E蛋白马赛克抗原包括SEQ ID NO:13～16所示的氨基酸序列。

9.一种权利要求6-8任一项所述的冠状病毒抗原在制备冠状病毒通用疫苗中的应用。

10.一种携带权利要求6-8任一项所述的冠状病毒抗原的冠状病毒通用疫苗，其特征在于，所述疫苗以pVAX1为DNA疫苗载体，或以删除E1和E3的复制缺陷性2型和5型腺病毒为疫苗载体。