CN112409496A - 一种跨膜表达新型冠状病毒抗原s2的融合蛋白、重组载体、重组树突状细胞及其应用 - Google Patents

一种跨膜表达新型冠状病毒抗原s2的融合蛋白、重组载体、重组树突状细胞及其应用 Download PDF

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CN112409496A
CN112409496A CN202011346373.6A CN202011346373A CN112409496A CN 112409496 A CN112409496 A CN 112409496A CN 202011346373 A CN202011346373 A CN 202011346373A CN 112409496 A CN112409496 A CN 112409496A
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焦顺昌
陈静远
汪坤福
袁翰
张艳玲
李斯慧
冯向辉
宋文静
吕鹏敏
钟宏东
胡坤
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Beijing Dingcheng Taiyuan Biotechnology Co ltd
Chinese PLA General Hospital
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Abstract

本发明提供了一种跨膜表达新型冠状病毒抗原S2的融合蛋白、重组载体、重组树突状细胞及其应用，属于全细胞疫苗技术领域，所述融合蛋白，包括顺次链接的CD4信号肽、新型冠状病毒抗原S2蛋白、Flag标签序列和CD4跨膜结构域；本发明将S2单独进行跨膜的细胞表达，避免了其他S蛋白表位可能导致的ADE风险，本发明提供的融合蛋白构建的细胞疫苗在小鼠体内可以诱发出更高的中和抗体滴度。

Description

一种跨膜表达新型冠状病毒抗原S2的融合蛋白、重组载体、重 组树突状细胞及其应用
技术领域
本发明属于全细胞疫苗技术领域，尤其涉及一种跨膜表达新型冠状病毒抗原S2的融合蛋白、重组载体、重组树突状细胞及其应用。
背景技术
新型冠状病毒(2019-nCoV，SARS-CoV-2)是一种β属的冠状病毒，感染该病毒后可导致患者出现发热、干咳、乏力等症状；部分患者会产生严重的肺炎，进而发展为急性呼吸窘迫综合征、脓毒症休克、出凝血功能障碍及多器官功能衰竭等，甚至死亡。SARS-CoV-2病毒通过其表面的囊膜蛋白吸附和进入宿主细胞，从而感染人的呼吸道上皮细胞及肺泡细胞。SARS-CoV-2的囊膜蛋白即刺突蛋白(Spike protein,S)是一种糖蛋白，主要作用于细胞粘附和细胞膜融合。
S蛋白由S1和S2两个亚基组成，其中S1亚基中包含受体结合结构域(ReceptorBinding Domain,RBD)介导吸附作用，S2亚基主要体现融合活性。RBD与细胞表面血管紧张素转换酶2(Angiotensin-converting enzyme 2,ACE2)结合，使病毒和受体相互作用，是病毒入侵细胞的第一步。S2介导病毒膜和人呼吸道水平细胞和肺泡细胞膜的融合，最终使病毒入侵宿主细胞。虽然RBD是宿主中和抗体的重要作用位点以及疫苗设计的关键靶点，但是RBD在不同冠状病毒之间变异大，不具有保守性，设计的疫苗不具有通用性。
由于目前没有效果非常好的药物、治疗性抗体、疫苗等用于SARS-CoV-2病毒感染的治疗和预防，因此开发针对SARS-CoV-2病毒的疫苗对于健康人的保护及应对病毒的再次爆发是至关重要的。然而，将病毒的全长蛋白作为临床使用的抗原可以引起部分对于病毒的预防作用，但也常见到临床报道有抗体依赖性增强(Antibody-dependent enhancement，简称ADE)作用，从而有可能产生比未用疫苗者更加严重的病毒感染。
发明内容
有鉴于此，本发明的目的在于提供一种跨膜表达新型冠状病毒抗原S2的融合蛋白、重组载体、重组树突状细胞及其应用；本发明将S2单独进行跨膜的细胞表达，避免了其他S蛋白表位可能导致的ADE风险，本发明提供了一种新的融合蛋白，所述融合蛋白构建的细胞疫苗在小鼠体内可以诱发出更高的中和滴度。同时，本发明以高保守性的S2为靶点(与RaTG13和SARS CoV S2进行比较，一致性为96.49％，见图8)，设计疫苗，具有靶向不同冠状病毒的通用性。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种跨膜表达新型冠状病毒抗原S2的融合蛋白，包括顺次链接的CD4信号肽、新型冠状病毒抗原S2蛋白、Flag标签序列和CD4跨膜结构域。
优选的，所述CD4信号肽、新型冠状病毒抗原S2蛋白、Flag标签序列和CD4跨膜结构域的氨基酸序列如SEQ ID No.1～SEQ ID No.4所示。
优选的，所述CD4跨膜结构域分别替换为CD80跨膜结构域或者CD86跨膜结构域。
优选的，所述CD80跨膜结构域的氨基酸序列如SEQ ID No.5所示；所述CD86跨膜结构域的氨基酸序列如SEQ ID No.6所示。
本发明提供了编码所述融合蛋白的基因，包括顺次连接的如SEQ ID No.7～SEQID No.10所示的编码CD4信号肽的核苷酸序列、编码新型冠状病毒抗原S2蛋白的核苷酸序列、Flag标签序列的核苷酸序列和编码CD4跨膜结构域的核苷酸序列。
本发明提供了编码所述融合蛋白的基因，编码CD80跨膜结构域的核苷酸序列如SEQ ID No.11所示；编码CD86跨膜结构域的核苷酸序列如SEQ ID No.12所示。
本发明还提供了一种跨膜表达新型冠状病毒抗原S2的融合蛋白的重组载体，包括初始载体和所述编码融合蛋白的基因。
优选的，所述初始载体为PB-713质粒或pCDH-K载体；所述编码融合蛋白的基因重组于PB-713质粒的Bsu36I和BmgBI酶切位点之间；所述编码融合蛋白的基因重组于pCDH-K载体的EcoRI和BamHI酶切位点之间。
本发明提供了一种跨膜表达新型冠状病毒抗原S2融合蛋白的重组树突状细胞，通过将所述的重组载体转入树突状细胞获得。
本发明提供了所述的融合蛋白、所述编码融合蛋白的基因、所述重组载体、所述的重组树突状细胞在制备新型冠状病毒疫苗中的应用。
本发明的有益效果：本发明提供了一种跨膜表达新型冠状病毒抗原S2的融合蛋白、重组载体、重组树突状细胞及其应用；本发明提供的冠状病毒刺突糖蛋白中S2亚基内部包含FP(Fusion Peptide)，SH(Stem Helix)和HR2(Heptad Repeat 2)等结构域在介导病毒膜与宿主细胞膜的融合过程起到关键作用；所述S2蛋白具有免疫原性强、在冠状病毒之间保守度高、在诱导中和抗体方面效果突出的优势，由S2蛋白诱导产生的中和抗体可以通过阻止病毒膜与宿主细胞膜的融合而防止病毒侵入。本发明通过将所述S2蛋白与不同跨膜蛋白结构(CD4、CD80和CD86)融合表达，实现了S2蛋白的高效跨膜表达，相对于天然的S蛋白三聚体，本发明中的融合蛋白跨膜表达能力更强。
本发明采用所述重组树突状细胞免疫动物获得的中和抗体滴度更高，远远高于以S2蛋白免疫动物或者以表达S2蛋白天然构象的细胞免疫动物获得的中和抗体滴度。
附图说明
图1为流式分析检测DC对照细胞中S2的表达量；
图2为流式分析检测DC-S细胞中S2的表达量；
图3为流式分析检测DC-S2细胞中S2的表达量；
图4流式分析检测DC-S2-CD4细胞中S2的表达量；
图5流式分析检测DC-S2-CD80细胞中S2的表达量；
图6流式分析检测DC-S2-CD86细胞中S2的表达量；
图7为抗体IgG的ELISA检测；其中A为以新型冠状病毒刺突蛋白全长蛋白进行ELISA检测；B为以新冠冠状病毒膜结合结构域S2进行ELISA检测；
图8为RaTG13、SARS-CoV和SARS-CoV2 S2的序列比对结果。
具体实施方式
本发明提供了一种跨膜表达新型冠状病毒抗原S2的融合蛋白，包括顺次链接的CD4信号肽、新型冠状病毒抗原S2蛋白、Flag标签序列和CD4跨膜结构域。
在本发明中，所述CD4信号肽、新型冠状病毒抗原S2蛋白、Flag标签序列和CD4跨膜结构域的氨基酸序列优选的如SEQ ID No.1～SEQ ID No.4所示；具体如下：
CD4信号肽：MNRGVPFRHLLLVLQLALLPAATQG(SEQ ID No.1)；
新型冠状病毒抗原S2蛋白：
SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ IDNo.2)；
Flag标签序列：DYKDDDDK(SEQ ID No.3)；
CD4跨膜结构域：
KIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRH(SEQ ID No.4)。
在本发明中，所述CD4跨膜结构域替换为CD80跨膜结构域或者CD86跨膜结构域。在本发明中，所述CD80跨膜结构域的氨基酸序列如SEQ ID No.5所示；所述CD86跨膜结构域的氨基酸序列如SEQ ID No.6所示；具体如下：
CD80跨膜结构域：
VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTY(SEQ ID No.5)；
CD86跨膜结构域：
APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKW(SEQ ID No.6)。
本发明提供了编码所述融合蛋白的基因，包括顺次连接的如SEQ ID No.7～SEQID No.10所示的编码CD4信号肽的核苷酸序列、编码新型冠状病毒抗原S2蛋白的核苷酸序列、Flag标签序列的核苷酸序列和编码CD4跨膜结构域的核苷酸序列。在本发明中，当CD4跨膜结构域替换为CD80跨膜结构域或CD86跨膜结构域时，编码CD80跨膜结构域的核苷酸序列如SEQ ID No.11所示；编码CD86跨膜结构域的核苷酸序列如SEQ ID No.12所示；具体如下：
编码CD4信号肽的核苷酸序列：atgaaccggggagtcccttttaggcacttgcttctggtgctgcaactggcgctcctcccagcagccactcaggga(SEQ ID No.7)；
编码新型冠状病毒抗原S2蛋白的核苷酸序列：tctgtggcaagccagagcatcattgcctacacaatgagtctgggagcagagaactctgtggcttacagcaacaacagcattgccatcccaaccaacttcaccatctctgtgaccacagagattctgcctgtgagtatgaccaagacctctgtggactgtacaatgtatatctgtggagacagcacagagtgtagcaacctgctgctccaatatggctccttctgtacccaacttaacagggctctgacaggcattgctgtggaacaggacaagaacacccaggaggtgtttgcccaggtgaagcagatttacaagacacctccaatcaaggactttggaggcttcaacttcagccagattctgcctgacccaagcaagccaagcaagaggtccttcattgaggacctgctgttcaacaaggtgaccctggctgatgctggcttcatcaagcaatatggagactgtctgggagacattgctgccagggacctgatttgtgcccagaagttcaatggactgacagtgctgcctccactgctgacagatgagatgattgcccaatacacctctgccctgctggctggcaccatcacctctggctggacctttggagcaggagcagccctccaaatcccatttgctatgcagatggcttacaggttcaatggcattggagtgacccagaatgtgctctatgagaaccagaaactgattgccaaccagttcaactctgccattggcaagattcaggactccctgtccagcacagcctctgccctgggcaaactccaagatgtggtgaaccagaatgcccaggctctgaacaccctggtgaagcaactttccagcaactttggagccatctcctctgtgctgaatgacatcctgagcagactggacaaggtggaggctgaggtccagattgacagactgattacaggcagactccaatccctccaaacctatgtgacccaacaacttatcagggctgctgagattagggcatctgccaacctggctgccaccaagatgagtgagtgtgtgctgggacaaagcaagagggtggacttctgtggcaagggctaccacctgatgagttttccacagtctgcccctcatggagtggtgttcctgcatgtgacctatgtgcctgcccaggagaagaacttcaccacagcccctgccatctgccatgatggcaaggctcactttccaagggagggagtgtttgtgagcaatggcacccactggtttgtgacccagaggaacttctatgaaccacagattatcaccacagacaacacctttgtgtctggcaactgtgatgtggtgattggcattgtgaacaacacagtctatgacccactccaacctgaactggactccttcaaggaggaactggacaaatacttcaagaaccacaccagccctgatgtggacctgggagacatctctggcatcaatgcctctgtggtgaacatccagaaggagattgacagactgaatgaggtggctaagaacctgaatgagtccctgattgacctccaagaactgggcaaatatgaacaatacatcaagtggccatggtacatctggctgggcttcattgctggactgattgccattgtgatggtgaccataatgctgtgttgtatgacctcctgttgttcctgtctgaaaggctgttgttcctgtggctcctgttgtaagtttgatgaggatgactctgaacctgtgctgaaaggagtgaaactgcactacacc(SEQ ID No.8)；
Flag标签序列：cgattacaag gatgacgacg ataag(SEQ ID No.9)；
编码CD4跨膜结构域的核苷酸序列：
acgcgtaaaatagacatcgtggtgctagctttccagaaggcctccagcatagtctataagaaagagggggaacaggtggagttctccttcccactcgcctttacagttgaaaagctgacgggcagtggcgagctgtggtggcaggcggagagggcttcctcctccaagtcttggatcacctttgacctgaagaacaaggaagtgtctgtaaaacgggttacccaggaccctaagctccagatgggcaagaagctcccgctccacctcaccctgccccaggccttgcctcagtatgctggctctggaaacctcaccctggcccttgaagcgaaaacaggaaagttgcatcaggaagtgaacctggtggtgatgagagccactcagctccagaaaaatttgacctgtgaggtgtggggacccacctcccctaagctgatgctgagcttgaaactggagaacaaggaggcaaaggtctcgaagcgggagaaggcggtgtgggtgctgaaccctgaggcggggatgtggcagtgtctgctgagtgactcgggacaggtcctgctggaatccaacatcaaggttctgcccacatggtcgaccccggtgcagccaatggccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttctgtgtcaggtgccggcac(SEQ ID No.10)；
编码CD80跨膜结构域的核苷酸序列：
gttatccacgtgaccaaggaagtgaaagaagtggcaacgctgtcctgtggtcacaatgtttctgttgaagagctggcacaaactcgcatctactggcaaaaggagaagaaaatggtgctgactatgatgtctggggacatgaatatatggcccgagtacaagaaccggaccatctttgatatcactaataacctctccattgtgatcctggctctgcgcccatctgacgagggcacatacgagtgtgttgttctgaagtatgaaaaagacgctttcaagcgggaacacctggctgaagtgacgttatcagtcaaagctgacttccctacacctagtatatctgactttgaaattccaacttctaatattagaaggataatttgctcaacctctggaggttttccagagcctcacctctcctggttggaaaatggagaagaattaaatgccatcaacacaacagtttcccaagatcctgaaactgagctctatgctgttagcagcaaactggatttcaatatgacaaccaaccacagcttcatgtgtctcatcaagtatggacatttaagagtgaatcagaccttcaactggaatacaaccaagcaagagcattttcctgataacctgctcccatcctgggccattaccttaatctcagtaaatggaatttttgtgatatgctgcctgacctac(SEQ ID No.11)；
编码CD86跨膜结构域的核苷酸序列：
cctctgaagattcaagcttatttcaatgagactgcagacctgccatgccaatttgcaaactctcaaaaccaaagcctgagtgagctagtagtattttggcaggaccaggaaaacttggttctgaatgaggtatacttaggcaaagagaaatttgacagtgttcattccaagtatatgggccgcacaagttttgattcggacagttggaccctgagacttcacaatcttcagatcaaggacaagggcttgtatcaatgtatcatccatcacaaaaagcccacaggaatgattcgcatccaccagatgaattctgaactgtcagtgcttgctaacttcagtcaacctgaaatagtaccaatttctaatataacagaaaatgtgtacataaatttgacctgctcatctatacacggttacccagaacctaagaagatgagtgttttgctaagaaccaagaattcaactatcgagtatgatggtgttatgcagaaatctcaagataatgtcacagaactgtacgacgtttccatcagcttgtctgtttcattccctgatgttacgagcaatatgaccatcttctgtattctggaaactgacaagacgcggcttttatcttcacctttctctatagagcttgaggaccctcagcctcccccagaccacattccttggattacagctgtacttccaacagttattatatgtgtgatggttttctgtctaattctatggaaatgg(SEQ ID No.12)。
本发明还提供了一种跨膜表达新型冠状病毒抗原S2的融合蛋白的重组载体，包括初始载体和所述编码融合蛋白的基因。在本发明中，所述初始载体优选为PB-713质粒或PCDH-K载体(购自SBI公司)；当所述初始载体为PB-713质粒时，所述编码融合蛋白的基因重组于PB-713质粒的Bsu36I和BmgBI酶切位点之间；当所述初始载体为PCDH-K载体时，所述编码融合蛋白的基因重组于PCDH-K载体的EcoRI和BamHI酶切位点之间。
在本发明中，所述重组载体的制备方法没有特殊限定，采用本领域常规的双酶切连接的方法即可，本发明对所述制备方法的具体参数没有特殊限定，参照本领域工具书或试剂盒说明即可。
本发明还提供了一种跨膜表达新型冠状病毒抗原S2融合蛋白的重组树突状细胞，通过将所述的重组载体转入树突状细胞获得。在本发明中，所述树突状细胞优选的自行制备获得，具体制备方法参见中国专利CN201911397010.2；在本发明中，所述重组载体转入树突状细胞的方法优选为电转化或慢病毒感染的方法。本发明在所述转入后，优选的进行嘌呤霉素筛选；当活细胞比例持续大于80％时，得到跨膜表达新型冠状病毒抗原S2融合蛋白的重组树突状细胞。
本发明还提供了所述的融合蛋白、所述编码融合蛋白的基因、所述重组载体、所述的重组树突状细胞在制备新型冠状病毒疫苗中的应用。在本发明中，以所述重组树突状细胞制备全细胞疫苗，本发明对所述全细胞疫苗的存在形式没有特殊限定，采用本领域常规的全细胞疫苗存在形式即可。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
质粒的构建：
融合基因CD4信号肽-S2蛋白-flag标签-CD4跨膜结构域的基因合成(委托金唯智生物科技有限公司)，具体序列为：
atgaaccggggagtcccttttaggcacttgcttctggtgctgcaactggcgctcctcccagcagccactcagggaagctttatcgaggacctgctgttcaacaaggtgaccctggccgatgccggcttcatcaagcagtatggcgattgcctgggcgacatcgcagccagggacctgatctgcgcccagaagtttaatggcctgaccgtgctgccacccctgctgacagatgagatgatcgcacagtacacaagcgccctgctggccggcaccatcacatccggatggaccttcggcgcaggagccgccctccagatcccctttgccatgcagatggcctataggttcaacggcatcggcgtgacccagaatgtgctgtacgagaaccagaagctgatcgccaatcagtttaactccgccatcggcaagatccaggacagcctgtcctctacagccagcgccctgggcaagctccaggatgtggtgaatcagaacgcccaggccctgaataccctggtgaagcagctgagcagcaacttcggcgccatctctagcgtgctgaatgacatcctgagccggctggacaaggtggaggcagaggtgcagatcgaccggctgatcaccggccggctccagagcctccagacctatgtgacacagcagctgatcagggccgccgagatcagggccagcgccaatctggcagcaaccaagatgtccgagtgcgtgctgggccagtctaagagagtggacttttgtggcaagggctatcacctgatgtccttccctcagtctgccccacacggcgtggtgtttctgcacgtgacctacgtgcccgcccaggagaagaacttcaccacagcccctgccatctgccacgatggcaaggcccactttccaagggagggcgtgttcgtgtccaacggcacccactggtttgtgacacagcgcaatttctacgagccccagatcatcaccacagacaacaccttcgtgagcggcaactgtgacgtggtcatcggcatcgtgaacaataccgtgtatgatccactccagcccgagctggacagctttaaggaggagctggataagtatttcaagaatcacacctcccctgacgtggatctgggcgacatcagcggcatcaatgcctccgtggtgaacatccagaaggagatcgaccgcctgaacgaggtggctaagaatctgaacgagagcctgatcgacctccaggagcgattacaaggatgacgacgataagacgcgtaaaatagacatcgtggtgctagctttccagaaggcctccagcatagtctataagaaagagggggaacaggtggagttctccttcccactcgcctttacagttgaaaagctgacgggcagtggcgagctgtggtggcaggcggagagggcttcctcctccaagtcttggatcacctttgacctgaagaacaaggaagtgtctgtaaaacgggttacccaggaccctaagctccagatgggcaagaagctcccgctccacctcaccctgccccaggccttgcctcagtatgctggctctggaaacctcaccctggcccttgaagcgaaaacaggaaagttgcatcaggaagtgaacctggtggtgatgagagccactcagctccagaaaaatttgacctgtgaggtgtggggacccacctcccctaagctgatgctgagcttgaaactggagaacaaggaggcaaaggtctcgaagcgggagaaggcggtgtgggtgctgaaccctgaggcggggatgtggcagtgtctgctgagtgactcgggacaggtcctgctggaatccaacatcaaggttctgcccacatggtcgaccccggtgcagccaatggccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttctgtgtcaggtgccggcac(SEQ ID No.13)。
合成基因后，将所述合成的基因通过上游Bsu36I和下游BmgBI双酶切位点，整合进入PB-713质粒(购自SBI公司)获得PB-S2-CD4质粒。
酶切体系：
Figure BDA0002799932120000051
37℃酶切15分钟后进行胶回收，然后进行连接，连接体系见下表：
Figure BDA0002799932120000052
16℃连接过夜。然后转化感受态stbl3细胞。将所述合成的基因通过上游EcoRI和下游BamHI双酶切位点插入pCDH-K载体(购自SBI公司)上获得pCDH-K-S2-CD4质粒。
酶切体系：
Figure BDA0002799932120000053
37℃酶切15min后进行胶回收，然后进行连接，连接体系见下表：
Figure BDA0002799932120000061
16℃连接过夜，然后转化感受态stbl3细胞。
由金唯智生物科技有限公司对制备成的质粒进行大量抽提和去除内毒素。
融合基因CD4信号肽-S2蛋白-flag标签-CD80跨膜结构域基因合成(委托金唯智生物科技有限公司)。
融合基因CD4信号肽-S2蛋白-flag标签-CD86跨膜结构域基因合成(委托金唯智生物科技有限公司)。
合成基因后，将所述合成的基因通过上游Bsu36I和下游BmgBI双酶切位点，整合进入PB-713质粒(购自SBI公司)，对应获得PB-S2-CD80质粒和PB-S2-CD86质粒。
将基因S2通过上游Bsu36I和下游BmgBI双酶切位点，整合进入PB-713质粒(购自SBI公司)，获得PB-S2质粒。
将基因S通过上游Bsu36I和下游BmgBI双酶切位点，整合进入PB-713质粒(购自SBI公司)，获得PB-S质粒；
将基因S通过上游EcoRI和下游BamHI双酶切位点，整合进入pCDH-K质粒(购自SBI公司)，获得pCDH-K-S质粒(委托金唯智生物科技有限公司构建)。
全长S核苷酸序列(SEQ ID No.14)：
atgtttgtgttcctggtgctgctgccactggtgtccagccagtgtgtgaacctgaccaccaggacccaacttcctcctgcctacaccaactccttcaccaggggagtctactaccctgacaaggtgttcaggtcctctgtgctgcacagcacccaggacctgttcctgccattcttcagcaatgtgacctggttccatgccatccatgtgtctggcaccaatggcaccaagaggtttgacaaccctgtgctgccattcaatgatggagtctactttgccagcacagagaagagcaacatcatcaggggctggatttttggcaccaccctggacagcaagacccagtccctgctgattgtgaacaatgccaccaatgtggtgattaaggtgtgtgagttccagttctgtaatgacccattcctgggagtctactaccacaagaacaacaagtcctggatggagtctgagttcagggtctactcctctgccaacaactgtacctttgaatatgtgagccaaccattcctgatggacttggagggcaagcagggcaacttcaagaacctgagggagtttgtgttcaagaacattgatggctacttcaagatttacagcaaacacacaccaatcaacctggtgagggacctgccacagggcttctctgccttggaaccactggtggacctgccaattggcatcaacatcaccaggttccagaccctgctggctctgcacaggtcctacctgacacctggagactcctcctctggctggacagcaggagcagcagcctactatgtgggctacctccaaccaaggaccttcctgctgaaatacaatgagaatggcaccatcacagatgctgtggactgtgccctggacccactgtctgagaccaagtgtaccctgaaatccttcacagtggagaagggcatctaccagaccagcaacttcagggtccaaccaacagagagcattgtgaggtttccaaacatcaccaacctgtgtccatttggagaggtgttcaatgccaccaggtttgcctctgtctatgcctggaacaggaagaggattagcaactgtgtggctgactactctgtgctctacaactctgcctccttcagcaccttcaagtgttatggagtgagcccaaccaaactgaatgacctgtgtttcaccaatgtctatgctgactcctttgtgattaggggagatgaggtgagacagattgcccctggacaaacaggcaagattgctgactacaactacaaactgcctgatgacttcacaggctgtgtgattgcctggaacagcaacaacctggacagcaaggtgggaggcaactacaactacctctacagactgttcaggaagagcaacctgaaaccatttgagagggacatcagcacagagatttaccaggctggcagcacaccatgtaatggagtggagggcttcaactgttactttccactccaatcctatggcttccaaccaaccaatggagtgggctaccaaccatacagggtggtggtgctgtcctttgaactgctccatgcccctgccacagtgtgtggaccaaagaagagcaccaacctggtgaagaacaagtgtgtgaacttcaacttcaatggactgacaggcacaggagtgctgacagagagcaacaagaagttcctgccattccaacagtttggcagggacattgctgacaccacagatgctgtgagggacccacagaccttggagattctggacatcacaccatgttcctttggaggagtgtctgtgattacacctggcaccaacaccagcaaccaggtggctgtgctctaccaggatgtgaactgtactgaggtgcctgtggctatccatgctgaccaacttacaccaacctggagggtctacagcacaggcagcaatgtgttccagaccagggctggctgtctgattggagcagagcatgtgaacaactcctatgagtgtgacatcccaattggagcaggcatctgtgcctcctaccagacccagaccaacagcccaaggagggcaaggtctgtggcaagccagagcatcattgcctacacaatgagtctgggagcagagaactctgtggcttacagcaacaacagcattgccatcccaaccaacttcaccatctctgtgaccacagagattctgcctgtgagtatgaccaagacctctgtggactgtacaatgtatatctgtggagacagcacagagtgtagcaacctgctgctccaatatggctccttctgtacccaacttaacagggctctgacaggcattgctgtggaacaggacaagaacacccaggaggtgtttgcccaggtgaagcagatttacaagacacctccaatcaaggactttggaggcttcaacttcagccagattctgcctgacccaagcaagccaagcaagaggtccttcattgaggacctgctgttcaacaaggtgaccctggctgatgctggcttcatcaagcaatatggagactgtctgggagacattgctgccagggacctgatttgtgcccagaagttcaatggactgacagtgctgcctccactgctgacagatgagatgattgcccaatacacctctgccctgctggctggcaccatcacctctggctggacctttggagcaggagcagccctccaaatcccatttgctatgcagatggcttacaggttcaatggcattggagtgacccagaatgtgctctatgagaaccagaaactgattgccaaccagttcaactctgccattggcaagattcaggactccctgtccagcacagcctctgccctgggcaaactccaagatgtggtgaaccagaatgcccaggctctgaacaccctggtgaagcaactttccagcaactttggagccatctcctctgtgctgaatgacatcctgagcagactggacaaggtggaggctgaggtccagattgacagactgattacaggcagactccaatccctccaaacctatgtgacccaacaacttatcagggctgctgagattagggcatctgccaacctggctgccaccaagatgagtgagtgtgtgctgggacaaagcaagagggtggacttctgtggcaagggctaccacctgatgagttttccacagtctgcccctcatggagtggtgttcctgcatgtgacctatgtgcctgcccaggagaagaacttcaccacagcccctgccatctgccatgatggcaaggctcactttccaagggagggagtgtttgtgagcaatggcacccactggtttgtgacccagaggaacttctatgaaccacagattatcaccacagacaacacctttgtgtctggcaactgtgatgtggtgattggcattgtgaacaacacagtctatgacccactccaacctgaactggactccttcaaggaggaactggacaaatacttcaagaaccacaccagccctgatgtggacctgggagacatctctggcatcaatgcctctgtggtgaacatccagaaggagattgacagactgaatgaggtggctaagaacctgaatgagtccctgattgacctccaagaactgggcaaatatgaacaatacatcaagtggccatggtacatctggctgggcttcattgctggactgattgccattgtgatggtgaccataatgctgtgttgtatgacctcctgttgttcctgtctgaaaggctgttgttcctgtggctcctgttgtaagtttgatgaggatgactctgaacctgtgctgaaaggagtgaaactgcactacacc
全长S氨基酸序列(SEQ ID No.15)：
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
由金唯智生物科技有限公司对制备成的质粒进行大量抽提和去除内毒素。
细胞的转染：
1.树突状细胞(DC)的培养
树突状细胞(DC)具体制备方法参见中国专利CN201911397010.2。
培养条件为：培养基：1640+10％(体积百分含量)FBS+200IU/mL IL-2。传代方法：维持细胞浓度在1×105～1×106/ml，每2～3天离心去除旧培养基，按密度将细胞悬浮于新鲜培养基。
2.使用Celetrix电转仪，按照使用说明书对树突状细胞进行质粒电转。取4×107个数的树突状细胞，在120μl电击管，1×107cells/管，分别加10μg的PB-S质粒、PB-S2质粒、PB-S2-CD4质粒、PB-S2-CD80质粒和PB-S2-CD86质粒，电转条件为：560V、30ms。随后取出细胞继续按原条件培养。
3.电转48h后，在原培养基基础上，加入嘌呤霉素到2μg/ml的浓度进行药筛分别获得DC-S、DC-S2、DC-S2-CD4、DC-S2-CD80和DC-S2-CD86细胞。一周后，通过流式分析检测DC-S、DC-S2、DC-S2-CD4、DC-S2-CD80和DC-S2-CD86细胞中S2的表达量；见图1～6，其中图1为DC对照中S2的表达量0.48％。
流式分析结果表明：DC细胞电转S2-CD4后，在用2μg/ml的嘌呤霉素药筛一周后DC-S2-CD4和DC-S2-CD80细胞S2表达量为100％，DC-S2-CD86 S2表达量98.7％，远超过全长S(52.3％)和DC-S2(66.6％)的表达量。
实施例2
材料：
细胞：DC细胞
培养基：1640完全培养基
ELISA抗体：
Biotinylated2019 n-Cov Spike S-His(KACTUS公司)
Spike S2(SARS-Cov-2)(HEK293)(北京义翘神州生物技术有限公司)
GoaAnti-Mouse IgG-HRP(1:10000)
小鼠：雌性Balb/c小鼠(5周，25g左右)
方法：
将DC-pCDH-K-S2-CD4和DC-PCDH-K-S2、DC-PCDH-K-S等三种细胞各取1×108cell，然后用1ml的PBS重悬细胞。
动物实验共分六组：①PBS组、②DC-S2-CD4组、③DC-PCDH-K-S2组、④DC-PCDH-K-S组，每组4只小鼠，每支只小鼠注射2×107cells。
14天后每只小鼠取200μl血分离血清：将采集的血清放37℃培养箱温浴1h，然后3000rpm离心20min，离心结束后，将上层血清转移到标记好的1.5ml离心管，然后进行ELISA检测。
ELISA包被：将S全长蛋白和S2蛋白按照1μg/ml的浓度分别包被3块ELISA板，100μl/孔，4℃过夜包被。
ELISA封闭：用TBST配置120ml的5％脱脂牛奶，200μl/孔，37℃培养箱孵育1h。
ELISA上样：将16管血清分别按照1:100、1:1000、1:10000稀释三个梯度，加入到封闭好的ELISA板中，每管血清做3次重复，100μl/孔，37℃培养箱孵育2～3h。
ELISA二抗：将GoaAnti-Mouse IgG-HRP抗体按照1:10000的比例用PBS稀释60ml，100μl/孔，室温孵育40min。
ELISA显色：每孔加入100μl TMB显色液避光显色5min后再加入100μl终止液。
ELISA检测小鼠血清抗体滴度值见表1和表2。
表1全长S蛋白进行ELISA检测小鼠血清抗体滴度值
名称 S S2 S2-CD4
1 1000 2000 5000
2 1090 2500 5900
3 787 2800 5100
4 1200 1900 3300
平均值 1019.25 2300 4825
标准差 87.55 212.1 546.8
表2 S2蛋白进行ELISA检测小鼠血清抗体滴度值
Figure BDA0002799932120000081
Figure BDA0002799932120000091
结果显示：与对照组相比，S2-CD4细胞可使小鼠体内产生S2抗体，在血清稀释比例为1:10000的条件下可以在DC-PCDH-K-S2-CD4组小鼠体内检测到S2抗体(10300)，抗体滴度较DC-PCDH-K-S(1027)、DC-PCDH-K-S2(1550)高，如图7所示，抗体滴度较对照组高。
由上述实施例可知，本发明提供的重组树突状细胞能够高效的跨膜表达S2蛋白，免疫动物获得的中和抗体滴度高，能够作为防治新型冠状病毒的全细胞疫苗应用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 焦顺昌
北京鼎成肽源生物技术有限公司
<120> 一种跨膜表达新型冠状病毒抗原S2的融合蛋白、重组载体、重组树突状细胞及其应用
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 1
Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gln Leu
1 5 10 15
Ala Leu Leu Pro Ala Ala Thr Gln Gly
20 25
<210> 2
<211> 588
<212> PRT
<213> Artificial Sequence
<400> 2
Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala
1 5 10 15
Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn
20 25 30
Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys
35 40 45
Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys
50 55 60
Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg
65 70 75 80
Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val
85 90 95
Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe
100 105 110
Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser
115 120 125
Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala
130 135 140
Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala
145 150 155 160
Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
165 170 175
Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu
180 185 190
Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala
195 200 205
Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
210 215 220
Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn
225 230 235 240
Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr
245 250 255
Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
260 265 270
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
275 280 285
Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala
290 295 300
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
305 310 315 320
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
325 330 335
Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser
340 345 350
Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
355 360 365
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
370 375 380
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly
385 390 395 400
Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His
405 410 415
Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr
420 425 430
Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val
435 440 445
Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
450 455 460
Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp
465 470 475 480
Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
485 490 495
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
500 505 510
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro
515 520 525
Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met
530 535 540
Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
545 550 555 560
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser
565 570 575
Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
580 585
<210> 3
<211> 8
<212> PRT
<213> Artificial Sequence
<400> 3
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 4
<211> 229
<212> PRT
<213> Artificial Sequence
<400> 4
Lys Ile Asp Ile Val Val Leu Ala Phe Gln Lys Ala Ser Ser Ile Val
1 5 10 15
Tyr Lys Lys Glu Gly Glu Gln Val Glu Phe Ser Phe Pro Leu Ala Phe
20 25 30
Thr Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp Gln Ala Glu
35 40 45
Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu Lys Asn Lys
50 55 60
Glu Val Ser Val Lys Arg Val Thr Gln Asp Pro Lys Leu Gln Met Gly
65 70 75 80
Lys Lys Leu Pro Leu His Leu Thr Leu Pro Gln Ala Leu Pro Gln Tyr
85 90 95
Ala Gly Ser Gly Asn Leu Thr Leu Ala Leu Glu Ala Lys Thr Gly Lys
100 105 110
Leu His Gln Glu Val Asn Leu Val Val Met Arg Ala Thr Gln Leu Gln
115 120 125
Lys Asn Leu Thr Cys Glu Val Trp Gly Pro Thr Ser Pro Lys Leu Met
130 135 140
Leu Ser Leu Lys Leu Glu Asn Lys Glu Ala Lys Val Ser Lys Arg Glu
145 150 155 160
Lys Ala Val Trp Val Leu Asn Pro Glu Ala Gly Met Trp Gln Cys Leu
165 170 175
Leu Ser Asp Ser Gly Gln Val Leu Leu Glu Ser Asn Ile Lys Val Leu
180 185 190
Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile Val Leu Gly
195 200 205
Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile Phe Phe Cys
210 215 220
Val Arg Cys Arg His
225
<210> 5
<211> 231
<212> PRT
<213> Artificial Sequence
<400> 5
Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys
1 5 10 15
Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp
20 25 30
Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn
35 40 45
Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn
50 55 60
Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr
65 70 75 80
Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His
85 90 95
Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser
100 105 110
Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys
115 120 125
Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn
130 135 140
Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu
145 150 155 160
Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr
165 170 175
Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn
180 185 190
Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn
195 200 205
Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile Phe
210 215 220
Val Ile Cys Cys Leu Thr Tyr
225 230
<210> 6
<211> 247
<212> PRT
<213> Artificial Sequence
<400> 6
Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Glu Thr Ala Asp Leu Pro
1 5 10 15
Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser Glu Leu Val Val
20 25 30
Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu Val Tyr Leu Gly
35 40 45
Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly Arg Thr Ser
50 55 60
Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn Leu Gln Ile Lys
65 70 75 80
Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro Thr Gly
85 90 95
Met Ile Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala Asn
100 105 110
Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu Asn Val
115 120 125
Tyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu Pro Lys
130 135 140
Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu Tyr Asp
145 150 155 160
Gly Val Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr Asp Val
165 170 175
Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Thr Ser Asn Met Thr
180 185 190
Ile Phe Cys Ile Leu Glu Thr Asp Lys Thr Arg Leu Leu Ser Ser Pro
195 200 205
Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp His Ile Pro
210 215 220
Trp Ile Thr Ala Val Leu Pro Thr Val Ile Ile Cys Val Met Val Phe
225 230 235 240
Cys Leu Ile Leu Trp Lys Trp
245
<210> 7
<211> 75
<212> DNA
<213> Artificial Sequence
<400> 7
atgaaccggg gagtcccttt taggcacttg cttctggtgc tgcaactggc gctcctccca 60
gcagccactc aggga 75
<210> 8
<211> 1764
<212> DNA
<213> Artificial Sequence
<400> 8
tctgtggcaa gccagagcat cattgcctac acaatgagtc tgggagcaga gaactctgtg 60
gcttacagca acaacagcat tgccatccca accaacttca ccatctctgt gaccacagag 120
attctgcctg tgagtatgac caagacctct gtggactgta caatgtatat ctgtggagac 180
agcacagagt gtagcaacct gctgctccaa tatggctcct tctgtaccca acttaacagg 240
gctctgacag gcattgctgt ggaacaggac aagaacaccc aggaggtgtt tgcccaggtg 300
aagcagattt acaagacacc tccaatcaag gactttggag gcttcaactt cagccagatt 360
ctgcctgacc caagcaagcc aagcaagagg tccttcattg aggacctgct gttcaacaag 420
gtgaccctgg ctgatgctgg cttcatcaag caatatggag actgtctggg agacattgct 480
gccagggacc tgatttgtgc ccagaagttc aatggactga cagtgctgcc tccactgctg 540
acagatgaga tgattgccca atacacctct gccctgctgg ctggcaccat cacctctggc 600
tggacctttg gagcaggagc agccctccaa atcccatttg ctatgcagat ggcttacagg 660
ttcaatggca ttggagtgac ccagaatgtg ctctatgaga accagaaact gattgccaac 720
cagttcaact ctgccattgg caagattcag gactccctgt ccagcacagc ctctgccctg 780
ggcaaactcc aagatgtggt gaaccagaat gcccaggctc tgaacaccct ggtgaagcaa 840
ctttccagca actttggagc catctcctct gtgctgaatg acatcctgag cagactggac 900
aaggtggagg ctgaggtcca gattgacaga ctgattacag gcagactcca atccctccaa 960
acctatgtga cccaacaact tatcagggct gctgagatta gggcatctgc caacctggct 1020
gccaccaaga tgagtgagtg tgtgctggga caaagcaaga gggtggactt ctgtggcaag 1080
ggctaccacc tgatgagttt tccacagtct gcccctcatg gagtggtgtt cctgcatgtg 1140
acctatgtgc ctgcccagga gaagaacttc accacagccc ctgccatctg ccatgatggc 1200
aaggctcact ttccaaggga gggagtgttt gtgagcaatg gcacccactg gtttgtgacc 1260
cagaggaact tctatgaacc acagattatc accacagaca acacctttgt gtctggcaac 1320
tgtgatgtgg tgattggcat tgtgaacaac acagtctatg acccactcca acctgaactg 1380
gactccttca aggaggaact ggacaaatac ttcaagaacc acaccagccc tgatgtggac 1440
ctgggagaca tctctggcat caatgcctct gtggtgaaca tccagaagga gattgacaga 1500
ctgaatgagg tggctaagaa cctgaatgag tccctgattg acctccaaga actgggcaaa 1560
tatgaacaat acatcaagtg gccatggtac atctggctgg gcttcattgc tggactgatt 1620
gccattgtga tggtgaccat aatgctgtgt tgtatgacct cctgttgttc ctgtctgaaa 1680
ggctgttgtt cctgtggctc ctgttgtaag tttgatgagg atgactctga acctgtgctg 1740
aaaggagtga aactgcacta cacc 1764
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 9
cgattacaag gatgacgacg ataag 25
<210> 10
<211> 693
<212> DNA
<213> Artificial Sequence
<400> 10
acgcgtaaaa tagacatcgt ggtgctagct ttccagaagg cctccagcat agtctataag 60
aaagaggggg aacaggtgga gttctccttc ccactcgcct ttacagttga aaagctgacg 120
ggcagtggcg agctgtggtg gcaggcggag agggcttcct cctccaagtc ttggatcacc 180
tttgacctga agaacaagga agtgtctgta aaacgggtta cccaggaccc taagctccag 240
atgggcaaga agctcccgct ccacctcacc ctgccccagg ccttgcctca gtatgctggc 300
tctggaaacc tcaccctggc ccttgaagcg aaaacaggaa agttgcatca ggaagtgaac 360
ctggtggtga tgagagccac tcagctccag aaaaatttga cctgtgaggt gtggggaccc 420
acctccccta agctgatgct gagcttgaaa ctggagaaca aggaggcaaa ggtctcgaag 480
cgggagaagg cggtgtgggt gctgaaccct gaggcgggga tgtggcagtg tctgctgagt 540
gactcgggac aggtcctgct ggaatccaac atcaaggttc tgcccacatg gtcgaccccg 600
gtgcagccaa tggccctgat tgtgctgggg ggcgtcgccg gcctcctgct tttcattggg 660
ctaggcatct tcttctgtgt caggtgccgg cac 693
<210> 11
<211> 693
<212> DNA
<213> Artificial Sequence
<400> 11
gttatccacg tgaccaagga agtgaaagaa gtggcaacgc tgtcctgtgg tcacaatgtt 60
tctgttgaag agctggcaca aactcgcatc tactggcaaa aggagaagaa aatggtgctg 120
actatgatgt ctggggacat gaatatatgg cccgagtaca agaaccggac catctttgat 180
atcactaata acctctccat tgtgatcctg gctctgcgcc catctgacga gggcacatac 240
gagtgtgttg ttctgaagta tgaaaaagac gctttcaagc gggaacacct ggctgaagtg 300
acgttatcag tcaaagctga cttccctaca cctagtatat ctgactttga aattccaact 360
tctaatatta gaaggataat ttgctcaacc tctggaggtt ttccagagcc tcacctctcc 420
tggttggaaa atggagaaga attaaatgcc atcaacacaa cagtttccca agatcctgaa 480
actgagctct atgctgttag cagcaaactg gatttcaata tgacaaccaa ccacagcttc 540
atgtgtctca tcaagtatgg acatttaaga gtgaatcaga ccttcaactg gaatacaacc 600
aagcaagagc attttcctga taacctgctc ccatcctggg ccattacctt aatctcagta 660
aatggaattt ttgtgatatg ctgcctgacc tac 693
<210> 12
<211> 738
<212> DNA
<213> Artificial Sequence
<400> 12
cctctgaaga ttcaagctta tttcaatgag actgcagacc tgccatgcca atttgcaaac 60
tctcaaaacc aaagcctgag tgagctagta gtattttggc aggaccagga aaacttggtt 120
ctgaatgagg tatacttagg caaagagaaa tttgacagtg ttcattccaa gtatatgggc 180
cgcacaagtt ttgattcgga cagttggacc ctgagacttc acaatcttca gatcaaggac 240
aagggcttgt atcaatgtat catccatcac aaaaagccca caggaatgat tcgcatccac 300
cagatgaatt ctgaactgtc agtgcttgct aacttcagtc aacctgaaat agtaccaatt 360
tctaatataa cagaaaatgt gtacataaat ttgacctgct catctataca cggttaccca 420
gaacctaaga agatgagtgt tttgctaaga accaagaatt caactatcga gtatgatggt 480
gttatgcaga aatctcaaga taatgtcaca gaactgtacg acgtttccat cagcttgtct 540
gtttcattcc ctgatgttac gagcaatatg accatcttct gtattctgga aactgacaag 600
acgcggcttt tatcttcacc tttctctata gagcttgagg accctcagcc tcccccagac 660
cacattcctt ggattacagc tgtacttcca acagttatta tatgtgtgat ggttttctgt 720
ctaattctat ggaaatgg 738
<210> 13
<211> 1954
<212> DNA
<213> Artificial Sequence
<400> 13
atgaaccggg gagtcccttt taggcacttg cttctggtgc tgcaactggc gctcctccca 60
gcagccactc agggaagctt tatcgaggac ctgctgttca acaaggtgac cctggccgat 120
gccggcttca tcaagcagta tggcgattgc ctgggcgaca tcgcagccag ggacctgatc 180
tgcgcccaga agtttaatgg cctgaccgtg ctgccacccc tgctgacaga tgagatgatc 240
gcacagtaca caagcgccct gctggccggc accatcacat ccggatggac cttcggcgca 300
ggagccgccc tccagatccc ctttgccatg cagatggcct ataggttcaa cggcatcggc 360
gtgacccaga atgtgctgta cgagaaccag aagctgatcg ccaatcagtt taactccgcc 420
atcggcaaga tccaggacag cctgtcctct acagccagcg ccctgggcaa gctccaggat 480
gtggtgaatc agaacgccca ggccctgaat accctggtga agcagctgag cagcaacttc 540
ggcgccatct ctagcgtgct gaatgacatc ctgagccggc tggacaaggt ggaggcagag 600
gtgcagatcg accggctgat caccggccgg ctccagagcc tccagaccta tgtgacacag 660
cagctgatca gggccgccga gatcagggcc agcgccaatc tggcagcaac caagatgtcc 720
gagtgcgtgc tgggccagtc taagagagtg gacttttgtg gcaagggcta tcacctgatg 780
tccttccctc agtctgcccc acacggcgtg gtgtttctgc acgtgaccta cgtgcccgcc 840
caggagaaga acttcaccac agcccctgcc atctgccacg atggcaaggc ccactttcca 900
agggagggcg tgttcgtgtc caacggcacc cactggtttg tgacacagcg caatttctac 960
gagccccaga tcatcaccac agacaacacc ttcgtgagcg gcaactgtga cgtggtcatc 1020
ggcatcgtga acaataccgt gtatgatcca ctccagcccg agctggacag ctttaaggag 1080
gagctggata agtatttcaa gaatcacacc tcccctgacg tggatctggg cgacatcagc 1140
ggcatcaatg cctccgtggt gaacatccag aaggagatcg accgcctgaa cgaggtggct 1200
aagaatctga acgagagcct gatcgacctc caggagcgat tacaaggatg acgacgataa 1260
gacgcgtaaa atagacatcg tggtgctagc tttccagaag gcctccagca tagtctataa 1320
gaaagagggg gaacaggtgg agttctcctt cccactcgcc tttacagttg aaaagctgac 1380
gggcagtggc gagctgtggt ggcaggcgga gagggcttcc tcctccaagt cttggatcac 1440
ctttgacctg aagaacaagg aagtgtctgt aaaacgggtt acccaggacc ctaagctcca 1500
gatgggcaag aagctcccgc tccacctcac cctgccccag gccttgcctc agtatgctgg 1560
ctctggaaac ctcaccctgg cccttgaagc gaaaacagga aagttgcatc aggaagtgaa 1620
cctggtggtg atgagagcca ctcagctcca gaaaaatttg acctgtgagg tgtggggacc 1680
cacctcccct aagctgatgc tgagcttgaa actggagaac aaggaggcaa aggtctcgaa 1740
gcgggagaag gcggtgtggg tgctgaaccc tgaggcgggg atgtggcagt gtctgctgag 1800
tgactcggga caggtcctgc tggaatccaa catcaaggtt ctgcccacat ggtcgacccc 1860
ggtgcagcca atggccctga ttgtgctggg gggcgtcgcc ggcctcctgc ttttcattgg 1920
gctaggcatc ttcttctgtg tcaggtgccg gcac 1954
<210> 14
<211> 3819
<212> DNA
<213> SARS-CoV-2
<400> 14
atgtttgtgt tcctggtgct gctgccactg gtgtccagcc agtgtgtgaa cctgaccacc 60
aggacccaac ttcctcctgc ctacaccaac tccttcacca ggggagtcta ctaccctgac 120
aaggtgttca ggtcctctgt gctgcacagc acccaggacc tgttcctgcc attcttcagc 180
aatgtgacct ggttccatgc catccatgtg tctggcacca atggcaccaa gaggtttgac 240
aaccctgtgc tgccattcaa tgatggagtc tactttgcca gcacagagaa gagcaacatc 300
atcaggggct ggatttttgg caccaccctg gacagcaaga cccagtccct gctgattgtg 360
aacaatgcca ccaatgtggt gattaaggtg tgtgagttcc agttctgtaa tgacccattc 420
ctgggagtct actaccacaa gaacaacaag tcctggatgg agtctgagtt cagggtctac 480
tcctctgcca acaactgtac ctttgaatat gtgagccaac cattcctgat ggacttggag 540
ggcaagcagg gcaacttcaa gaacctgagg gagtttgtgt tcaagaacat tgatggctac 600
ttcaagattt acagcaaaca cacaccaatc aacctggtga gggacctgcc acagggcttc 660
tctgccttgg aaccactggt ggacctgcca attggcatca acatcaccag gttccagacc 720
ctgctggctc tgcacaggtc ctacctgaca cctggagact cctcctctgg ctggacagca 780
ggagcagcag cctactatgt gggctacctc caaccaagga ccttcctgct gaaatacaat 840
gagaatggca ccatcacaga tgctgtggac tgtgccctgg acccactgtc tgagaccaag 900
tgtaccctga aatccttcac agtggagaag ggcatctacc agaccagcaa cttcagggtc 960
caaccaacag agagcattgt gaggtttcca aacatcacca acctgtgtcc atttggagag 1020
gtgttcaatg ccaccaggtt tgcctctgtc tatgcctgga acaggaagag gattagcaac 1080
tgtgtggctg actactctgt gctctacaac tctgcctcct tcagcacctt caagtgttat 1140
ggagtgagcc caaccaaact gaatgacctg tgtttcacca atgtctatgc tgactccttt 1200
gtgattaggg gagatgaggt gagacagatt gcccctggac aaacaggcaa gattgctgac 1260
tacaactaca aactgcctga tgacttcaca ggctgtgtga ttgcctggaa cagcaacaac 1320
ctggacagca aggtgggagg caactacaac tacctctaca gactgttcag gaagagcaac 1380
ctgaaaccat ttgagaggga catcagcaca gagatttacc aggctggcag cacaccatgt 1440
aatggagtgg agggcttcaa ctgttacttt ccactccaat cctatggctt ccaaccaacc 1500
aatggagtgg gctaccaacc atacagggtg gtggtgctgt cctttgaact gctccatgcc 1560
cctgccacag tgtgtggacc aaagaagagc accaacctgg tgaagaacaa gtgtgtgaac 1620
ttcaacttca atggactgac aggcacagga gtgctgacag agagcaacaa gaagttcctg 1680
ccattccaac agtttggcag ggacattgct gacaccacag atgctgtgag ggacccacag 1740
accttggaga ttctggacat cacaccatgt tcctttggag gagtgtctgt gattacacct 1800
ggcaccaaca ccagcaacca ggtggctgtg ctctaccagg atgtgaactg tactgaggtg 1860
cctgtggcta tccatgctga ccaacttaca ccaacctgga gggtctacag cacaggcagc 1920
aatgtgttcc agaccagggc tggctgtctg attggagcag agcatgtgaa caactcctat 1980
gagtgtgaca tcccaattgg agcaggcatc tgtgcctcct accagaccca gaccaacagc 2040
ccaaggaggg caaggtctgt ggcaagccag agcatcattg cctacacaat gagtctggga 2100
gcagagaact ctgtggctta cagcaacaac agcattgcca tcccaaccaa cttcaccatc 2160
tctgtgacca cagagattct gcctgtgagt atgaccaaga cctctgtgga ctgtacaatg 2220
tatatctgtg gagacagcac agagtgtagc aacctgctgc tccaatatgg ctccttctgt 2280
acccaactta acagggctct gacaggcatt gctgtggaac aggacaagaa cacccaggag 2340
gtgtttgccc aggtgaagca gatttacaag acacctccaa tcaaggactt tggaggcttc 2400
aacttcagcc agattctgcc tgacccaagc aagccaagca agaggtcctt cattgaggac 2460
ctgctgttca acaaggtgac cctggctgat gctggcttca tcaagcaata tggagactgt 2520
ctgggagaca ttgctgccag ggacctgatt tgtgcccaga agttcaatgg actgacagtg 2580
ctgcctccac tgctgacaga tgagatgatt gcccaataca cctctgccct gctggctggc 2640
accatcacct ctggctggac ctttggagca ggagcagccc tccaaatccc atttgctatg 2700
cagatggctt acaggttcaa tggcattgga gtgacccaga atgtgctcta tgagaaccag 2760
aaactgattg ccaaccagtt caactctgcc attggcaaga ttcaggactc cctgtccagc 2820
acagcctctg ccctgggcaa actccaagat gtggtgaacc agaatgccca ggctctgaac 2880
accctggtga agcaactttc cagcaacttt ggagccatct cctctgtgct gaatgacatc 2940
ctgagcagac tggacaaggt ggaggctgag gtccagattg acagactgat tacaggcaga 3000
ctccaatccc tccaaaccta tgtgacccaa caacttatca gggctgctga gattagggca 3060
tctgccaacc tggctgccac caagatgagt gagtgtgtgc tgggacaaag caagagggtg 3120
gacttctgtg gcaagggcta ccacctgatg agttttccac agtctgcccc tcatggagtg 3180
gtgttcctgc atgtgaccta tgtgcctgcc caggagaaga acttcaccac agcccctgcc 3240
atctgccatg atggcaaggc tcactttcca agggagggag tgtttgtgag caatggcacc 3300
cactggtttg tgacccagag gaacttctat gaaccacaga ttatcaccac agacaacacc 3360
tttgtgtctg gcaactgtga tgtggtgatt ggcattgtga acaacacagt ctatgaccca 3420
ctccaacctg aactggactc cttcaaggag gaactggaca aatacttcaa gaaccacacc 3480
agccctgatg tggacctggg agacatctct ggcatcaatg cctctgtggt gaacatccag 3540
aaggagattg acagactgaa tgaggtggct aagaacctga atgagtccct gattgacctc 3600
caagaactgg gcaaatatga acaatacatc aagtggccat ggtacatctg gctgggcttc 3660
attgctggac tgattgccat tgtgatggtg accataatgc tgtgttgtat gacctcctgt 3720
tgttcctgtc tgaaaggctg ttgttcctgt ggctcctgtt gtaagtttga tgaggatgac 3780
tctgaacctg tgctgaaagg agtgaaactg cactacacc 3819
<210> 15
<211> 1273
<212> PRT
<213> SARS-CoV-2
<400> 15
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 Leu
1010 1015 1020
Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val
1025 1030 1035 1040
Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala
1045 1050 1055
Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu
1060 1065 1070
Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His
1075 1080 1085
Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val
1090 1095 1100
Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr
1105 1110 1115 1120
Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr
1125 1130 1135
Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu
1140 1145 1150
Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp
1155 1160 1165
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1170 1175 1180
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
1185 1190 1195 1200
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile
1205 1210 1215
Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile
1220 1225 1230
Met 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 Val
1250 1255 1260
Leu Lys Gly Val Lys Leu His Tyr Thr
1265 1270

Claims (10)

1.一种跨膜表达新型冠状病毒抗原S2的融合蛋白，其特征在于，包括顺次链接的CD4信号肽、新型冠状病毒抗原S2蛋白、Flag标签序列和CD4跨膜结构域。
2.根据权利要求1所述的融合蛋白，其特征在于，所述CD4信号肽、新型冠状病毒抗原S2蛋白、Flag标签序列和CD4跨膜结构域的氨基酸序列如SEQ ID No.1～SEQ ID No.4所示。
3.根据权利要求1所述的融合蛋白，其特征在于，所述CD4跨膜结构域替换为CD80跨膜结构域或者CD86跨膜结构域。
4.根据权利要求3所述的融合蛋白，其特征在于，所述CD80跨膜结构域的氨基酸序列如SEQ ID No.5所示；所述CD86跨膜结构域的氨基酸序列如SEQ ID No.6所示。
5.编码权利要求1或2所述融合蛋白的基因，其特征在于，包括顺次连接的如SEQ IDNo.7～SEQ ID No.10所示的编码CD4信号肽的核苷酸序列、编码新型冠状病毒抗原S2蛋白的核苷酸序列、Flag标签序列的核苷酸序列和编码CD4跨膜结构域的核苷酸序列。
6.编码权利要求3或4所述融合蛋白的基因，其特征在于，编码CD80跨膜结构域的核苷酸序列如SEQ ID No.11所示；编码CD86跨膜结构域的核苷酸序列如SEQ ID No.12所示。
7.一种跨膜表达新型冠状病毒抗原S2的融合蛋白的重组载体，其特征在于，包括初始载体和权利要求5或6所述编码融合蛋白的基因。
8.根据权利要求7所述的重组载体，其特征在于，所述初始载体为PB-713质粒或pCDH-K载体；当所述初始载体为PB-713质粒时，所述编码融合蛋白的基因重组于PB-713质粒的Bsu36I和BmgBI酶切位点之间；当所述初始载体为pCDH-K载体时，所述编码融合蛋白的基因重组于pCDH-K载体的EcoRI和BamHI酶切位点之间。
9.一种跨膜表达新型冠状病毒抗原S2融合蛋白的重组树突状细胞，其特征在于，通过将权利要求8所述的重组载体转入树突状细胞获得。
10.权利要求1～4任意一项所述的融合蛋白、权利要求5或6所述编码融合蛋白的基因、权利要求7或8所述的重组载体、权利要求9所述的重组树突状细胞在制备新型冠状病毒疫苗中的应用。
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CN113621646A (zh) * 2021-08-10 2021-11-09 杭州医学院 一种新型冠状病毒侵染细胞的筛选系统及其应用
WO2021224429A1 (en) * 2020-05-06 2021-11-11 Biovelocita S.R.L Exosome-anchoring coronavirus fusion proteins and vaccines
WO2021254270A1 (zh) * 2020-06-15 2021-12-23 上海市公共卫生临床中心 基于细胞膜展示冠状病毒免疫原以诱导中和抗体的方法
CN113929786A (zh) * 2021-04-13 2022-01-14 武汉大学 新型冠状病毒突变株s蛋白及其亚单位疫苗
CN113943375A (zh) * 2021-10-01 2022-01-18 中国科学院昆明动物研究所 一类来源于新型冠状病毒s2蛋白hr区域的重组融合蛋白及其应用
CN114829608A (zh) * 2020-11-20 2022-07-29 北京震旦鼎泰生物科技有限公司 融合基因及一种重组新型冠状病毒高效免疫dna疫苗及其构建方法和应用
WO2022257237A1 (zh) * 2021-06-07 2022-12-15 扬州大学 一种新冠病毒SARS-CoV-2广谱多肽抗原及其特异性中和抗体和应用
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