CN114196701A - 一种sars-cov-2的二价重组新城疫病毒载体及相应疫苗株和制备方法 - Google Patents

一种sars-cov-2的二价重组新城疫病毒载体及相应疫苗株和制备方法 Download PDF

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CN114196701A
CN114196701A CN202111363444.8A CN202111363444A CN114196701A CN 114196701 A CN114196701 A CN 114196701A CN 202111363444 A CN202111363444 A CN 202111363444A CN 114196701 A CN114196701 A CN 114196701A
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周孟云
毛水花
孙慧敏
陈瑞婷
宋家升
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Zhejiang Difu Runsi Biotechnology Co ltd
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Abstract

本发明提供了一种SARS‑COV‑2的二价重组新城疫病毒载体及相应疫苗株和制备方法，属于生物制品技术领域。本发明以NDV LaSota病毒株为骨架载体，S2P蛋白基因***到NDV病毒载体P基因和M基因之间，deltaRBD‑Fc融合蛋白基因***M基因和F基因之间，利用病毒反向遗传学技术拯救获得具有限制感染性NDV毒株用于疫苗株生产。相对于目前单个新冠S抗原或RBD抗原疫苗，本发明引入原始株S蛋白突变体S2P的编码序列和含Delta变异株S蛋白受体结合域RBD的融合蛋白编码序列共同作为抗原形成二价疫苗株，动物试验结果显示无论是经鼻腔接种亦或肌肉注射免疫途径，该疫苗株都能达到优异的免疫效果。

Description

一种SARS-COV-2的二价重组新城疫病毒载体及相应疫苗株和 制备方法
技术领域
本发明属于生物制品技术领域，具体涉及一种SARS-COV-2的二价重组新城疫病毒载体及相应疫苗株和制备方法。
背景技术
新型冠状病毒肺炎(Corona Virus Disease 2019，COVID-19)，简称"新冠肺炎"，世界卫生组织命名为"2019冠状病毒病"，是指2019新型冠状病毒感染(SARS-COV-2)导致的肺炎。目前，SARS-COV-2已有四种变异株(分别是Alpha、Beta、Gamma和Delta)被WHO列为需关注毒株，其中Delta变异株传播最广，相较于最原始新冠毒株，其传染性提高约75％。
预防病毒感染的有效途径是接种疫苗。目前，上市的新冠疫苗主要分为以下几个种类：mRNA类，全病毒灭活疫苗，腺病毒载体疫苗以及重组蛋白亚单位疫苗。然而，这些疫苗多少存在一些不足，且随着疫苗接种慢慢凸显。具体表现在以下几个方面：(1)注射类疫苗(mRNA，重组蛋白等)，可以降低发病，但是无法刺激机体产生局部粘膜免疫，无法阻止感染，携带和传播；(2)制造工艺复杂(mRNA)、高生物安全等级厂房要求(灭活)、高成本等因素制约疫苗在发展中国家普及；(3)腺病毒载体类受预先存在载体抗体影响，无法进行多次(2次以上)免疫，而新冠疫苗需要持续接种。(4)对德尔塔变异株的效率逐渐下降。因此，综合考虑疫苗有效性，安全性，工艺成熟，成本可控，适合大规模普及接种的新型疫苗仍然是急需的。
发明内容
有鉴于此，本发明的目的在于提供一种SARS-COV-2的二价重组新城疫病毒载体及相应疫苗株和制备方法，具有针对Delta变异株具有优异的免疫效果和较高的安全性。
本发明提供了一种新冠病毒SARS-COV-2的二价重组新城疫病毒载体，在新城疫病毒载体的基础上***S2P蛋白基因和deltaRBD-Fc融合蛋白基因；所述S2P蛋白是在原始新冠病毒株S蛋白基础上进行K986P和V987P突变得到；
所述S2P蛋白基因***在新城疫病毒的基因P和M基因之间；
所述deltaRBD-Fc融合蛋白基因***在M基因和F基因之间。
优选的，所述S2P蛋白的氨基酸序列如SEQ ID NO:1所示。
优选的，所述deltaRBD-Fc融合蛋白由N端到C端依次包括人血清白蛋白信号肽(HSA信号肽)、Delta毒株S蛋白的受体结合域RBD片段和人IgG Fc片端。
优选的，所述deltaRBD-Fc融合蛋白的氨基酸序列如SEQ ID NO:3所示。
优选的，所述S2P蛋白基因或deltaRBD-Fc融合蛋白基因的5'端依次添加转录终止信号、转录起始信号和kozak序列，所述S2P蛋白基因或deltaRBD-Fc融合蛋白基因的3'端依次添加转录终止信号、转录起始信号。
优选的，所述新城疫病毒载体包括新城疫LaSota病毒株载体。
本发明提供了一种新冠病毒SARS-COV-2的二价新城疫病毒粒子或疫苗株，由所述新冠病毒SARS-COV-2的二价重组新城疫病毒载体经病毒拯救获得。
本发明提供了所述二价重组新城疫病毒粒子或疫苗株的制备方法，包括以下步骤：
1)PCR扩增S2P蛋白基因、deltaRBD-Fc融合蛋白基因和NDV载体基因序列；
2)将扩增的S2P蛋白基因片段***NDV载体的P基因和M基因之间，将deltaRBD-Fc融合蛋白基因片段***载体的M基因和F基因，形成重组载体；
3)将所述重组载体与病毒拯救辅助质粒共转染BHK21-T7细胞，经病毒拯救获得二价重组新城疫病毒粒子或疫苗株。
本发明提供了一种新冠病毒SARS-COV-2的二价新城疫病毒疫苗，包括所述二价新城疫病毒粒子或疫苗株。
本发明提供了所述重组二价新城疫病毒粒子或疫苗株在制备防控新冠肺炎的疫苗中的应用。
本发明提供了一种新冠病毒SARS-COV-2的二价重组新城疫病毒载体，在新城疫病毒载体的基础上***S2P蛋白基因和deltaRBD-Fc融合蛋白基因；所述新冠病毒S2P蛋白基因***在新城疫病毒P基因和M基因之间；所述deltaRBD-Fc融合蛋白基因***在M基因和F基因之间。本发明利用病毒反向遗传学技术，将S2P蛋白基因和以融合蛋白形式Delta毒株RBD抗原***新城疫病毒载体中，基于新城疫病毒在人体中安全性和病毒易复制获得特性，使制备的载体疫苗株具有较高的免疫安全性和容易生产特点；同时，将所述重组新城疫病毒载体拯救的疫苗株免疫动物，无论是经鼻腔接种亦或肌肉注射免疫途径，与单一S蛋白抗原免疫效果相比，均能显著提高疫苗株的免疫效果。本发明以人IgG Fc片段与Delta毒株RBD形成融合蛋白，与其他蛋白多肽(TBSV、Ferritin)形成的融合蛋白相比，具有更优异的免疫原性。
附图说明
图1为本发明构建的新冠病毒SARS-COV-2的二价重组新城疫病毒载体结构示意图；
图2为用ELISA检测重组NDV载体病毒株感染Vero细胞后RBD的表达量结果；
图3为本发明采用候选疫苗株免疫动物程序示意图；
图4为免疫后血清IgG抗体滴度，其中图4a为新冠S蛋白IgG抗体滴度；图4b为NDV载体IgG抗体滴度；
图5为构建病毒株反转录PCR凝胶鉴定电泳图，泳道1：NDV-S2P扩增片段，泳道2：NDV-S2P-P2A-deltaRBD-Fc扩增片段，泳道3-4：NDV-S2P-deltaRBD-Fc扩增片段，泳道5-6：NDV-S2P-deltaRBD-Ferrtin扩增片段，泳道7-8：NDV-S2P-deltaRBD-TBSV扩增片段；泳道1，2，3，5，7由鉴定引物NDV-F3153和NDV-R3454扩增得到，泳道4，6，8由鉴定引物NDV-M-F928和NDV-F-R24扩增得到；
图6为本发明构建的各疫苗株免疫小鼠后IgG抗体滴度的比较；
图7为二价与单抗原靶标的重组NDV载体疫苗株免疫小鼠后，NDV载体IgG抗体滴度的比较，****P<0.0001。
具体实施方式
本发明提供了一种新冠病毒SARS-COV-2的二价重组新城疫病毒载体，在新城疫病毒载体的基础上***S2P蛋白基因和deltaRBD-Fc融合蛋白基因；所述S2P蛋白是在原始新冠病毒株S蛋白基础上进行K986P和V987P突变得到；所述S2P蛋白基因***在新城疫病毒的P基因和M基因之间；所述deltaRBD-Fc融合蛋白基因***在M基因和F基因之间。
在本发明中，所述S2P蛋白氨基酸序列优选如SEQ ID NO:1(MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT*)所示。S2P蛋白中K986P和V987P突变使表达的S2P蛋白构象发生变化，成为预融合构象，更容易被识别。所述载体中，***的S2P蛋白基因是在S2P蛋白对应的核苷酸序列基础上进行密码子优化得到的。
在本发明中，所述deltaRBD-Fc融合蛋白由N端到C端依次包括HSA信号肽、deltaRBD片段和人IgG Fc片端。其中Delta毒株RBD区域优选为Delta毒株S蛋白R317-S585氨基酸对应区域，Delta毒株RBD区域的氨基酸序列优选如SEQ ID NO:2所示(RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS)。所述HSA信号肽的氨基酸序列如SEQ ID NO:4(MKWVTFISLLFSSAYS)所示。人IgG Fc的氨基酸序列如SEQ ID NO:5(DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK)所示。通过在Delta毒株RBD区域的C端连接人IgG Fc片段，有利于帮助RBD形成正确的折叠，提高其与抗原呈递细胞的结合能力，提高抗原免疫原性。并且经过实验结果证明，人IgG Fc片段与其他多肽相比，对于提高RBD抗原的免疫原性有显著优势。
在本发明中，所述deltaRBD-Fc融合蛋白中Delta毒株RBD的C端优选通过蛋白linker与人IgG Fc连接。本发明对所述蛋白linker的种类没有特殊限制，采用本领域所熟知的蛋白linker即可。在本发明实施例中，所述蛋白linker的氨基酸序列优选如SEQ IDNO:6(GGSGGS)所示。所述蛋白linker有利于保持融合蛋白中各蛋白活性。本发明所述deltaRBD-Fc融合蛋白的氨基酸序列见SEQ ID NO:3(MKWVTFISLLFSSAYSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSGGSGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPGVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK)所示。
在本发明中，所述S2P蛋白基因或deltaRBD-Fc融合蛋白基因的5'端优选依次添加转录终止信号、转录起始信号和kozak序列，，所述S2P蛋白基因或deltaRBD-Fc融合蛋白基因的3'端优选依次添加转录终止信号和转录起始信号。所述转录终止信号的核苷酸序列优选为TTAGAAAAAA(SEQ ID NO:8)。所述转录起始信号的核苷酸序列优选为ACGGGTAGAA(SEQID NO:9)。所述kozak序列的核苷酸序列为GCCACC所示。通过在两抗原基因的5'和3'端连接上述顺式作用元件，使两蛋白抗原单独表达，同时提高表达效率，从而提高后续免疫动物时抗原的免疫原性。
在本发明中，所述新城疫病毒载体优选包括新城疫LaSota病毒株载体。
其中新城疫LaSota病毒株是在申请号202010542477.8、名称为一种重组新城疫病毒载体新型冠状病毒疫苗候选株及其构建方法和应用的专利公开的新城疫LaSota病毒株。
本发明提供了一种新冠病毒SARS-COV-2的二价重组新城疫病毒粒子或疫苗株，由所述新冠病毒SARS-COV-2的二价重组新城疫病毒载体经病毒拯救获得。
本发明提供了所述新冠病毒SARS-COV-2的二价重组新城疫病毒粒子或疫苗株的构建方法，包括以下步骤：
1)PCR扩增S2P蛋白基因、deltaRBD-Fc融合蛋白基因和NDV载体基因序列；
2)将扩增得到的两种抗原基因序列以及载体序列通过同源重组的方式连接起来，形成重组新城疫病毒载体；
3)将所述重组载体与辅助质粒经病毒拯救获得二价重组新城疫病毒粒子或疫苗株。
在本发明中，具体PCR扩增过程优选如下：
首先PD-S2P1-F和PD-S2P681-R进行PCR扩增S2P蛋白基因，用PD3-NDV-S2P-opt-F、NDV-R15186和NDV-F15139、S-opt14-R进行PCR扩增载体基因，同源重组将S蛋白克隆到P基因和M基因位置，得到NDV-S2P。所用到的引物序列如下：
引物名称 引物序列5'-3'
PD-S2P1-F CATGTTCGTGTTTCTGGTGCTG(SEQ ID NO:10)
PD-S2P681-R TTAAACTCAGGTGTAGTGCAGTTTCACG(SEQ ID NO:11)
NDV-R15186 ACCAAACAAAGATTTGGTGAATG(SEQ ID NO:12)
NDV-F15139 CAATTATTCTTGAGTGTAGTCTCGTCATTCACCAAATCTT(SEQ ID NO:13)
S-opt14-R AGAAACACGAACATGGTG(SEQ ID NO:14)
PD3-NDV-S2P-opt-F TGAGTTTAAACTTAGAAAAAATACGGGTAGAATTG(SEQ ID NO:15)
在NDV-S2P基础上，使用PD1-HSA-RBD-Fc-F1和PD1-HSA-DTRBD-Fc-R引物对扩增deltaRBD-Fc融合蛋白基因，使用PD2-F-15186-F、NDV-R15186和NDV-F15139、ZT-NDV-S2P-opt-R3引物对扩增NDV载体基因，将融合蛋白基因克隆进入M基因和F基因位置之间，得到NDV-S2P-deltaRBD-Fc克隆。所用到的引物序列如下：
Figure BDA0003360104430000041
Figure BDA0003360104430000051
在本发明中，所述同源重组克隆方式优选按照说明书推荐的体系和程序进行即可。
得到重组新城疫病毒载体后，本发明将所述重组新城疫病毒载体转染细胞，经病毒拯救，获得二价重组新城疫病毒粒子或疫苗株。
本发明对所述病毒拯救的方法没有特殊限制，采用本领域所熟知病毒拯救的方法即可。在本发明实施例中，将所述二价新城疫病毒载体和病毒拯救辅助质粒pCI-NP、pCI-P和pCI-L混合，共同转染细胞，经细胞培养和冻融，收集细胞上清液，接种SPF鸡胚，收集鸡胚尿囊液测定血凝效价后，得到二价新城疫病毒。结果表明，初始拯救病毒的HA效价一般为(2～6)log2，经过传代适应后，病毒效价可显著提高，其效价可达到(8～11)log2。
本发明提供了一种新冠病毒SARS-COV-2的二价新城疫病毒疫苗，包括所述二价新城疫病毒粒子或疫苗株。
在本发明中，将所述疫苗按照滴鼻免疫和注射免疫动物后，该疫苗株都能取得优异的免疫效果，可见，所述疫苗能够针对新冠病毒具有较强的免疫效果。
下面结合实施例对本发明提供的一种SARS-COV-2的二价重组新城疫病毒载体及相应疫苗株和制备方法进行详细的说明，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
候选疫苗株NDV-S2P-deltaRBD-Fc病毒载体的构建方法
对原始新冠病毒株的S蛋白抗原进行K986P和V987P突变，形成S2P序列(SEQ IDNO:1)，经过密码子优化后得到S2P基因片段。在新冠病毒Delta病毒株受体结合区RBD(aa317-585，SEQ ID NO:2)序列的N端添加信号肽HSA(SEQ ID NO:4)，C端添加人IgG Fc片段(SEQ ID NO:5)，使用Linker(SEQ ID NO:6，GGSGGS)连接RBD区域与融合蛋白Fc序列，形成deltaRBD-Fc序列(SEQ ID NO:3)。对以上序列分别进行密码子优化后得到S2P基因片段和融合蛋白基因片段deltaRBD-Fc。对两个片段5'端分别添加转录终止信号、转录起始信号和kozak序列，3'端分别添加转录终止信号、转录起始信号，然后委托金唯智生物有限公司进行基因合成。
基因合成后，对以上合成片段和NDV-LaSota载体分别进行PCR扩增，PCR扩增反应体系和反应程序按照Primer STAR酶说明书要求进行扩增，同源重组过程按照
Figure BDA0003360104430000052
HiFi DNA Assembly试剂盒说明书要求进行。将S2P基因序列通过常规的同源重组的方式克隆到NDV载体P-M基因之间，形成病毒载体质粒NDV-S2P。在病毒载体质粒NDV-S2P基础上，将合成的deltaRBD-Fc基因序列利用同源重组克隆方式***NDV Lasota株M-F基因之间(见图1)。将同源重组产物的病毒载转化进入感受态细胞中，挑取单克隆测序鉴定后，使用LB培养基扩大培养，使用TIANGEN无内毒素质粒提取试剂盒进行质粒抽提。最终构建得到NDV-S2P-deltaRBD-Fc。
实施例2
候选疫苗株病毒拯救方法
将实施例1构建的重组病毒载体NDV-S2P-deltaRBD-Fc和病毒拯救辅助质粒pCI-NP、pCI-P和pCI-L，经Nano drop测定质粒浓度后，转染BHK21-T7细胞，具体转染过程为：
将BHK21-T7细胞铺六孔板，待细胞密度达到70％～80％时，按照LipofectaminLTX说明书取500μL Opti-MEM于1.5mL离心管中，随后按照1:1:1:1的比例分别加入实施例1中的重组NDV病毒载体以及pCI-NP、pCI-P和pCI-L辅助质粒，每管质粒总量为4μg，供1个孔使用。向上述各溶液中分别加入3μL Plus reagent，室温作用5min后各加入9μLLipofectamine LTX，轻轻混匀，室温放置30min；随后取出待转染的BHK21-T7细胞，弃去培养基，无菌PBS洗2次，加入1mL Opti-MEM培养基，将上述转染复合物逐滴滴加到培养皿中，37℃二氧化碳培养箱孵育6～8h后换液，24hr后加入含0.08μg/ml TPCK的opti-MEM培养基继续培养。72h后，反复冻融细胞3次，并将混合物接种9～11日龄SPF鸡胚，0.2～0.3mL/胚，37℃培养24h。弃去24h内的死胚，收获72h内收获存活的鸡胚尿囊液。逐个测定血凝效价。血凝价测定方法为：将收取的NDV候选病毒疫苗株稀释至后接种9～11日龄鸡胚，将鸡胚封口后置34℃培养，72小时后，取尿囊液100μl置于96孔V型血凝板，用PBS将尿囊液做10个梯度系列稀释，同时取50μl PBS设阴性对照，即可读取血凝价。初始拯救NDV-S2P-RBD-Fc病毒不同克隆的血凝价在(5.5～7)log2，经过传代1代适应后，病毒血凝价最高到9.5log2。最后，收取有血凝价且存活的鸡胚尿囊液。离心后分装，0.5～1ml/管，冻存于-80℃冰箱。取其中200μL收获的病毒，利用AXYGEN病毒基因组RNA提取试剂盒提取病毒基因组RNA，而后利用HiScript III 1st Strand cDNA Synthesis Kit试剂盒反转录，合成首链cDNA。合成后，进行PCR产物电泳及测序鉴定，PCR反应体系和反应条件按照喏唯赞HiscriptII One StepRT-PCR kit说明书要求设置。PCR鉴定及测序合格后，进行下一步实验。
实施例3
重组NDV载体病毒疫苗株体外评价
将Vero-E6细胞传代到6孔板培养皿中长至单层备用，加入实施例2中所拯救重组NDV载体病毒NDV-S2P-DeltaRBD-Fc，同时设置NDV-COVS对照组。NDV-COVS病毒株即在NDVLaSota载体P-M位置***原始新冠病毒株S蛋白序列(参见专利CN202010542477.8有详细构建方法)的疫苗株。对长至单层的细胞板，用PBS洗两遍后加入上述病毒液，500μL/孔，每孔补加1500μL含0.1μg/mL TPCK的opti-MEM。放于37℃，5％CO2培养箱中培养至36h，取上清样品检测RBD浓度。
RBD浓度的测定使用双抗体夹心酶联免疫法，按照义翘神州SARS-CoV-2(2019-nCoV)Spike RBD ELISA Kit试剂盒说明书要求，配制1×洗涤液和1×稀释液，对标准品进行复溶，并梯度稀释标准品。同时，将细胞吹打悬浮，取150μL液体反复冻融3次，对待测的细胞上清液进行100倍稀释，按照说明书要求对标准品和待测的细胞冻融液进行抗原孵育、洗涤、酶标检测抗体孵育、洗涤、显色、终止等操作后，在20分钟在450nm波长下读取光吸收值OD450nm。每个样品平行3次取平均值。
结果如图2。由图2可知，相对于NDV-COVS，双抗原疫苗株RBD抗原表达量显著升高。
实施例4
小鼠免疫实验
使用实施例3验证后的双抗原疫苗株NDV-S2P-deltaRBD-Fc对4～5周龄Balb\c雌性小鼠进行免疫，测试新冠S蛋白IgG抗体滴度，同时检测NDV载体本身产生的IgG抗体滴度。
免疫途径分别为滴鼻免疫和肌肉注射，麻醉使用5％水合氯醛腹腔注射，每20g体重小鼠腹腔注射0.1mL。麻醉后，用移液器进行滴鼻免疫，25μL/鼻孔或用0.3mL微量注射器对小鼠后腿肌肉注射100μL病毒液。共进行两次免疫，首免后7天进行二次加强免疫。首免后14d，采集血清检测新冠S蛋白结合抗体以及NDV病毒相关蛋白IgG抗体。免疫程序如图3所示。免疫所使用病毒疫苗株血凝价约为6Log2。
免疫小鼠血清S蛋白IgG抗体滴度按照义翘神州(SARS-CoV-2(2019-nCoV))SpikeS1抗体滴度检测试剂盒说明书进行检测，重组NDV病毒疫苗株小鼠血清新城疫抗体滴度按照爱德士新城疫病毒抗体检测试剂盒说明书进行检测。
结果如图4a所示，首次免疫后14天，与NDV疫苗株相比，无论滴鼻还是注射，血清新冠S1蛋白IgG抗体都显著增加，同时，NDV载体本身IgG抗体也相应增加(图4b)。这说明该疫苗株可以产生特异性免疫，并且免疫效果良好。
实施例5
构建不同新城疫病毒株比较免疫原性的差异。
一.用于比较的疫苗株NDV-S2P、NDV-S2P-P2A-deltaRBD-Fc、NDV-S2P-deltaRBD-TBSV和NDV-S2P-HSA-deltaRBD-Ferritin病毒载体设计与构建
在新冠病毒受体结合区RBD序列N端添加人血清白蛋白分泌信号肽HSA(MKWVTFISLLFSSAYS)，C端添加蛋白TBSV和Ferritin等，使用蛋白Linker(GGGGSGGGGS，SEQID NO:26)和(GGGSGGGS，SEQ ID NO:27)分别连接deltaRBD和蛋白多肽序列TBSV和Ferritin，设计形成deltaRBD-TBSV(SEQ ID NO:20)和deltaRBD-Ferritin(SEQ ID NO:21)，对以上序列进行密码子优化并由金唯智生物有限公司进行基因合成。
在NDV-S2P载体基础上，将以上合成的抗原片段分别克隆***到载体NDV-S2P-deltaRBD-Fc中，代替deltaRBD-Fc融合蛋白序列，分别构建形成NDV-S2P-deltaRBD-TBSV和NDV-S2P-deltaRBD-Ferritin质粒。其中PCR扩增按照Primer STAR酶说明书要求进行扩增，同源重组按照
Figure BDA0003360104430000071
HiFi DNA Assembly试剂盒说明书要求进行。
NDV-S2P构建方法如实施例1记载。在NDV-S2P载体基础上，删除S2P的3'端终止密码子，添加P2A基因序列(P2A对应的氨基酸序列ATNFSLLKQAGDVEENPGP，SEQ ID NO:7)，通过同源重组克隆方式在其后***deltaRBD-Fc，构建形成NDV-S2P-P2A-deltaRBD-Fc病毒载体质粒。另外，将同源重组产物的病毒载转化进入感受态细胞中，挑取单克隆测序鉴定后，使用LB培养基扩大培养，使用TIANGEN无内毒素质粒提取试剂盒进行质粒抽提。
二、用于比较的疫苗株病毒拯救
方法同实施例2，本发明病毒株及比较例毒株所用到的鉴定引物如下表1。
表1鉴定引物具体信息
Figure BDA0003360104430000072
PCR扩增用的反应程序和反应体系按照喏唯赞HiscriptII One Step RT-PCR kit说明书要求设置结果表明，凝胶电泳显示条带与目的片段大小匹配(图5)。
三、用于比较的重组NDV载体病毒疫苗株动物实验
小鼠免疫实验方法同实施例4。
结果可见，本发明保护的NDV-S2P-deltaRBD-Fc疫苗株在免疫原性方面，效果优于NDV-S2P、NDV-S2P-P2A-deltaRBD-Fc、NDV-S2P-deltaRBD-TBSV和NDV-S2P-deltaRBD-Ferritin疫苗株。特别是以滴鼻方式进行的免疫，本发明所保护疫苗株免疫效果超过单个抗原疫苗株NDV-S2P效果近3倍(图6)。同时，S2P和deltaRBD-Fc二价的引入，与单个S2P重组NDV载体疫苗株相比极显著地降低了NDV载体本身的抗体滴度(图7)，说明该疫苗株可以有效降低载体毒副作用，有利于双抗原免疫原性的充分发挥。
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> 浙江迪福润丝生物科技有限公司
<120> 一种SARS-COV-2的二价重组新城疫病毒载体及相应疫苗株和制备方法
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
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 Pro Pro 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
<210> 2
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
210 215 220
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
225 230 235 240
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
245 250 255
Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys
260 265 270
Ser
<210> 3
<211> 522
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Ser Ser Ala Tyr Ser
1 5 10 15
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
20 25 30
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
35 40 45
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
50 55 60
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
65 70 75 80
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
85 90 95
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
100 105 110
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
115 120 125
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
130 135 140
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
145 150 155 160
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys
165 170 175
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
180 185 190
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
195 200 205
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
210 215 220
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
225 230 235 240
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
245 250 255
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
260 265 270
Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys
275 280 285
Ser Gly Gly Ser Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys
290 295 300
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
305 310 315 320
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Gly Val Thr Cys
325 330 335
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
340 345 350
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
355 360 365
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
370 375 380
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
385 390 395 400
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
405 410 415
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
420 425 430
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
435 440 445
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
450 455 460
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
465 470 475 480
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
485 490 495
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
500 505 510
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
515 520
<210> 4
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Ser Ser Ala Tyr Ser
1 5 10 15
<210> 5
<211> 227
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Gly Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 6
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gly Gly Ser Gly Gly Ser
1 5
<210> 7
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 8
<211> 10
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ttagaaaaaa 10
<210> 9
<211> 10
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
acgggtagaa 10
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
catgttcgtg tttctggtgc tg 22
<210> 11
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ttaaactcag gtgtagtgca gtttcacg 28
<210> 12
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
accaaacaaa gatttggtga atg 23
<210> 13
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
caattattct tgagtgtagt ctcgtcattc accaaatctt 40
<210> 14
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
agaaacacga acatggtg 18
<210> 15
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tgagtttaaa cttagaaaaa atacgggtag aattg 35
<210> 16
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
cgggtagaag ccaccatgaa atgggtcacc tttatcag 38
<210> 17
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gttttttgtg tcatgtcatt tgccggggct cagag 35
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
catgacacaa aaaactaatc tg 22
<210> 19
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ggtggcttct acccgtattt tttctaaatg atctgggtga gtgggcggag cg 52
<210> 20
<211> 325
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
20 25 30
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
35 40 45
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
50 55 60
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
65 70 75 80
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
85 90 95
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
100 105 110
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
115 120 125
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
130 135 140
Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn
145 150 155 160
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
165 170 175
Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
180 185 190
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
195 200 205
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
210 215 220
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
225 230 235 240
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
245 250 255
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
260 265 270
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
275 280 285
Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Asn His
290 295 300
Val Gly Gly Thr Gly Gly Ala Ile Met Ala Pro Val Ala Val Thr Arg
305 310 315 320
Gln Leu Val Gly Ser
325
<210> 21
<211> 472
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
20 25 30
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
35 40 45
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
50 55 60
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
65 70 75 80
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
85 90 95
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
100 105 110
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
115 120 125
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
130 135 140
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
145 150 155 160
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
165 170 175
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
180 185 190
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
195 200 205
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
210 215 220
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
225 230 235 240
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
245 250 255
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
260 265 270
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
275 280 285
Pro Cys Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Ser Gln Val Arg
290 295 300
Gln Gln Phe Ser Lys Asp Ile Glu Lys Leu Leu Asn Glu Gln Val Asn
305 310 315 320
Lys Glu Met Asn Ser Gln Asn Leu Tyr Met Ser Met Ser Ser Trp Cys
325 330 335
Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala
340 345 350
Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu
355 360 365
Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys
370 375 380
Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu Gln
385 390 395 400
His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys Ser
405 410 415
Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala Glu Gln
420 425 430
His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu
435 440 445
Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys
450 455 460
Gly Ile Ala Lys Ser Arg Lys Ser
465 470
<210> 22
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
aaggtccaac tctccaagcg g 21
<210> 23
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gtcctcctta ctatcagtcc aca 23
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
caggtggcca agatactctg 20
<210> 25
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
cttggtagaa ggtctggag 19
<210> 26
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
<210> 27
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Gly Gly Gly Ser Gly Gly Gly Ser
1 5

Claims (10)

1.一种新冠病毒SARS-COV-2的二价重组新城疫病毒载体，其特征在于，在新城疫病毒载体的基础上***S2P蛋白基因和deltaRBD-Fc融合蛋白基因；所述S2P蛋白是在原始新冠病毒株S蛋白基础上进行K986P和V987P突变得到的；
所述S2P蛋白基因***新城疫病毒载体的P基因和M基因之间；
所述deltaRBD-Fc融合蛋白基因***新城疫病毒载体M基因和F基因之间。
2.根据权利要求1所述新冠病毒SARS-COV-2的二价重组新城疫病毒载体，其特征在于，所述S2P蛋白的氨基酸序列如SEQ ID NO:1所示。
3.根据权利要求1所述新冠病毒SARS-COV-2的二价重组新城疫病毒载体，其特征在于，所述deltaRBD-Fc融合蛋白由N端到C端依次包括人血清白蛋白信号肽、新冠delta毒株S蛋白的受体结合域RBD片段和人IgG Fc片端。
4.根据权利要求3所述新冠病毒SARS-COV-2的二价重组新城疫病毒载体，其特征在于，所述deltaRBD-Fc融合蛋白的氨基酸序列如SEQ ID NO:3所示。
5.根据权利要求1所述新冠病毒SARS-COV-2的二价重组新城疫病毒载体，其特征在于，所述S2P蛋白基因或deltaRBD-Fc融合蛋白基因的5'端依次添加转录终止信号、转录起始信号和kozak序列，所述S2P蛋白基因或deltaRBD-Fc融合蛋白基因的3'端依次添加转录终止信号和转录起始信号。
6.根据权利要求1～5任意一项所述新冠病毒SARS-COV-2的二价重组新城疫病毒载体，其特征在于，所述新城疫病毒载体包括新城疫LaSota病毒株载体。
7.由权利要求1～6任意一项所述二价重组新城疫病毒载体通过反向遗传学技术制备得到的二价重组新城疫病毒粒子或疫苗株。
8.权利要求7所述二价重组新城疫病毒粒子或疫苗株的制备方法，其特征在于，包括以下步骤：
1)PCR扩增S2P蛋白基因、deltaRBD-Fc融合蛋白基因和NDV载体基因序列；
2)将扩增的S2P蛋白基因片段***NDV载体的P基因和M基因之间，将deltaRBD-Fc融合蛋白基因片段***载体的M基因和F基因，形成重组载体；
3)将所述重组载体与病毒拯救辅助质粒共转染BHK21-T7细胞，经病毒拯救获得二价重组新城疫病毒粒子或疫苗株。
9.一种新冠病毒SARS-COV-2的二价新城疫病毒疫苗，其特征在于，包括权利要求7所述重组二价新城疫病毒粒子或疫苗株。
10.权利要求7所述重组二价新城疫病毒粒子或疫苗株在制备防控新冠肺炎的疫苗中的应用。
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