文献类型：期刊文献

中文题名：融合表达新冠病毒S1与N蛋白的非复制型重组腺病毒的构建与鉴定

英文题名：Construction and Identification of A Non-Replicating Recombinant Adenovirus Expressing the S1 and N Proteins of SARS-CoV-2

作者：毛彤瑶[1];张鹏[1,2];姜苏芮[2];李丹地[1];段招军[1]

第一作者：毛彤瑶

机构：[1]传染病溯源预警与智能决策全国重点实验室,国家卫生健康委医学病毒和病毒病重点实验室,中国疾病预防控制中心病毒病预防控制所,北京102206;[2]甘肃中医药大学公共卫生学院,兰州730000

第一机构：传染病溯源预警与智能决策全国重点实验室,国家卫生健康委医学病毒和病毒病重点实验室,中国疾病预防控制中心病毒病预防控制所,北京102206

年份：2024

卷号：40

期号：2

起止页码：225

中文期刊名：病毒学报

外文期刊名：Chinese Journal of Virology

收录：CSTPCD;;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；PubMed;

基金：国家重点研发计划(项目号:2022YFC2304302),题目:呼吸道疾病非注射剂型疫苗研发及靶向递送技术体系研究。

语种：中文

中文关键词：新型冠状病毒;腺病毒载体疫苗;体液免疫;融合表达

外文关键词：Severe acute respiratory syndrome coronavirus-2;Adenovirus-based vector vaccine;Humoral immunity;Fusion expression

摘要：体外构建融合表达新型冠状病毒(SARS-CoV-2)S1抗原与N抗原的复制缺陷型重组腺病毒疫苗,为深入开展该疫苗免疫原性的研究提供基础。利用Overlap PCR和同源重组方法构建插入SARS-CoV-2的S1-N基因的重组腺病毒质粒pKAd5-S1-N,通过琼脂糖凝胶电泳、多酶切、测序等方法鉴定重组质粒正确性;重组质粒转染HEK293A细胞获得病毒毒种AdV5-S1-N并扩增,氯化铯密度梯度离心法纯化病毒,有限稀释分析法测病毒滴度,Western blot方法验证AdV5-S1-N重组腺病毒S1-N融合蛋白表达情况;将AdV5-S1-N疫苗通过肌肉注射途径免疫BALB/c小鼠,第14d采集颌下静脉血,ELISA法检测血清针对S1和N蛋白抗原的特异性抗体滴度。成功获得滴度为9.45×10^(11)IU/mL的重组腺病毒AdV5-S1-N。Western blot结果发现:AdV5-S1-N感染细胞后,融合蛋白可正常表达,与抗S1蛋白及抗N蛋白抗体均有特异结合。ELISA结果显示免疫重组腺病毒的小鼠可产生针对S1和N蛋白的特异性IgG抗体。应用复制缺陷型腺病毒载体成功获得融合表达SARS-CoV-2 Omicron突变株S1-N蛋白的高滴度重组腺病毒,该重组病毒能在小鼠诱导针对S1和N蛋白的特异性免疫应答,为后续深入研究和评价该候选疫苗奠定了基础。
We aimed to construct a replication-deficient recombinant adenovirus vaccine fused with the Sl antigen and N antigen of severe acute respiratory syndrome coronavirus(SARS-CoV-2),and provide a basis for further research on the immunogenicity of the vaccine.The recombinant adenovirus plasmid pKAd5-S1-N inserted into the S1-N gene of SARS-CoV-2 was constructed by overlap polymerase chain reaction and homologous recombination.The recombinant plasmid was transfected into HEK293A cells to obtain the virus species AdV5-S1-N and amplified.The virus was purified by density ,gradient centrifugation using cesium chloride.The virus titer was measured by limited dilution.Western blotting was used to verify expression of the S1-N fusion protein of the AdV5-S1-N recombinant adenovirus.Immunization of BALB/c mice with AdV5-S1-N vaccine was through intramuscular injection.On day 14,we collected blood from the submandibular vein,and measured the specific antibody titers of serum against the antigens of S1 and N proteins using ELISAs.Titers with 9.45×10^(11)IU/mL of AdV5-S1-N were obtained.Western blotting showed that,after infection of AdV5-S1-N cells,the fusion protein could be expressed normally and bind specifically to anti-S1 protein and anti-N protein antibodies.ELISAs showed that mice immunized with recombinant adenovirus could produce specific IgG antibodies targeting S1 and N proteins.A replication-deficient adenovirus vector was used to obtain a hightiter recombinant adenovirus that fused and expressed the S1-N protein of the SARS-CoV-2 Omicron mutant strain.The recombinant virus could induce specific immune responses against S1 and N proteins in mice,laying the foundation for further research and evaluation of this candidate vaccine.