新型冠状病毒RBD区重组流感病毒的拯救及初步鉴定

Rescue and preliminary identification of recombinant influenza virus in RBD region of SARS-CoV-2

目的利用流感反向遗传技术,以B型流感病毒B/Yamagata/16/88株作为骨架,表达新型冠状病毒(SARSCoV-2)S蛋白的受体结合域(RBD),构建以B型流感病毒为载体的新型冠状病毒疫苗候选株。方法基因合成新冠病毒参考株S蛋白上的RBD基因片段(318-541aa),对B型流感病毒B/Yamagata/16/88/的NS1片段进行设计改造并插入RBD序列,构建重组质粒NS110-RBD。将NS110-RBD与其余7个骨架株重组质粒共转染293T细胞和MDCK细胞,拯救表达新型冠状病毒RBD区重组流感病毒,对拯救出的毒株进行形态学、分子生物学鉴定及病毒滴度和Western blot检测。结果成功拯救出重组流感病毒株并命名为rIBV-NS110-RBD。PCR鉴定RBD目的基因大小正确,测序表明拯救病毒株的序列正确,在透射电镜下观察到拯救病毒株的病毒粒子具有流感病毒粒子的典型特征。经测定,拯救株rIBV-NS110-RBD在MDCK细胞上的病毒滴度为10^(5.5) TCID_(50)/ml,在鸡胚上的病毒滴度为10^(6.5) EID_(50)/ml,血凝效价最高达2_(5);Western Blot检测到RBD的表达,其分子质量为35ku。结论成功拯救出高表达新型冠状病毒RBD蛋白的高血凝效价的重组流感病毒株,该病毒具有流感病毒粒子的典型特征,为以B型流感病毒为载体作为新型冠状病毒疫苗的研发提供了新思路。

Objective Using influenza reverse genetics,the receptor binding domain(RBD)of S protein of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was expressed by B/Yamagata/16/88strain as the vaccine backbone.Influenza B virus used as a vaccine vector when constructed a novel SARS-CoV-2vaccine candidate.Methods Firstly,the RBD gene fragment(318-541aa)on the S protein of the reference strain of SARS-CoV-2was synthesized.NS1 fragment of B/Yamagata/16/88/was designed and inserted into the RBD sequence to construct the recombinant plasmid NS110-RBD.The recombinant plasmid NS110-RBD and other 7gene plasmids were co-transfected into 293Tcells and MDCK cells to rescue the recombinant influenza virus expressing the RBD region of SARS-CoV-2.The rescued virus was identified in the morphology,molecular biology,virus titer and Western Blot.Results We have successfully rescued the recombinant influenza virus strain and named rIBV-NS110-RBD.The result of PCR identification showed that the RBD gene was correct,and the sequence of the rescued virus was correct.The virus particles of the rescued virus had typical characteristics of influenza virus particles.The virus titer of rIBV-NS110-RBD was 10^(5.5) TCID_(50)/ml in MDCK cells and 10^(6.5) EID_(50)/ml in chicken embryo eggs.The hemagglutination titer of this rescued virus could reach 2^(5).The expression of RBD protein was detected by Western Blot.Conclusion The recombinant influenza B virus vector that expressed the SARS-CoV-2Spike RBD protein was rescued successfully and provides a novel strategy for the development of a novel SARS-CoV-2vaccine.