表达新冠病毒S基因重组狂犬病病毒的构建及其免疫原性  

Construction and immunogenicity of recombinant rabies virus expressing S gene of SARS-CoV-2

作　　者：罗均[1] 刘青 张博越 张月 陈昱彤 郭霄峰[1] LUO Jun;LIU Qing;ZHANG Boyue;ZHANG Yue;CHEN Yutong;GUO Xiaofeng(College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,Guangdong,China)

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《微生物学通报》2022年第12期5256-5265,共10页Microbiology China

基　　金：国家自然科学基金(31772742);国家重点研发计划(2022YFD1800100)。

摘　　要：【背景】新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)在全球流行已近3年,除对人类造成了巨大伤害,也影响了人类的伴侣动物。人的COVID-19疫苗已在全球应用,但动物用的新冠病毒疫苗却鲜有报道。【目的】研制兽用新冠病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)和狂犬病病毒(rabies virus,RABV)的二联苗。【方法】将合成的SARS-CoV-2 S基因和S1基因分别克隆至RABV弱毒疫苗株rHEP-Flury基因组G与L基因间,并将2个重组质粒分别与辅助质粒共转染至BHK-21细胞中,拯救重组病毒rHEP-nCOV-S和rHEP-nCOV-S1。通过RT-PCR、Western blotting和荧光抗体染色,验证重组病毒、确证S和S1蛋白在RABV中成功表达。再将重组病毒接种NA细胞及成年小白鼠,测定病毒的体外生长特性、重组病毒的致病性及免疫原性。【结果】免疫荧光结果显示,转染7 d后细胞上清均出现了绿色免疫荧光,表明已成功拯救嵌合SARS-CoV-2 S和S1基因的重组病毒RABV rHEP-nCOV-S和rHEP-nCOV-S1,并且rHEP-nCOV-S1的增殖和扩散能力强于亲本株rHEP-Flury,但rHEP-nCOV-S与亲本株无显著差异。Western blotting结果显示,在目的位置处均出现72 kDa和144 kDa特异性条带,表明S和S1蛋白在重组RABV中高效表达。重组病毒免疫6周KM小鼠后,小鼠的体重变化与亲本RABV基本一致,重组病毒诱导小鼠产生狂犬中和抗体。【结论】本研究拯救出了嵌合SARS-CoV-2 S/S1基因的重组RABV,为动物COVID-19载体疫苗的研发奠定了基础。[Background]The coronavirus disease 2019(COVID-19)pandemic has lasted for nearly three years in the globe,which has not only caused serious harm to humans but also affected companion animals.The COVID-19 vaccines for human have been used globally,while those for animals are rarely reported.[Objective]To develop a bivalent vaccine against both severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and rabies virus(RABV)for animal use.[Methods]We cloned the S and S1 genes of SARS-CoV-2 into the region between G and L genes of the attenuated RABV vaccine strain rHEP-Flury to construct the recombinant plasmids pHEP-nCOV-S and pHEP-nCOV-S1,respectively.The two plasmids were respectively co-transfected into BHK-21 cells with the helper plasmids and finally the recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 were rescued.The recombinant viruses were confirmed by RT-PCR and direct fluorescent antibody staining against RABV N protein.Western blotting was employed to detect the expression of S and S1 proteins in the cells infected with the recombinant viruses.The growth curves,pathogenicity,and immunogenicity of recombinant viruses were confirmed in NA cells and mice.[Results]The rescued recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 respectively carrying the S and S1 genes of SARS-CoV-2 were confirmed by direct fluorescent antibody assay based on the green fluorescence from the supernatants 7 days post infection.rHEP-nCOV-S1 rather than rHEP-nCOV-S showed stronger proliferation and diffusion abilities than the parental virus rHEP-Flury in NA cells.The specific bands at 72 kDa and 144 kDa in the Western blotting confirmed the efficient expression of S and S1 in the recombinant viruses,respectively.The mice vaccinated with the recombinant viruses did not show significant changes in the body weight compared with those vaccinated with rHEP-Flury,and the recombinant viruses induced the production of neutralizing antibody against RABV in mice.[Conclusion]The production of the recombinant RABV carrying the S/S1 gene of SA

关 键 词：新型冠状病毒 狂犬病病毒 S基因 疫苗 免疫原性 

分 类 号：S859.797[农业科学—临床兽医学]