SARS-CoV基因重组质粒的构建与表达

目的　针对SARS冠状病毒 (SARS CoV)M、N、S和E蛋白 ,构建 4种重组真核表达质粒pVAX1 M、pVAX1 N、pVAX1 S和pVAX1 E ,为研制SARSDNA疫苗奠定基础。方法　根据GenBank中SARS病毒基因序列分别设计M、N、S和E引物 ,用RT PCR方法从感染SARS病毒的Vero E6细胞中扩增得到M、N、S和E片段 ,构建重组质粒pVAX1 M、pVAX1 N、pVAX1 S和pVAX1 E。并以重组质粒转染COS 7细胞 ,用免疫细胞化学和Westernblot方法鉴定重组质粒体外的表达。结果　经酶切鉴定及DNA序列测定证实重组质粒构建正确 ,细胞转染实验表明 ,构建的 4种重组质粒均能在COS 7细胞内表达。结论　成功构建 4种SARS CoV 4种蛋白抗原真核表达质粒 ,并能在真核细胞内获得表达。

Construction and expression of SARS-CoV gene recombinant plasmids

Objective To construct a recombinant eukaryotic expression plasmids pVAX1/SARS-CoV M, N, S, E based on their protein coding-gene, COS-7 cells were transfected by these recombinant plasmids, which provide scientific evidence for preparing SARS DNA vaccine. Methods SARS-CoV M, N, S, E genes were amplified from Vero-E6 cells infected with SARS-CoV by RT-PCR. They were inserted into the eukaryotic expression vectors called pVAX1-M, pVAX1-N, pVAX1-S, pVAX1-E;COS-7 cells were transfected with recombinant plasmids;M, N, S, E protein were detected by Immunocytochemisty method and Western blot. Results Recombinant plasmids were confirmed by restriction and DNA sequencing;M, N, S, E proteins were detected in COS-7 cells. Conclusion All recombinant eukaryotic expression plasmids have been successfully constructed.