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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。
Objective To construct four recombinant eukaryotic expression plasmids pVAX1 M, pVAX1 N, pVAX1 S and pVAX1 E against SARS-CoV SARS CoV M, N, S and E proteins and lay the foundation for the development of SARS DNA vaccine. Methods M, N, S and E primers were designed according to the sequence of SARS virus in GenBank. M, N, S and E fragments were amplified from Vero E6 cells infected with SARS by RT PCR. The recombinant plasmid pVAX1 M was constructed. pVAX1 N, pVAX1 S and pVAX1 E. The recombinant plasmids were transfected into COS 7 cells and the expression of recombinant plasmids was identified by immunocytochemistry and Western blot. Results The recombinant plasmids were confirmed by restriction enzyme digestion and DNA sequence analysis. The transfection experiments showed that all four recombinant plasmids could be expressed in COS 7 cells. Conclusion The eukaryotic expression plasmids of 4 kinds of SARS CoV protein antigens were successfully constructed and expressed in eukaryotic cells.