SARS冠状病毒核蛋白与mGM-CSF融合质粒的构建及表达

Construction and Expression Plasmid Co-expression mGM-CSF and Nucleocapsid Protein of SARS-CoV

目的构建SARS冠状病毒(SARS-COV)核蛋白(N)与mGM-CSF的融合真核表达质粒,借助mGM-CSF的免疫佐剂作用,提高SARSDNA疫苗的免疫效果。方法采用PCR方法扩增N和mGM-CSF基因片段,用分子克隆和基因融合的方法将目的片段定向插入真核表达载体中,构建融合表达质粒pVAX1/N-mGM-CSF,并以融合质粒转染细胞,用免疫细胞化学和WesternBlot的方法鉴定融合质粒在体外的表达。结果经酶切鉴定及DNA序列测定证实融合质粒构建正确,细胞转染实验表明,融合质粒能在COS-7细胞中表达。结论成功构建SARS-COV核蛋白与mGM-CSF的融合表达质粒,重组质粒能在真核细胞内表达。

Objective To construct plasmid co-expressing mGM-CS and nucleocapsid protein of SARS-COV and study the expression of plasmid in vitro. Methods The mGM-CSF and N DNA fragment were amplified by PCR and inserted into eukaryotic expression vector called pVAX1/ N-GM-CS. COS-7 cells was transfected with recombinant plasmid, the fusion protein was detected by immunocytochemistry and Western blot method.Results The fuse plasmid was confirmed by enzyme digestion and DNA sequence, the fuse protein was detected in COS-7 cells. Conclusion The recombinant eukaryotic expression plasmid has been constructed successfully, and the fuse protein can express in COS-7 cellls.