SARS病毒M蛋白真核表达载体的构建与表达

目的 构建SARS病毒M蛋白片段真核表达载体,并检测其在Vero细胞中的表达.方法 在PCR引物下游引入Flag序列,以PCR从质粒pGEX-6P-1-SARS-M中扩增M蛋白编码基因片段,将酶切后的PCR产物克隆至pcDNA3.1(+)中,构建并鉴定重组质粒pcDNA3.1(+)-SARS-M.重组质粒经Superfect转染Vero细胞,Western blot检测基因表达情况.结果 重组质粒经酶切鉴定和基因测序显示构建正确;Western blot检测表明重组M蛋白片段在Vero细胞中获得正确表达.结论 成功构建SARS病毒M蛋白片段真核表达载体,并在Vero细胞中获得正确表达,为进一步研究M蛋白的功能奠定了基础.

Construction and expression of eukaryotic expression vector of SARS-CoV M protein

Objective To construct eukaryotic expression vector of SARS-CoV M protein,and detect its expression in Vero cell.Methods A pair of PCR primers with a Flag sequence,ligated downstream,were designed to amplify gene fragments,encoding M protein,from plasmid pGEX-6P-1+SARS-M.PCR products,digested by enzymes,were cloned into expression vector pcDNA3.1(+) to construct recombinant plasmid pcDNA3.1(+)-SARS-M.Recombinant plasmid was transfected into Vero cell with Superfect.The expression of recombinant M protein in Vero cell was detected by Western blot.Results Recombinant plasmid was confirmed correct with enzymes digestion and gene sequence analysis.Western blot demonstrated that M protein fragments could be correctly expressed in Vero cell.Conclusion Eukaryotic expression vector of SARS-CoV M protein was construct successfully,and recombinant M protein could be expressed correctly in Vero cell,which laid a foundation for investigating the function of M protein.