摘要
目的:克隆B19病毒XA株VP1u基因,构建真核重组表达载体。方法:从已构建好的B19病毒XA株原核表达载体中获得VP1u基因,将其克隆入真核表达载体pIRES2-EGFP中,经酶切鉴定并测序验证后,获得真核表达载体pIRES2-EGFP-VP1u。将其转染至HeLa细胞,提取细胞总蛋白,用Western blot技术检测VP1u蛋白的表达。结果:成功构建了携带人B19病毒VP1u基因的真核表达载体pIRES2-EGFP-VP1u,荧光显微镜下可见pIRES2-EGFP-VP1u转染HeLa细胞后表达EGFP蛋白而发出绿色荧光,Western blot证明VP1u蛋白在HeLa细胞中表达。结论:成功构建了携带人B19病毒VP1u基因的真核表达载体pIRES2-EGFP-VP1u并在HeLa细胞中正确表达,为今后B19病毒VP1u基因疫苗的研究奠定基础。
Objective：To clone VP1-unique region of parvovirus B19-XA and to construct eukaryotic expression plasmid.Methods：VP1-unique region of parvovirus B19-XA was acquired from the prokaryotic expression plasmid pQE30-VP1u,and was cloned into vec-tor pIRES2-EGFP.The recombinant plasmids were identified by sequencing.The recombinant plasmid were transfected into HeLa cells.Then Western blot and fluorescence microscopy were used to identify the expression of VP1u and green fluorescence protein.Results：The VP1u gene was cloned and pIRES2-EGFP-VP1u vector was build.The VP1u gene expressed successfully in Hela cells.Conclusion：The recombinant plasmid containing parvovirus B19-XA VP1u gene was successfully constructed and expressed exactly in HeLa cells,which will be helpful in investigating the DNA vaccines of parvovirus B19.
出处
《现代生物医学进展》
CAS
2010年第23期4401-4403,共3页
Progress in Modern Biomedicine
基金
国家自然科学基金资助项目(81070543)
