iNOS基因和iNOS-VP1融合基因真核表达载体的构建及初步表达

目的 构建pcDNA3．1介导的诱导性一氧化氮合成酶（iNOS）的基因表达载体和pcDNA3．1介导的iNOS与柯萨奇病毒B组3型（CVB3）结构蛋白VP1融和基因的表达载体。方法 应用PCR扩增和DNA重组技术构建pcDNA3．1-iNOS和pcDNA3．1-iNOS—VP1表达载体；应用真核细胞转染技术及间接免疫荧光技术进行所构建的真核表达载体的初步表达和鉴定。结果 经PCR扩增技术用特异引物从质粒pKSiNOS分离编码iNOS开放阅读框架的cDNA，TA克隆于pMD19-T载体，根据设计引物时加入的酶切位点将插入片断亚克隆于表达载体pcDNA3．1；经PCR扩增技术用特异引物从质粒pCR2．1-VP1分离编码CVB3VP1结构蛋白的cDNA，TA克隆于pMD19-T载体，根据设计引物时加入的酶切位点将VP1亚克隆于iNOS的表达载体pcDNA3．1-iNOS，从而构建含有iNOS和VP1融合基因的真核表达载体。限制性内切酶分析、PCR鉴定和测序证实重组体peDNA3．1-iNOS和peDNA3．1-iN—OS—VP1插入片断的大小和方向正确且开放阅读框架的读码框不变；重组质粒peDNA3．1-iNOS和peDNA3．1-iNOS．VP1在HeLa细胞中均有表达，但表达效率较低。结论 获得含iNOS基因和iNOS—VP1融合基因的真核表达载体，并将重组质粒进行了初步表达，为体外iNOS抗CVB3作用的研究奠定了物质基础。

Construction and initial expression of iNOS gene and iNOS-VP1 fused gene eukaryotic expression vector mediated by pcDNA3.11

Objective To construct inducible nitric-oxide synthase (iNOS) gene as well as iNOS and coxsackievirus B type 3 VP1(capsid protein 1) fused gene eukaryotic expression vector mediated by pcDNA3.1. Methods Polymerase chain reaction (PCR) and recombinant DNA were used, The transfection technology and indirect immunofluorescence were used in initial expression and identification of the recombinant plasmids.Results The full-length cDNA encoding iNOS was obtained from plasmid pKSiNOS by PCR , cloned to T vector pMD-19 and then subcloned to eukaryotic expression vector pcDNA3.1. The CVB3 VP1 sequence was obtained from the pCR2.1-VP1, cloned to pMD19-T vector and then subcloned to pcDNA3.1iNOS. The pcDNA3.1iNOS contains iNOS open reading frame and the pcDNA3.1iNOS-VP1 contains simultaneously the iNOS gene and VP1 gene, namely, the fused gene iNOS-VP1. The authenticity of insertion size and orientation of iNOS sequence was verified by restriction mapping, PCR and sequence analysis with iNOS gene-specific primers. The same methods were used in the authenticity of VP1 in the pcDNA3.1iNOS-VP1, The recombiniant plasmid pcDNA3.1iNOS and pcDNA3.1iNOS-VP1 all expression in HeLa cell on low level. Conclusion Eukaryotic expression vector carrying iNOS cDNA as well as iNOS and VP1 fused gene were obtained, which provide a material to investigate the effect of the iNOS protein against the CVB3 in vitro.