人呼吸道合胞病毒RNA聚合酶复合体蛋白N、P、L和M2-1重组质粒的构建和鉴定

摘要

目的 构建可表达人呼吸道合胞病毒(RSV)转录延长/终止抑制因子M2-1(简称M2-1)、核蛋白(N)、磷蛋白(P)和大蛋白(L)的真核表达载体并鉴定蛋白表达情况.方法 采用合成T7启动子Oligo DNA及PCR方法获得含有T7启动子的表达盒,通过体外连接方法克隆入载体px8δT,制备表达载体px8δT-PT.设计3对PCR引物,PCR扩增出RSV的M2-1、N和P基因,并克隆至px8δT-PT,构建px8δT-PT-M2-1、px8δT-PT-N和px8δT-PT-P.同时利用已有的质粒pcDNA3.1-L构建px8δT-PT-L.用重组质粒转染 BSR T7/5细胞,72 h后再用Western blot法鉴定蛋白的表达.结果 成功实现px8δT的改造,构建的4种重组质粒px8δT-PT-M2-1、px8δT-PT-N、px8δT-PT-P和px8δT-PT-L,经限制性内切酶分析与预期一致,经Western blot法分析证实M2-1、N及P蛋白被表达.结论 获得以T7为启动子的表达载体px8δT-P,成功构建了含有M2-1、N及P编码基因的表达载体,为利用反向遗传学技术进一步改造RSV奠定了基础.%Objective To construct and identify the eukaryotic expression vector encoding human respiratory syncy-tial virus ( RSV ) transcription elongation/antitermination factor M2-1 ( M2-1 ), nucleoprotein ( N ), phosphoprotein ( P ) and large protein ( L ). Methods Three pairs of PCR primers were designed to amplify M2-1, N and P genes. The PCR products were cloned into expression vector of px88T-PT, which was controlled by T7 promoter to construct recombinant plasmids of px88T-PT-M2-l, px88T-PT-N and px88T-PT-P. Meanwhile, px88T-PT-L was constructed from pcDNA3. 1 -L. The resultant recombinant plasmids were transfected into BSR T7/5 cells and trans-gene expression was identified by Western blot at 72 h after transfection. Results By restriction endonuclease a-nalysis, four plasmids encoding N, P, L and M2-1 were constructed successfully, and the expression of three genes of N, P, and M2-1 was confirmed by Western blot. Conclusion The T7 promoter-based expression vector encoding M2-1, N and P genes, respectively, are successfully constructed, which are competented for modifying RSV genetically by reverse genetics technique.