O型口蹄疫病毒样颗粒编码基因的真核表达载体构建及表达

Construction and Expression of Eukaryotic Expression Vector of O Type Foot-and-mouth Disease Virus-like Particles Encoding Gene

为了构建O型口蹄疫病毒(FMDV)病毒样颗粒编码基因的真核表达载体,将FMDV结构蛋白VP0、VP1、VP3的基因分别克隆到真核表达载体pVAX1中,再分别通过带有BsmBⅠ、EcoRⅠ和SalⅠ酶切位点的引物,分别对重组质粒pVAX-VP0、pVAX-VP1和pVAX-VP3扩增后进行酶切,用T4连接酶连接得到重组的真核表达载体pVAX-VP0VP1VP3。对重组真核表达载体pVAX-VP0VP1VP3进行PCR鉴定并测序。用脂质体将重组真核表达载体pVAX-VP0VP1VP3转染BHK-21细胞,用间接免疫荧光试验(IFA)及Western blot检测重组真核表达载体pVAX-VP0VP1VP3在细胞中的表达情况。结果表明,成功构建了重组真核表达载体pVAX-VP0VP1VP3,并在BHK-21细胞中得到表达。

To construct and express the eukaryotic expression vector of serotype O foot-and-mouth disease virus-like particles encoding gene,VP0,VP1 and VP3 genes were cloned into the eukaryotic expression vector pVAX1 through the known FMDV structural protein gene sequences in this paper.Then,the recombinant plasmids pVAX-VP0,pVAX-VP1,pVAX-VP3 were amplified by using primers with Bsm B Ⅰ, Eco R Ⅰ and Sal Ⅰ restriction sites respectively,the recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was obtained by ligating with T4 ligase.The recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was identified by PCR and sent to Suzhou Jinzhizhi Biotechnology Co.,Ltd. for sequencing.The recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was transfected into BHK cells by lipofectin transfection method and the expression of recombinant eukaryotic expression vector pVAX-VP0VP1VP3 in cells was detected by indirect immunofluorescence assay test (IFAT) and Western blot.The results showed that the recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was constructed and expressed successfully in BHK-21 cells.