PPVSC-1株VP2基因的定点突变及真核表达载体的构建  

Construction and Eukaryotic Expression of Site-directed Mutagenesis of VP2 Gene of Porcine Parvovirus Strain SC-1

作　　者：胡秋炅[1] 徐志文[1] 郭万柱[1] 朱玲[1] 王印[1] 王小玉[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014

出　　处：《四川农业大学学报》2008年第3期258-262,共5页Journal of Sichuan Agricultural University

基　　金：国家自然科学基金项目(30500019);四川省科技厅科技攻关项目。

摘　　要：以PPV VP2为基础,利用重叠延伸PCR技术分别将VP2蛋白中第402位(A)和214位(B)的半胱氨酸突变为精氨酸,并将突变体克隆入pMD19-T Simple载体中;经酶切、PCR和测序鉴定后,利用KpnⅠ和BamHⅠ酶切位点,定向克隆入真核表达载体pPI-2.EGFP中,构建突变体与报告基因EGFP融合表达的pPI-2.EGFP.VP2(A)和pPI-2.EGFP.VP2(B)真核表达载体;利用脂质体法将重组质粒分别转染COS-7细胞。结果:成功构建了突变体的真核表达载体;在转染后的荧光观察中发现,pPI-2.EGFP.VP2(A)和pPI-2.EGFP.VP2(B)质粒转染细胞后,两者表达后的荧光信号不同,A样品的荧光呈颗粒状分布于细胞中,而B样品的荧光信号均匀分布于细胞中。提示,突变是引起表达差异的主要原因。Based on the porcine parvovirus （PPV） VP2 gene, by using overlap extension PCR, the site-directed mutation, Cys to Arg, was imported in the site 402 （A） and 214 （B） of VP2 protein. The fragments containing the mutation site were cloned into the pMD19-T Simple vector, and the recombinant plasmids were confirmed by restriction enzyme, PCR and sequencing. Then the mutants were directly cloned into the eukaryotic expression vector pPI-2. EGFP and recombinant plasmids pPI-2. EGFP. VP2 （A） and pPI-2. EGFP. VP2 （B） were obtained. Under the fluorescence microscope, some differences were observed between the cells transfected by pPI-2. EGFP. VP2 （A） and pPI-2. EGFP. VP2 （B） respectively. The fluorescence produced by pPI-2. EGFP. VP2 （A） scattered in the cell, whereas the fluorescence produced by pPI-2. EGFP. VP2 （B） spread in the cell. The introduction of mutant was the main reason for the different expression of plasmids.

关 键 词：猪细小病毒 VP2 基因 定点突变 

分 类 号：S852.659.2[农业科学—基础兽医学] Q786[农业科学—兽医学]