四川农业大学学报 ›› 2008, Vol. 26 ›› Issue (03): 258-262.doi: 10.16036/j.issn.1000-2650.2008.03.011

• 研究论文 • 上一篇    下一篇

PPVSC-1株VP2基因的定点突变及真核表达载体的构建

胡秋炅, 徐志文, 郭万柱, 朱玲, 王印, 王小玉   

  1. 四川农业大学 动物医学院, 四川 雅安 625014
  • 收稿日期:2008-01-16 出版日期:2008-09-30 发布日期:2017-03-03
  • 基金资助:
    国家自然科学基金项目(30500019);四川省科技厅科技攻关项目

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

HU Qiu-jiong, XU Zhi-wen, GUO Wan-zhu, ZHU Ling, WANG Yin, WANG Xiao-yu   

  1. College of Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, Sichuan, China
  • Received:2008-01-16 Online:2008-09-30 Published:2017-03-03

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。

关键词: 猪细小病毒, VP2, 基因, 定点突变

Abstract: 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.

Key words: PPV, VP2, gene, site-directed mutation

中图分类号: 

  • S852.659.2