| PPVSC-1株VP2基因的定点突变及真核表达载体的构建 |
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| 引用本文： | 胡秋炅,徐志文,郭万柱,朱玲,王印,王小玉.PPVSC-1株VP2基因的定点突变及真核表达载体的构建[J].四川农业大学学报,2008,26(3). |
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| 作者姓名： | 胡秋炅 徐志文 郭万柱 朱玲 王印 王小玉 |
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| 作者单位： | 四川农业大学,动物医学院,四川,雅安,625014 |
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| 基金项目： | 国家自然科学基金，四川省科技厅资助项目 |
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| 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。
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| 关 键 词： | 猪细小病毒 VP2 基因 定点突变 |
Construction and Eukaryotic Expression of Site-directed Mutagenesis of VP2 Gene of Porcine Parvovirus Strain SC-1 |
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| HU Qiu-jiong,XU Zhi-wen,GUO Wan-zhu,ZHU Ling,WANG Yin,WANG Xiao-yu.Construction and Eukaryotic Expression of Site-directed Mutagenesis of VP2 Gene of Porcine Parvovirus Strain SC-1[J].Journal of Sichuan Agricultural University,2008,26(3). |
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| Authors: | HU Qiu-jiong XU Zhi-wen GUO Wan-zhu ZHU Ling WANG Yin WANG Xiao-yu |
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| 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. |
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| Keywords: | PPV VP2 gene site-directed mutation |
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