真核重组荧光质粒pEGFP-N1-Et HP的构建及高效转染细胞的筛选  

Construction and Screening of Eukaryotic Recombinant Fluorescent Plasmid pEGFP-N1-Et HP in Highly Transfected Cells

作　　者：郭许情 王黎霞[2] 张榕珍 张健 张天宇 石梦云 王诗琪 赵小涵 张建军[1] 安健[1] GUO Xu-qing;WANG Li-xia;ZHANG Rong-zhen;ZHANG Jian;ZHANG Tian-yu;SHI Meng-yun;WANG Shi-qi;ZHAO Xiao-han;ZHANG Jian-jun;AN Jian(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China;College of Animal Science and Veterinary Medicine,Beijing Vocatioeal College of Agriculture,Beijing 102442,China)

机构地区：[1]北京农学院动物科学技术学院,北京昌平102206 [2]北京农业职业学院畜牧兽医系,北京房山102442

出　　处：《中国兽医杂志》2022年第4期45-48,55,共5页Chinese Journal of Veterinary Medicine

基　　金：北京市特色高水平骨干专业群项目-技术平台与社会服务建设项目(PXM2020-157102-000060-15);北京农业职业学院院级科研项目(XY-JT-03)。

摘　　要：为了构建真核重组荧光质粒pEGFP-N1-Et HP,并筛选出Lipofectamine 2000介导的高效转染细胞,本试验将柔嫩艾美尔球虫假定蛋白(Et HP)基因扩增后插入真核荧光表达载体pEGFP-N1,构建真核重组荧光质粒pEGFP-N1-Et HP;测序成功后,用Lipofectamine 2000介导pEGFP-N1-Et HP转染4种细胞即Vero、DF-1、BHK、HD-11;用Western blot检测确定Et HP蛋白在4种细胞内的表达,并观察转染48 h后荧光表达情况,计算5个视野内带有绿色荧光的细胞数占细胞总数的比例,从而比较质粒与Lipofectamine 2000比例不同时4种细胞的转染效率[转染效率/%=(带有绿色荧光的细胞数/细胞总数)×100%]。测序结果显示,pEGFP-N1-Et HP构建成功。Western blot检测显示,Et HP蛋白在4种细胞中均成功表达;荧光显微镜观察到4种细胞转染后48 h均出现绿色荧光,表明重组质粒pEGFP-N1-Et HP成功表达。通过转染效率的比较,发现DF-1细胞转染效率均显著高于其他细胞(P<0.05);当质粒与Lipofectamine 2000比例为1∶2时,DF-1细胞转染效率最高。因此,重组质粒pEGFP-N1-Et HP可高效转染DF-1细胞。本试验结果为进一步探索Et HP蛋白在柔嫩艾美尔球虫入侵寄生过程中的生物学功能提供有效的试验工具。In order to construct the eukaryotic recombinant fluorescent plasmid pEGFP-N1-Et HP,and screen for the cells with high transfection efficiency mediated by Lipofectamine 2000,Eimeria tenella hypothetical protein(Et HP)gene was amplified and inserted into the eukaryotic fluorescent expression vector pEGFP-N1,and the eukaryotic recombinant fluorescent plasmid pEGFP-N1-Et HP was constructed.After sequencing confirmation,PEGFP-N1-Et HP was transfected via Lipofectamine 2000 mediation into Vero cells,DF-1 cells,BHK cells and HD-11 cells.The expression of Et HP protein in four kinds of cells was determined by Western blot,the fluorescence expression was observed after transfection for 48 h,and the ratio of the number of cells with green fluorescence to the total number of cells in five visual fields was calculated to compare the transfection efficiency[Transfection efficiency/%=(Number of cells with green fluorescence/Total number of cells)×100%]of four kinds of cells when the ratio of plasmid to Lipofectamine 2000 was different.The result showed that pEGFP-N1-Et HP was successfully constructed.Western blot analysis showed that Et HP protein was successfully expressed in four kinds of cells.Green fluorescence was observed 48 h after transfection by fluorescence microscope,which indicated that the recombinant plasmid pEGFP-N1-Et HP was successfully expressed.By comparing the transfection efficiency,it was found that the transfection efficiency of DF-1 cells was significantly higher than that of other cells(P<0.05),and DF-1 cells had the highest transfection efficiency when the ratio of plasmid to Lipofectamine 2000 was 1∶2.Thus,the recombinant plasmid pEGFP-N1-Et HP could be transfected into DF-1 cells efficiently.The results of this experiment provide a useful tool for further investigation on the biological function of Et HP protein during E.tenella invasion and parasitism.

关 键 词：柔嫩艾美尔球虫假定蛋白 真核重组荧光质粒 DF-1细胞 转染效率 

分 类 号：S855.9[农业科学—临床兽医学]