猪瘟病毒E0蛋白在PK-15中的表达与鉴定
Expression and Identification of E0 Protein of Classical Swine Fever Virus in PK-15
周景明 1刘芳冰 1祁艳华 1张改平 2栗宁 1王爱萍1
作者信息
- 1. 郑州大学生命科学学院,河南郑州450001
- 2. 郑州大学生命科学学院,河南郑州450001;河南农业大学,河南郑州450002
- 折叠
摘要
[目的]研究猪瘟病毒(CSFv)E0基因在PK-15细胞中的表达特性.[方法]通过PCR方法克隆了E0基因全长681 bp的片段,连接到真核表达载体pEGFP-C1上,构建出重组质粒并进行双酶切鉴定.[结果]成功构建出重组质粒pEGFP-E0.通过双酶切鉴定,PCR鉴定和测序确定了目的基因大小一致,插入位置完全正确后,利用Lipofecta mine 2000转染试剂盒将重组质粒转染到猪肾细胞PK-15中,转染48 h后在荧光显微镜下观察,可以看到大多数细胞呈现出绿色荧光,说明转染成功.通过G418筛选后,在荧光显微镜下仍能观察到部分细胞能够呈现出绿色荧光,这表明重组融合蛋白pEGFP-E0在PK-15细胞中得到了表达.[结论]获得的能够表达重组融合蛋白的细胞克隆,为进一步大量表达与纯化E0糖蛋白、制备其单抗以及研究猪瘟病毒E0糖蛋白的生物学功能奠定了基础.
Abstract
[Objective] The aim was to study the characteristic how gene EO of classical swine fever virus (CSFV) expressed in PK-15.[Method] E0 gene whose full length was 681 bp fragment was cloned by PCR,and then a recombinant plasmid was constructed with eukaryotic expression vector of pEGFP-C1,double enzyme digestion was conducted.[Result] A recombinant plasmid pEGFP-E0 was sucessfully constructed,by restriction enzyme digestion,PCR identification and sequencing,the size of the target gene and insertion position was determined and was right,then recombinant plasmid were transformed into pig kidney-derived cells PK-15 by the Lipofecta mine 2000 transfection kit,after transfection 48h,many cells showed green fluorescence under a fluorescence microscope,which indicated the transfection was successful.After screening by geneticin,there were still green fluorescent cells under the fluorescence microscope,which indicated that the recombinant fusion protein of pEGFP-E0 was expressed in the cell PK-15.[Conclusion] The obtain of expressing recombinant fusion protein of cell clones lay a foundation for further mass production of soluble glycoprotein E0,preparing its monoclonal antibody epitope screening and researching the biological function of protein E0.
关键词
猪瘟病毒/E0蛋白/增强型绿色荧光蛋白/PK-15细胞Key words
CSFV/E0 protein/Enhanced green fluorescent protein/PK-15 cell引用本文复制引用
出版年
2016
国家科技期刊平台
NSTL
维普
万方数据