期刊论文

Construction of Recombinant Expression Vector and Establishment of Stable Expression Cell Line of N Gene of Porcine Epidemic Diarrhea Virus Strain CH-HuN1301

中文

中国畜牧兽医

1671-7236

2017

44

8

2261-2268

10.16431/j.cnki.1671-7236.2017.08.007

国家自然科学基金青年基金（31101837） 衡阳师范学院***专项项目（16D20） 湖南省教育厅优秀青年项目（17B038）

本校为第一机构

本研究旨在建立猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）CH-HuN1301株N蛋白稳定表达细胞系。采用RT-PCR扩增PEDV新流行毒株CH-HuN1301株N基因,分别克隆至原核表达载体pET28a和真核表达载体pEGFP-C1构建重组表达载体,所测定的序列采用Mega 5.0软件进行进化分析。将原核表达的N蛋白免疫家兔制备多克隆抗体,重组真核表达载体pEGFP-PEDV-N转染HEK293细胞,经用G418筛选获得稳定的表达细胞系。荧光倒置显微镜观察细胞荧光,免疫过氧化物酶单层细胞染色法（IPMA）检测N蛋白的表达。结果表明,PEDV HuN1301-14毒株N基因大小为1 323bp,原核表达重组N蛋白分子质量约60ku,其多克隆抗体与PE...

This study was aimed to construct stable cell line expressing N protein coded by porcine epidemic diarrhea virus(PEDV)strain CH-HuN1301.N gene of this virus strain was amplified by RT-PCR,which was then cloned into prokaryotic expression vector pET28a and eukaryotic expression vector pEGFP-C1,respectively.Recombinant plasmids were sequenced and analyzed by software Mega 5.0.To prepare polyclonal antibody of N protein,the prokaryotic expression recombinant N protein was used as antigen to immunize rabbits.HEK293 transfected with recombinant eukaryotic expression plasmids pEGFP-PEDV-N was select...