猪流行性腹泻病毒非结构蛋白Nsp14真核表达载体构建及蛋白表达鉴定  

Construction of Eukaryotic Expression Vector and Protein Expression of PEDV Nsp14 Gene

作　　者：武峰峰 高振秋 郑亮[1,3] 牛欣雨 魏佳琪 吴志军 张华 曹宏伟 Wu Fengfeng;Gao Zhenqiu;Zheng Liang;Niu Xinyu;Wei Jiaqi;Wu Zhijun;Zhang Hua;Cao Hongwei(College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319;School of Pharmacy,Yancheng Teachers University;College of Animal Science and Technology,Heilongjiang Bayi Agricultural University)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,大庆163319 [2]盐城师范学院药学院 [3]黑龙江八一农垦大学动物科技学院

出　　处：《黑龙江八一农垦大学学报》2023年第5期72-77,共6页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省自然科学基金联合引导项目(LH2019C046);江苏省盐土生物资源研究重点实验室开放课题(JKLBS2020005);大学生创新训练计划项目(202210324271H)。

摘　　要：猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)Nsp14蛋白是PEDV的一种非结构蛋白,由于它同时具有核酸外切酶活性和N7-甲基转移酶活性,在病毒的生命周期中发挥重要作用。为了对PEDV Nsp14蛋白进行更加深入的研究,从病变猪小肠中提取总RNA,并通过PCR扩增获取PEDV Nsp14基因,经过双酶切与连接试验,最终将其连接到PRK5-Myc载体上。通过构建PEDV Nsp14真核重组表达载体,并通过Western Blot技术和免疫荧光技术在真核细胞中验证其蛋白表达情况和细胞内定位情况,为后续研究PEDV Nsp14蛋白的功能奠定研究基础。Porcine epidemic diarrhea virus(PEDV)Nsp14 protein was a non-structural protein of PEDV,which played an important role in the life cycle of the virus due to its simultaneous exonuclease and N7-methyltransferase activities.In order to conduct a more deep study on PEDV Nsp14 protein,total RNA was extracted from the small intestine of diseased pigs,and the PEDV Nsp14 gene was obtained by PCR amplification,after double enzyme digestion and ligating test,it was finally connected to PRK5-Myc vector.It aimed to establish PEDV Nsp14 eukaryotic recombinant expression vector,and verify its protein expression and intracellular localization in eukaryotic cells by Western Blot technology and immunofluorescence technology,so as to lay a research foundation for the subsequent study of PEDV Nsp14 protein function.

关 键 词：猪流行性腹泻病毒 Nsp14 真核表达载体 蛋白表达 

分 类 号：S852.659.6[农业科学—基础兽医学]