表达PRRSV N蛋白稳定细胞系的构建与鉴定  

Construction and characterization of a stable cell line expressing PRRSV N protein

作　　者：付宁宁 肖长广 陈梦莉 邱亚峰[1] 李宗杰 李蓓蓓[1] 刘珂[1] 邵东华[1] 马志永[1] 魏建超[1] FU Ningning;XIAO Changguang;CHEN Mengli;QIU Yafeng;LI Zongjie;LI Beibei;LIU Ke;SHAO Donghua;MA Zhiyong;WEI Jianchao(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2023年第12期105-110,共6页Animal Husbandry & Veterinary Medicine

基　　金：上海科技兴农项目(2022-02-08-00-12-F01195,2021-02-08-00-12-F00770);上海科委农业领域项目(20392002400)。

摘　　要：旨在通过慢病毒表达系统构建稳定表达猪繁殖与呼吸综合征病毒(PRRSV) N蛋白的Marc-145细胞系。以PRRSV SH1株感染性克隆质粒为模板,通过PCR扩增N基因,并将其克隆到慢病毒载体中,获得重组慢病毒质粒pSin-Ires-Puro-N,将重组质粒pSin-Ires-Puro-N与辅助质粒psPAS2、pMD2.G、pRSV-Rev共转染293T细胞进行慢病毒包装,获得表达N蛋白的重组慢病毒颗粒并将其感染Marc-145细胞,嘌呤霉素初步筛选阳性细胞,筛选几代后的细胞采用有限稀释法和终点稀释法获得稳定表达N蛋白的Marc-145细胞系。通过PCR鉴定表明细胞系中存在N蛋白基因,通过间接免疫荧光(IFA)和Western blot试验验证N蛋白能在细胞系中能稳定表达。本研究成功构建了稳定表达PRRSV N蛋白的Marc-145细胞系,为研制PRRSV新型复制缺陷型疫苗奠定基础。The aim of this study was to construct a Marc-145 cell line with stable expression of porcine reproductive and respiratory syndrome virus(PRRSV) N protein by a lentivirus expression system.The N gene was amplified by PCR using PRRSV SH1 strain infectious cloning plasmid as template.Then,the N gene was cloned into the Lentiviral vector,resulting in a recombinant lentivirus plasmid pSin-Ires-Puro-N.The recombinant plasmid pSin-Ires-Puro-N and auxiliary plasmids psPAS2,pMD2.G and pRSV-Rev were co-transfected into 293T cells for lentivirus packaging in order to obtain recombinant lentiviral particles expressing N protein,and the particles were used to infected Marc-145 cells.Next,positive cells were preliminarily screened with puromycin;and after several generations of screening,the Marc-145 cell line with stable expression of N protein was obtained by limited dilution and end-point dilution.PCR amplification of N gene showed that there was N protein gene sequence in the cell line,IFA and Western blot assays verified that N protein could be stably expressed in the cell line.Therefore,we successfully constructed a Marc-145 cell line with stable expression of PRRSV N protein.

关 键 词：猪繁殖与呼吸综合征病毒 N蛋白 细胞系 稳定表达 慢病毒载体 

分 类 号：S852[农业科学—基础兽医学]