猪繁殖与呼吸综合征病毒CH-1a株核蛋白基因在原核系统中的高效表达与初步应用  

High-level Expression and Primary Application of Nucleocapsid Gene of PRRSV CH-1a Strain in Prokaryotic Expression System

作　　者：周艳君[1] 童光志[1] 薛强[1] 仇华吉[1] 王云峰[1] 赵永刚[1] 王玫[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2002年第6期401-404,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：获国家重点基础研究发展规划项目 (973)资助(G19990 1190 2 )

摘　　要：将猪繁殖与呼吸综合征病毒CH_1a株核蛋白基因克隆至原核表达载体pET30a的多克隆位点中 ,经鉴定后得到重组质粒pET30a_N ,将此重组质粒转化到受体菌BL2 1中 ,用诱导剂IPTG分别以不同浓度进行诱导 ,并在不同诱导时间进行采样 ,经处理后做SDS_PAGE、Western_blot分析 ,并以此为包被抗原进行ELISA检测 ,结果发现以终浓度为 1mmol/L的IPTG进行诱导 ,4小时后表达可达到高峰 ,其大小约为 2 3kD ,薄层扫描结果表明该蛋白表达量约占菌体蛋白总量的 32 % ,该蛋白不仅与PRRSV阳性血清能发生特异性反应 ,而且可被PRRSV抗N蛋白单抗所识别 ,证实此大量表达的蛋白为核蛋白。将此表达产物应用ProBondTM纯化系统进行纯化 ,其纯度可达到 91% ,纯化后的蛋白以 5 0 μg/孔包被 96孔板 ,检测来自不同地区送检的猪血清 ,同时以全病毒ELISA为对照 ,结果发现二者的符合率高达 93.3% ,而应用该蛋白进行检测其反应更为敏感 ,且背景低 ,制备过程简单 ,这将为今后应用该蛋白作诊断抗原检测PRRSV奠定了基础。The recombinant expression vector pET30a_N was constructed by cloning nucleuocapsid gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) strain CH_1a into a prokaryotic expression plasmid pET30a. The recombinant vector was transformed into E. coli. BL21 (DE3) strain. Samples were collected at different induction time after induction with IPTG. The specificity of the expressed protein was identified with SDS_PAGE, Western Blotting and ELISA. And the protein is about 23kDa in size. The optimal amount of expressed protein is 32% of total bacterial protein after induced with IPTG at 1mmol/L concentration for 4 hours. The expressed protein was purified by ProBondTM purification system and the degree of purity is about 91%. The purified protein was coated on 96_well plate at 50μg/well and used for identification of PRRSV_positive sera from different regions. The results are 93.3% identical to the wells coated by PRRS virions. But this method is more sensitive and easier to perform. High efficient expression of PRRSV N protein will benefit for diagnosis of PRRS in practice.

关 键 词：猪 CH-1A株 核蛋白基因 原核系统 应用 PRRSV 繁殖呼吸综合征病毒 基因表达 

分 类 号：S858.28[农业科学—临床兽医学] S852.65[农业科学—兽医学]