脑心肌炎病毒VP1结构蛋白的可溶性表达及鉴定  

Soluble expression and identification for VP1 protein of encephalomyocarditis virus

作　　者：李向茸[1,2] 冯若飞[1,3] 凡静静[2] 张海霞[2] 韦鹏建[2] 樊得英[1] 马忠仁 

机构地区：[1]西北民族大学生物工程与技术国家民委重点实验室,甘肃兰州730030 [2]西北民族大学生命科学与工程学院,甘肃兰州730030 [3]甘肃省动物细胞工程技术研究中心,甘肃兰州730030

出　　处：《中国兽医科学》2011年第6期557-561,共5页Chinese Veterinary Science

基　　金：国家民委科研项目(10XB03);甘肃省教育厅科研项目(1018B-04);人畜共患病诊断技术平台建设校企合作项目(H2009-15)

摘　　要：为获得可溶性表达的脑心肌炎病毒(EMCV)结构蛋白VP1片段,将EMCV VP1基因克隆到原核表达载体pGEX-4T-1中,并转化大肠杆菌BL21(DE3)表达菌中。采用不同温度、不同IPTG浓度及不同诱导时间诱导目的蛋白表达,以筛选VP1融合蛋白可溶性表达的最佳条件,并通过Western-blotting检测最佳条件下表达蛋白的免疫活性。PCR、酶切鉴定及测序结果表明,VP1基因已克隆入pGEX-4T-1质粒。经优化,确定VP1融合蛋白可溶性表达的最佳条件为:当D600nm值为0.4～0.6时,以0.25mmol/L IPTG于25℃条件下诱导表达12h。Western-blotting结果显示,诱导表达的蛋白具有免疫活性。To express soluble VP1 in prokaryotic expression system,the gene encoding VP1 of encephalomyocarditis virus（EMCV） VP1 was cloned into the expression vector pGEX-4T-1,and the recombinant plasmid was transformed into Escherichia coli BL21（DE3）.The expression conditions,including additives,IPTG concentration,temperature and induction duration were optimized.Then,Western-blotting was used to identify the immunoreactivity of the expressed protein.In result,recombinant prokaryotic expression vector pGEX-4T-1-VP1 was constructed successfully.Based on the optimization experiments,it was concluded that the best soluble expression conditions for the fusion VP1 protein involved incubation to an D600 nm from 0.4 to 0.6,addition of IPTG to a final concentration of 0.25 mmol/L and then further incubation for 12 h at 25 ℃.Western-blotting result showed that the fusion VP1 protein possessed immunoreactivity.

关 键 词：脑心肌炎病毒 VP1基因 可溶性表达 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学]