猪A组轮状病毒VP4全基因在大肠杆菌中的表达与检测  

Expression of VP4 gene of porcine group A rotavirus in Escherichia coli

作　　者：王春凤[1] 沈明浩[2] 王会岩[1] 刘尚高[3] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林农业大学食品科学院,吉林长春130118 [3]中国农业大学动物医学院,北京100094

出　　处：《中国预防兽医学报》2005年第5期344-347,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家863计划(No.2003AA24112002);吉林省科技厅项目(NO.20020220);吉林省杰出青年基金(NO.20030118);中国博士后科学基金项目(NO.2002031156);吉林农业大学青年教师启动基金(NO.2002-QQN-002)

摘　　要：以猪轮状病毒mRNA为模板,应用反转录聚合酶链式反应(RT_PCR)技术,扩增了2331bp的VP4全基因,通过T_A克隆技术,将PCR产物克隆至pGEM_T Vector中,成功地构建出克隆质粒pGEM_T_VP4。以BamHI和SalI双酶切pGEM_T_VP4和pGEX_6P_1,并将纯化的VP4基因亚克隆至融合型表达载体pGEX_6P_1中,构建出原核表达质粒pGEX_6P_VP4。将pGEX_6P_VP4转化至感受态E.coliBL21(DE3)中,经IPTG诱导和SDS_PAGE分析,可见约111.47 ku融合蛋白带。Western blot分析发现,该蛋白具有轮状病毒抗原性,从而为进一步研究轮状病毒的亚单位疫苗及DNA疫苗奠定基础。The antigenic determinants of VP4 gene of porcine group A rotavirus were amplified from cell infected with rotavirus by the reverse transcfiption-polymerase chain reaction （RT-PCR）. and the PCR product was approximately 2331bp DNA segment. Using T- A cloning technique, the PCR product was cloned into pGEM-T vector and cloning plasmid pGEM-T-VP4 was thus constructed successfully. pGEM-T-VP4 and pGEX-6P-1 were digested by BamH I and Sa/I double enzymes.The purified VP4 gene was subcloned into the expression vector pGEX-6P-1, and the porkaryotic expression vector pGEX-6P-VP4 was thus constructed. Plasmid containing pGEX-6P-VP4 was transformed into competence E. coli BL21 （DE3）. The activity of VP4 gene of Porcine Rotavirus. The results showed that the bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE, approximately 111.47 ku exogenous protein was observed on the SDS,-PAGE. The protein was analyzed using Western-blot. The results indicate that the protein is of antigenic expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of VP4 gene in their prevention of porcine rotavirus.

关 键 词：轮状病毒 VP4基因 克隆与原核表达 

分 类 号：S852.659.3[农业科学—基础兽医学] Q786[农业科学—兽医学]