猪繁殖与呼吸综合征病毒核衣壳蛋白基因的克隆与原核表达  

Cloning of Nucleocapsid Protein Gene of Porcine Reproductive and Respiratory Syndrome Virus and Its Procaryotic Expression

作　　者：刘成倩[1] 肖长峰[2] 易建中[3] 关平原[1] 吴双林[3] 陈斌[3] 张天庆[3] 

机构地区：[1]内蒙古农业大学动物科学与医学学院,内蒙古呼和浩特010018 [2]上海水产大学生命学院,上海200090 [3]上海市农业科学院畜牧兽医研究所,上海201106

出　　处：《安徽农业科学》2009年第22期10429-10430,10513,共3页Journal of Anhui Agricultural Sciences

基　　金：上海市科技兴农重点攻关项目[沪农科攻字(2007)第12-1号]

摘　　要：[目的]克隆猪繁殖与呼吸综合征病毒核衣壳蛋白基因并在原核表达系统中表达。[方法]根据GenBank上登录的猪繁殖与呼吸综合征病毒(PRRSV)核酸序列设计1对引物,用RT-PCR方法扩增出PRRSV的核衣壳蛋白(N)基因,并将其克隆到原核表达载体pGEX-4T-1上,得到重组质粒pGEX-4T-1-N。将重组质粒转化大肠杆菌BL31(DE3)感受态细胞,经TPTG诱导,SDS-PAGE电泳检测。[结果]PRRSV肺组织所提取的RNA,经RT-PCR扩增,得到一段392 bp的片段,与预期结果一致。SDS-PAGE电泳分析,所得重组蛋白的分子量约39.866 Da,与预期结果相同。重组菌体超声裂解后,经10%的SDS-PAGE电泳分析,结果显示该重组蛋白为可溶性蛋白。[结论]该研究为重组蛋白的纯化奠定了基础。[ Objective] The aim was to clone nucleocapsid protein gene of porcine reproductive and respiratory syndrome virus （PRRSV） and express it in procaryotic expression system. [ Method ] According to the nucleic acid sequence of PRRSV addressed in GenBank, a pair of primers was designed for amplifying the nucleoprotein （N） gene with RT-PCR. The N gene was cloned into the procaryotic expression vector pGEX-4T-1 and the recombinant plasmid pGEX-4T-1-N was obtained, then the recombinant plasmid was transformed into competent cell of Escherichia coli BL31 （DE3）, after TPTG induction, it was detected by SDS-PAGE electrophoresis. [ Result] A fragment of 392 bp was am- plified from PRRSV RNA extracted from lung by RT-PCR, which was consistent with anticipated result. The analysis on SDS-PAGE electro- phoresis showed that the molecular weight of the obtained recombinant protein was about 39. 866 Da, which was consistent with anticipated resuit. After ultrasonic degradation, the recombinant was analyzed by 10% SDS-PAGE eleetrophoresis and the result showed that the recombi- nant protein was soluble. [ Conclusion] The research laid the foundation for the purification of the recombinant protein.

关 键 词：猪繁殖与呼吸综合征病毒 核衣壳蛋白 克隆 表达 

分 类 号：S828[农业科学—畜牧学]