羊地方性鼻内肿瘤病毒SU基因的克隆与原核表达  

Cloning and prokaryotic expression of SU gene of enzootic nasal tumor virus from goat

作　　者：谢智勇[1] 马新玥 万莉[1] 马磊[1] 张琦[1] 杨映[1] 颜其贵[1,2] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]四川农业大学动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医科学》2013年第11期1175-1179,共5页Chinese Veterinary Science

基　　金：教育部“长江学者和创新团队发展计划”创新团队项目(IRT0848);科技部科技人员服务企业行动计划项目(2009GJF00049)

摘　　要：为了研究羊地方性鼻内肿瘤病毒(ENTV)SU蛋白的功能,采用RT-PCR方法从ENTV感染羊鼻内分泌物中扩增获得SU基因片段,将其克隆到pMD19-T Simple载体;测序验证后,再对克隆载体进行EcoRⅠ和SalⅠ双酶切并亚克隆到pET-32a上,将重组质粒转化至表达宿主菌Rosetta中进行IPTG诱导表达,对表达产物用SDS-PAGE筛选最适条件。结果显示,表达的重组菌融合蛋白大小约60.14ku,在IPTG终浓度0.75mmol/L,37℃诱导5h表达效果最好。表达产物通过Ni柱纯化后可得到纯度较高的目的蛋白。经Western-blot分析,所纯化的蛋白能与兔抗羊ENTV阳性血清进行特异性的免疫印迹反应,证明表达的SU蛋白有较好的反应原性。In order to explore the function of SU protein of enzootic nasal tumor virus（ENTV）, SU gene of ENTV was amplified by RT-PCR from nasal secretion of goat and cloned into the pMD19-T Simple vector. After sequencing, the SU gene containing vector was digested by EcoR I and Sal I and subcloned in- to the pET-32a vector. The recombinant plasmid was transformed into Escherichia coli Rosetta. The trans- formed bacteria were induced by IPTG, and the best condition for the expressed product was selected by SDS-PAGE. The results showed that the recombinant proteins had 60.14 ku and the high production was obtained under the following conditions.0.75 mmol/L IPTG,37 ℃ and 5 h of induction time. Pure recombi- nant protein would be produced after Ni^2＋ column purification. The purified protein could react with the rabbit positive serum against ENTV using Western-blot, which showed that the expressed protein had strong reactinogenicity.

关 键 词：羊地方性鼻内肿瘤病毒 克隆 原核表达 纯化 免疫印迹 

分 类 号：S852.659.3[农业科学—基础兽医学]