羊流产嗜衣原体ompA基因的克隆与表达  

Cloning and Prokaryotic Expression of the ompA Gene of Goat Chlamydophila abortus

作　　者：龙冲冲[1] 江楠[1] 杨细桃[1] 周碧君[1,2] 罗意[1] 文明[1,2] 

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究室,贵州贵阳550025

出　　处：《生物技术》2012年第4期25-28,共4页Biotechnology

基　　金：贵州省农业攻关项目("山羊繁殖障碍性疫病防控关键技术研究与示范";黔科合NY字[2011]3105号);贵州省重点实验室项目("贵州省动物疫病与兽医公共卫生重点实验室培育";黔科合计Z字[2011]4008号)资助

摘　　要：目的:克隆羊流产嗜衣原体ompA基因,获得原核表达ompA蛋白。方法:以前期构建的质粒pMD-18T-ompA为模板,扩增出无信号肽无终止子ompA基因1 101bp条带,亚克隆至原核表达载体pet-32a,转化大肠杆菌BL21(DE3),重组质粒稳定性测试合格后经1mmol/L IPTG诱导表达4h,并采用SDS-PAGE和Western-blot检测重组蛋白的表达结果。结果:本试验成功获得60kDa重组ompA蛋白,该重组蛋白主要以包涵体的形式存在,免疫印迹显示可与山羊流产嗜衣原体阳性血清反应,具有免疫原性。结论:成功建立ompA基因的原核表达方法,为后续ompA蛋白的功能研究奠定基础。Objective:Cloned and prokaryotic expression the ompA gene.Method:The ompA gene 1 101bp fragment that lacking the N-terminal signal peptide was obtained from the previous constructed plasmid pMD-18T-ompA by PCR.Inserted it into the bacterial plasmid pET-32a vector and then transformed into E.coli BL21(DE3) induced 4h by 1mmol/L IPTG for expressing after plasmid stability testing.Result:SDS-PAGE analysis showed that the ompA protein with 60 kDa in molecular mass was successfully expressed in insoluble protein form.The recombinant protein ompA purified with purification Resin could reacted with positive sera against goat C.abortus by Western-blot,indicating that ompA protein had reactogenicity.Conclusion:A method for prokaryotic expression was successfully established,which lays a foundation for the ompA functional research.

关 键 词：流产嗜衣原体 ompA基因 克隆 原核表达 稳定性 

分 类 号：S852.67[农业科学—基础兽医学] Q78[农业科学—兽医学]