钩端螺旋体鞭毛蛋白B(FlaB)基因flaB在大肠杆菌内的表达  

Expression of the leptospiral flagellar protein B gene in E.coli

作　　者：寇志华[1] 张毅[1] 郭瀛军[1] 陈祖欢[1] 施柯[1] 孙树汉[1] 

机构地区：[1]第二军医大学医学遗传学教研室,上海200433

出　　处：《中国人兽共患病杂志》2004年第3期180-182,共3页Chinese Journal of Zoonoses

基　　金：国家"8 63"计划资助项目 ( 2 0 0 1AA2 13 111)

摘　　要：目的　在大肠杆菌中高效表达钩端螺旋体内鞭毛蛋白 (FlaB)用于钩体病的诊断和预防。方法　将目的基因flaB定向克隆至原核表达载体 pGEX - 5T ,构建重组融合表达质粒 pGF ;Western -blot鉴定其特异性 ,ELISA法判定其用于钩体病诊断的可行性。结果　GST -FlaB主要以包涵体形式表达 ,表达量约占菌体总蛋白的 4 0 % ;Western -blot结果显示该蛋白与FlaB抗血清能呈现明显的单一条带 ;包涵体经TritonX - 10 0及尿素纯化后 ,纯度可达 90 % ,并且能溶于包被缓冲液中 ;ELISA检测显示纯化后的GST -FlaB具有较好的特异性。结论　钩端螺旋体FlaB可以高效表达并可能用于ELISA检测及钩体病的预防。To investigate the gene encoding the highly expressed leptospiral flagellar protein B(FlaB) in E.coli,the desired gene flaB was amplified by PCR and cloned into the prokaryotic expression vector pGEX-5T to reconstruct the recombinant fusion expression plasmid pGF.Western blotting analysis was used to identify the specificity,and ELISA assay was employed to determine the possibility for the diagnosis of leptospirosis.It was found that the GST-FlaB was expressed in E.coli mainly in the form of inclusion bodies,and Western blotting analysis revealed the GST-FlaB fusion protein exhibiting a specific reactive band.The fusion protein was purified to about 90% purity by extracting the inclusion bodies with Triton X-100 and urea,and it could be dissolved in the coating buffer solution.ELISA assay demonstrated that the GST-FlaB after purification showed definite specificity.It concludes that highly expressed fusion protein GST-FlaB could be expressed in E.coli and it could be used in ELISA testing and for the purpose of prevention of leptospirosis.

关 键 词：钩端螺旋体 鞭毛蛋白B 基因 flaB 大肠杆菌 表达 

分 类 号：R377[医药卫生—病原生物学]