问号钩端螺旋体fliN基因原核表达及其产物的鉴定  

Prokargotic expression of Leptospira interrogans fliN gene and identification of the expressed product

作　　者：孙琦[1] 陈蔚青[2] 孙爱华[1] 严杰[3] 

机构地区：[1]浙江医学高等专科学校,杭州310053 [2]浙江树人大学生物与环境工程学院,杭州310015 [3]浙江大学医学院紫金港校区,杭州310058

出　　处：《中国人兽共患病学报》2008年第1期30-32,62,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金面上项目资助(30370072)

摘　　要：目的克隆问号钩端螺旋体(简称钩体)黄疸出血群赖型赖株鞭毛相关基因fliN并构建其原核表达系统,鉴定表达产物的免疫性。方法提取钩体株基因组DNA,高保真PCR扩增全长fliN基因片断,T-A克隆后测序。按常规方法构建fliN基因原核表达系统,并用IPTG诱导目的重组蛋白rFliN表达。采用SDS-PAGE结合Bio-Rad凝胶图象分析系统检测rFliN表达量,Ni-NTA亲和层析法提纯rFliN。采用兔抗问号钩体黄疸出血群赖株全菌抗血清的Western blot鉴定rFliN及其免疫反应性。结果与报道的相应序列比较,所克隆的fliN基因核苷酸和氨基酸序列相似性均为100%。IPTG诱导原核表达系统pET32a-fliN-E.coliBL21DE3的rFliN表达量约占细菌总蛋白的30%,rFliN提纯后仅见单一的蛋白条带。rF-liN能与抗血清发生免疫结合反应。结论本研究成功地构建了高效表达目的重组蛋白的问号钩体fliN基因原核表达系统,RFliN有良好的免疫反应性,为深入研究FliN致病及免疫保护作用奠定了基础。The aim of this study is to clone a flagellar-associated fliN gene of Leptospira interrogans, and to construct its prokaryotic expression system so as to identify the immunogenicity of the expressed products. The genomic DNA was extracted from Leptospira interrogans serogroup Icterohaemorrhagiae serovar lai strain Lai. By using high fidelity PCR, the entire fliN gene was amplified for sequencing after T-A cloning. A prokaryotic expression system of fliN gene was constructed by using routine method. IPTG was applied to induce the target recombinant protein rFliN expression and SDS-PAGE plus BioRad Agarose Image Analyser was performed to determine the output of rFliN. Then the rFliN was extracted and purified by Ni-NTA affinity chromatography. Western blot assays with rabbit antiserum against the whole cell of L. interrogans serogroup Icterohaemorrhagiae strain Lai was used to identify rFliN and its immunoreactivity. According to the sequencing results, the similarity of both the nucleotide and putative amino acid sequences of the cloned fliN gene was 100% compared to the reported corresponding sequences. The rFliN output expressed by the prokaryotic expression system pET32a-fliN-E, coliBL21DE3 was approximate 30% of the total bacterial proteins. The purified rFliN only showed a single fragment in the gel. The recombinant protein rFliN was able to combine with the antiserum. All these results lead us to have a conclusion that a prokaryotic expression system with high efficiency of L. interrogans fliN gene is successfully constructed in this study and the recombinant protein rFliN shows a fine immunoreactivity, which lay a foundation for further study of pathogenic and immunoprotective effects of fliN.

关 键 词：问号钩端螺旋体 fliN基因 克隆 原核表达 鉴定 

分 类 号：R377[医药卫生—病原生物学]