问号钩端螺旋体赖株基因组中一个新溶血基因的表达及功能鉴定  

Expression and functional identification of a novel hemolytic gene in Leptospira interrogans strain Lai

作　　者：杨杨[1] 秦金红[1] 杨宏亮[1] 钟怡[1] 何平[1] 胡宝瑜[1] 郭晓奎[1] 

机构地区：[1]上海交通大学医学院病原生物学教研室,上海200025

出　　处：《中国人兽共患病学报》2007年第5期462-465,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(30670102;30370071)

摘　　要：目的对问号钩端螺旋体赖株基因组中LA0202基因进行克隆表达并对重组蛋白的溶血活性进行初步鉴定。方法以问号钩端螺旋体赖株基因组DNA为模板,PCR扩增LA0202基因并重组到原核表达载体pET28b(+),重组质粒经限制性核酸内切酶酶切并经测序鉴定后,在大肠埃希菌BL21中诱导表达重组蛋白。制备绵羊血平板对表达的重组蛋白进行溶血活性鉴定。RT-PCR检测钩端螺旋体赖株体外培养时LA0202基因的转录。结果在大肠埃希菌中成功表达LA0202基因,表达的重组蛋白具有溶血活性。RT-PCR结果显示钩端螺旋体赖株体外培养时LA0202基因发生了转录。结论问号钩端螺旋体赖株体外培养时LA0202基因发生了转录,LA0202可能为一个新的溶血素基因。The genomic DNA of Leptospira interrogans serovar Lai strain Lai was used as template to amplify gene LA0202 by PCR and the amplified product was then linked to prokaryotie expression vector pET28b（＋）. The recombinant plasmid was digested with BarnHI and HindIII and then transformed to E. coli BL21 cells to express the recombinant protein. The recombinant protein LA0202 was identified with SDS-PAGE and the hemolytic activity of this protein was determined using sheep blood agar plate method. RT-PCR assay was used to detect the transcription of LA0202 gene by culturing L. interrogans strain Lai in vitro. It was demonstrated that the recombinant protein LA0202 was successfully expressed in E. coli BL21 cells and its hemolytic activity could be confirmed using the sheep blood agar plate method. In addition, the transcription of the LA0202 gene was identified in vitro by RT-PCR assay. It is evident that LA0202 gene transcribed in vitro, and it may be the novel hemolytic gene in the genome of L. interrogans.

关 键 词：问号钩端螺旋体 溶血活性 溶血素基因 蛋白表达 

分 类 号：R377[医药卫生—病原生物学]