副溶血弧菌tlh基因缺失株的构建  

Construction of tlh Gene Deleted Mutant of Vibrio parahaemolyticus

作　　者：赵永刚[1,2] 唐小千[1] 战文斌[1] 

机构地区：[1]中国海洋大学教育部海水养殖重点实验室,青岛266003 [2]中国动物卫生与流行病学中心,青岛266032

出　　处：《上海交通大学学报（农业科学版）》2011年第1期10-15,共6页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：国家"863"项目(2006AA100306)和(2006AA100307)

摘　　要：副溶血弧菌(Vibrio parahaemolyticuss,VP)是我国海水养殖鱼、虾、贝类的重要病原,其主要致病因子热不稳定性溶血素(thermolabile hemolysin,TLH)的功能和致病性仍不十分清楚。本研究利用PCR技术从VP临床分离株的基因组DNA中扩增出tlh基因,利用同源重组技术,构建了副溶血弧菌tlh基因打靶载体;利用PCR方法筛选tlh基因缺失株,并在卵磷脂存在条件下进行溶血性检测,结果在5%兔血平板上能够引起溶血,5%的马血琼脂平板未出现溶血现象,说明tlh基因敲除成功。本研究成功构建了副溶血弧菌tlh基因缺失株,为进一步研究tlh基因致病性和溶血机制提供了必要的实验菌株,为副溶血弧菌其他基因的敲除提供了可行性依据。Vibrio parahaemolyticus（VP） is listed as one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis,and shellfish in coastal areas of China.TLH（thermolabile hemolysin） is one of major virulence factors produced by VP.The information regarding the biological function and nosogenesis remains completely lacking.In this study,we cloned tlh gene from the genome DNA of VP by polymerase chain reaction（PCR）.Targeting vector of tlh gene was constructed by homologous recombination and gene deletion mutant was identified by PCR.Hemolysis of tlh gene deleted mutant was separately identified on 5% rabbit blood agar plate and 5% horse blood agar plate,The tlh gene deleted mutant in the addition of phmphatidyehdine,could induce hemolysis on rabbit blood agar plate but not on horse blood agar plate.The result suggested that tlh gene was knockouted successfully.Conclusively,tlh gene deleted mutant was constructed by homologous recombination in order to discuss molecular mechanism of nosogenesis and hemolysis.At the same time,an effective method in which unknown functional genes in VP genome would researched.

关 键 词：副溶血弧菌 tlh基因 同源重组 基因敲除 溶血活性 

分 类 号：R378.3[医药卫生—病原生物学]