| 摘要: |
| 利用PCR 方法从VP 基因组DNA 中扩增出 trh 溶血素基因, 构建了大肠杆菌原核表达载体, 对 trh 进行了表达和纯化, 经溶血活性检测, 复性蛋白具有溶血活性。同时还构建了 trh 基因缺失株, 对 trh 基因进行了基因敲除研究。结果表明, 单独敲除 trh 基因并不能够影响菌株的溶血活性, 说明副溶血弧菌还存在其它溶血素基因。 |
| 关键词: VP, TRH, 克隆, 表达, 基因敲除 |
| DOI：10.11693/hyhz201301006006 |
| 分类号: |
| 基金项目:国家高技术研究发展(863)计划项目: 海水养殖动物疾病高通量诊断技术, 2006AA100306 号和浅海养殖扇贝流行病控制技术, 2006AA100307 号。 |
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| CLONING, EXPRESSING AND CONSTRUCTION OF GENE DELETED MUTANT OF TRH OF VIBRIO PARAHAEMOLYTICUS |
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ZHAO Yong-Gang1,2, TANG Xiao-Qian1, ZHAN Wen-Bin1
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1.Key Laboratory of Mariculture, Ministry of Education, Ocean University of China;2.China Animal Health and Epidemiology Center
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| Abstract: |
| Vibrio parahaemolyticus (VP) is a Gram-negative, facultative anaerobic marine bacteria, which is an important pathogen in aquaculture. TRH (TDH-related hemolysin) is one of major virulence factors produced by VP. The biological function and nosogenesis of trh gene remains completely unknown. In this study, we cloned trh gene from the genome DNA of VP by polymerase chain reaction (PCR). The PCR product was purified and inserted into prokaryotic expression vector pET-28a(+). The recombinant TRH was successfully expressed in E. coli strain BL21(DE3)and purified by Ni-IDA affinity chromatography. The recombinant TRH was activated and showed the hemolytic activity after renaturation. Targeting vector of trh gene was constructed by homologous recombination and gene deletion mutant was identified by PCR. Hemolysis was identified on 5% rabbit blood agar plate, The gene deleted mutant could induce hemolysis. It indicated that knockout of single gene had no influence on hemolysis of VP. In summary, the results have laid a good foundation for further exploration of the pathogenicity of VP from sea food isolates. |
| Key words: Vibrio parahaemolyticus, TRH, Cloning, Expression, Gene knockout |
