| 副溶血弧菌trh基因的克隆、表达及基因缺失株的构建 |
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| 引用本文： | 赵永刚,唐小千,战文斌.副溶血弧菌trh基因的克隆、表达及基因缺失株的构建[J].海洋与湖沼,2013,44(1):38-42. |
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| 作者姓名： | 赵永刚 唐小千 战文斌 |
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| 作者单位： | 1. 中国海洋大学教育部海水养殖重点实验室 青岛 266003;中国动物卫生与流行病学中心 青岛 266032
2. 中国海洋大学教育部海水养殖重点实验室 青岛 266003 |
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| 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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| 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.
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| 关 键 词： | VP TRH 克隆 表达 基因敲除 |
| 收稿时间： | 2011/12/31 0:00:00 |
| 修稿时间： | 2012/2/28 0:00:00 |
CLONING, EXPRESSING AND CONSTRUCTION OF GENE DELETED MUTANT OF TRH OF VIBRIO PARAHAEMOLYTICUS |
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| ZHAO Yong-Gang,TANG Xiao-Qian and ZHAN Wen-Bin.CLONING, EXPRESSING AND CONSTRUCTION OF GENE DELETED MUTANT OF TRH OF VIBRIO PARAHAEMOLYTICUS[J].Oceanologia Et Limnologia Sinica,2013,44(1):38-42. |
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| Authors: | ZHAO Yong-Gang TANG Xiao-Qian and ZHAN Wen-Bin |
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| Institution: | Key Laboratory of Mariculture, Ministry of Education, Ocean University of China; China Animal Health and Epidemiology Center;Key Laboratory of Mariculture, Ministry of Education, Ocean University of China;Key Laboratory of Mariculture, Ministry of Education, Ocean University of China |
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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. |
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| Keywords: | Vibrio parahaemolyticus TRH Cloning Expression Gene knockout |
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