创伤弧菌溶细胞素基因vvhA的表达和其融合蛋白对细胞的毒性作用

发布时间：2018-03-12 19:15

本文选题：创伤弧菌　切入点：溶细胞素　出处：《浙江大学》2006年硕士论文　论文类型：学位论文

【摘要】：背景和目的创伤弧菌(Vibrio vulnificus)是一种有荚膜的革兰阴性嗜盐弧菌,存在于海水及海产品中,由Farmer等于1979年首先报道。该菌具有强烈毒性,主要引起原发性败血症和严重的创口感染。败血症多与生食含菌的牡蛎等贝类海产品有关,尤其好发于肝硬化、血色素沉着症、地中海贫血等体内荷铁量过多的人群中,病死率超过50%。皮肤创口受海水中的创伤弧菌感染,常于24小时内迅速导致严重的组织坏死。创伤弧菌的确切致病机理至今尚未完全明了。溶细胞素是由结构基因vvhA编码的一种相对分子质量为50851的细胞外蛋白质,具有水溶性,由大多数致病菌株产生,能溶解哺乳动物红细胞及中国仓鼠卵巢(CHO)细胞,是创伤弧菌向胞外释放的唯一细胞毒素,一直被认为是创伤弧菌重要的致病因子,且因其具有穿孔特性而颇受关注。目前对创伤弧菌溶细胞素的细胞毒性机制已有广泛研究和报道,但其确切机制尚未完全明了。为了给今后研究探讨创伤弧菌溶细胞素的致病机制以及制备创伤弧菌基因工程疫苗和免疫诊断试剂盒等奠定实验基础,本研究在我们实验室以往工作基础上,用工程菌BL21DE3~(pET-32a(+)-vvhA)表达vvhA融合蛋白,对其进行纯化、复性;通过台盼蓝染料排斥试验检测vvhA融合蛋白对细胞活力的影响;分别以2,
[Abstract]:Background and objective Vibrio vulnificus (Vibrio vulnificus) is a gram-negative halophilic vibrio with capsule, which is found in seawater and seafood, first reported by Farmer in 1979. Mainly causing primary sepsis and severe wound infection. Septicemia is associated with shellfish such as oyster, which is raw and contains bacteria, especially in people with liver cirrhosis, hemochromatosis, thalassemia and other people with excessive amounts of iron in their bodies. The mortality rate is more than 50%. The skin wound is infected by Vibrio vulnificus in seawater. The exact pathogenicity of Vibrio vulnificus is not fully understood. Cytolysin is an extracellular protein with a relative molecular weight of 50851 encoded by the structural gene vvhA, which is water-soluble. Produced by most pathogenic bacteria strains and capable of dissolving mammalian erythrocytes and Chinese hamster ovary Cho cells, it is the only cytotoxin released by Vibrio vulnificus to extracellular, and has been regarded as an important pathogenic factor of Vibrio vulnificus. The cytotoxicity mechanism of Vibrio vulnificus cytolysin has been widely studied and reported, but its exact mechanism has not been fully understood. In order to lay an experimental foundation for further study on the pathogenicity of vibrio vulnificus lytic cytokine and the preparation of genetic engineering vaccine and immunological diagnostic kit of Vibrio vulnificus, this study was based on the previous work in our laboratory. The vvhA fusion protein was expressed by BL21DE3, pET-32a (TvvhA), purified and renatured. The effect of vvhA fusion protein on cell viability was detected by trypan blue dye exclusion assay.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R378

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