创伤弧菌溶细胞素致病机制与机体免疫应答的研究

发布时间：2018-05-16 04:40

  本文选题：创伤弧菌 + vvhA　； 参考：《大连医科大学》2017年硕士论文

【摘要】：目的:创伤弧菌(V.vulfinicus)溶细胞素VvhA蛋白是由V.vulfinicus的vvhA基因所编码的一种外分泌蛋白。它通过在靶细胞膜表面形成小孔发挥其生物作用,具有溶血活性和细胞毒性,既往研究认为该基因的编码产物在创伤弧菌感染人体中发挥了重要的作用。目前国内外尚缺乏对vvhA基因及其编码产物VvhA蛋白免疫应答的研究报道。本文以V.vulfinicus临床分离菌株Y066为研究对象,通过分子生物学、原核重组表达技术、生物信息学、动物模型和流式细胞术等手段对vvhA基因及其编码产物的机体免疫应答规律进行了探究,这为深入研究其在V.vulfinicus致病机制中的地位及相关疫苗的制备奠定了基础。方法:1.将V.vulfinicus Y066于哥伦比亚血琼脂平板中培养,采用普通生化反应联合16S rRNA序列分析法对其进行鉴定。采用PCR手段,以V.vulfnicus Y066菌株基因组为模板,采用特异性引物扩增vvhA基因片段,采用原核重组表达技术构建vvhA基因的原核表达载体,并送测序进行验证;2.原核表达载体经测序后,推导其编码产物的氨基酸序列。采用生物信息学分析方法获得vvhA基因推导蛋白的基本理化性质、细胞定位、信号肽及空间结构等相关信息;3.以IPTG诱导E.coli Rosetta(DE3)宿主表达融合蛋白VvhA。用Ni2+-NTA亲和层析柱对融合蛋白进行纯化和鉴定。以纯化后的VvhA为抗原,通过免疫Balb/c小鼠制备抗-VvhA多克隆抗体,通过ELISA方法检测抗体效价并检测多克隆抗血清是否具有保护作用;4.采用同源重组的方法构建vvhA基因敲除质粒pBlueKM40-△vvhA::kan,采用电转化法将其转入V.vulfinicus Y066感受态细胞中以获得vvhA基因缺失株;5.通过构建动物感染模型,腹腔注射V.vulfinicus Y066野生株及vvhA基因缺失株,检测小鼠脾脏Th1/Th2细胞亚群的变化,同时分离小鼠血清检测IFN-γ的含量,以探讨VvhA蛋白所引起的免疫应答类型;同时取小鼠的肝脏、肾脏、小肠、肺脏、心脏和脑等组织,进行固定及染色以检测其组织病理学变化,分离小鼠血清检测相关炎症因子含量。结果:1.以V.vulfnicusY066为模板,成功构建了 vvhA基因的原核表达载体pET28a-vvhA;2.生物信息学分析显示,vvhA基因全长1416bp。该基因可编码分子量为52.87kDa的由471个氨基组成的蛋白质。该蛋白的等电点为7.55,属于亲水性的稳定蛋白,该蛋白在1-20个氨基酸处存在信号肽,其细胞定位也预测该蛋白为外分泌蛋白;3.原核表达载体pET28a-vvhA经诱导纯化后获得了高纯度的VvhA蛋白。通过免疫小鼠获得了效价为1:51200的鼠抗-VvhA多克隆抗体,并显示出保护性作用;4.成功构建了vvhA 基因的敲除载体pBlueKM40-△vvhA::kan,并获得了 vvhA基因缺失株;5.通过动物感染模型、流式细胞术等免疫学手段检测了小鼠脾脏Th1/Th2细胞亚群的变化,结果显示,创伤弧菌野生株组小鼠脾脏Th1细胞亚群应答明显增强,Th2细胞亚群无明显变化,其血清IFN-γ浓度也显著高于缺失组。病理学检测结果表明,小鼠肝组织受损最严重,且野生株肝脏损伤程度明显重于缺失株,血清学检测结果也与组织损伤结果一致(野生株:ALT 160U/L,AST 717 U/L;缺失株:ALT41U/L,AST147U/L)。结论:1.成功构建了 vvhA基因的原核表达载体pET-28a-vvhA(1353bp)。2.VvhA蛋白是一种亲水性高、稳定性强的外分泌蛋白,有较强的抗原性,适合抗体的制备。该蛋白含有杀白细胞素和RICIN超家族,与该蛋白的细胞毒性作用密切相关。3.获得了目的蛋白和高效价的鼠源VvhA多克隆抗体,动物实验证实该多克隆抗体具有中和毒素的保护作用。4.成功构建了vvhA基因缺失株。5.VvhA蛋白能引起小鼠Th1型细胞免疫应答并对组织引起损伤,这可为VvhA蛋白免疫致病机制的深入研究和疫苗的制备提供了基础。
[Abstract]:Objective: the lysin VvhA protein of Vibrio vulnificus (V.vulfinicus) is an exocrine protein encoded by the vvhA gene of V.vulfinicus. It plays its biological action through the formation of small pores on the surface of the target cell membrane, which has hemolytic activity and cytotoxicity. At present, there is still a lack of research on the immune response to the vvhA gene and its encoding product VvhA protein at home and abroad. In this paper, the vvhA gene and its encoding production by means of molecular biology, Prokaryotic Recombinant Expression Technology, bioinformatics, animal model and flow cytometry were used to study the immune response of the vvhA gene and its encoded product, Y066. The immune response law of the body was explored, which laid the foundation for the in-depth study of its status in the pathogenesis of V.vulfinicus and the preparation of the related vaccines. Method: 1. V.vulfinicus Y066 was cultured in Columbia blood agar plate, and the common biochemical reaction combined with 16S rRNA sequence analysis was used to identify them. PCR hands were used. The genome of V.vulfnicus Y066 strain was used as a template, the vvhA gene fragment was amplified by specific primers, and the prokaryotic expression vector of the vvhA gene was constructed by prokaryotic recombinant expression technology, and the sequence was verified. 2. prokaryotic expression vector was sequenced and the amino acid sequence of the encoding product was derived. The bioinformatics analysis method was used to obtain vv. The hA gene derives the basic physical and chemical properties of the protein, cell location, signal peptide and spatial structure and other related information. 3. IPTG induced E.coli Rosetta (DE3) host expression fusion protein VvhA. is purified and identified by Ni2+-NTA affinity chromatography column. The purified VvhA is the antigen, and the anti -VvhA polyclones are prepared by immune Balb/c mice. Antibody titer was detected by ELISA method and the protective effect of polyclonal antisera was detected. 4. vvhA gene knockout plasmid pBlueKM40- Delta vvhA:: Kan was constructed by homologous recombination method, and it was transferred into V.vulfinicus Y066 receptive cells by electric transformation to obtain vvhA gene deletion strain; 5. through the construction of animal infection model. Type, intraperitoneal injection of V.vulfinicus Y066 wild strain and vvhA gene deletion strain to detect the changes of spleen Th1/Th2 cell subgroup in mice, and detect the content of IFN- gamma in mice serum in order to explore the type of immune response caused by VvhA protein, and take the liver, kidney, small intestine, lungs, heart and brain tissue of mice to be fixed and stained. In order to detect the histopathological changes, the content of related inflammatory factors was detected in the serum of mice. Results: 1. the prokaryotic expression vector pET28a-vvhA of vvhA gene was constructed with V.vulfnicusY066 as a template. 2. bioinformatics analysis showed that the total length of vvhA gene, 1416bp., was composed of 471 amino groups of the encoding molecular weight of 52.87kDa. Protein. The isoelectric point of the protein is 7.55, which belongs to the hydrophilic stable protein, the protein has signal peptide at 1-20 amino acids, and its cell location also predicts the protein as exocrine protein; 3. prokaryotic expression vector pET28a-vvhA has been purified and purified VvhA egg white. The mice were immunized to get the mice of 1:51200. Anti -VvhA polyclonal antibody and showed protective effect. 4. vvhA gene knockout carrier pBlueKM40- Delta vvhA:: Kan was successfully constructed and vvhA gene deletion strain was obtained; 5. through the animal infection model and flow cytometry, the changes of the spleen Th1/Th2 cell subgroup in mice were detected, and the results showed that the wild strains of Vibrio vulnificus were found. The subgroup response of Th1 cells in the spleen of mice was obviously enhanced, and the Th2 cell subgroup had no obvious change, and the serum IFN- gamma concentration was also significantly higher than that of the missing group. The pathological examination showed that the liver tissue was the most serious damage in mice, and the degree of liver damage in the wild plant was significantly heavier than that of the missing strain, and the results of serological detection were also in accordance with the tissue damage results (wild strain: ALT 160U/L, AST 717 U/L, missing strain: ALT41U/L, AST147U/L). Conclusion: 1. the prokaryotic expression vector of vvhA gene is successfully constructed, pET-28a-vvhA (1353bp).2.VvhA protein is a high hydrophilic, stable exocrine protein with strong antigenicity and suitable for preparation of antibodies. The protein contains the superfamily of leukocyte and RICIN, and the protein. The cytotoxic effect closely related to.3. obtained the target protein and the high price mouse VvhA polyclonal antibody. The animal experiment proved that the polyclonal antibody had the protective effect of neutralizing toxin..4. successfully constructed the.5.VvhA protein of the vvhA gene deletion strain, which could cause the immune response of the mouse type Th1 cell and damage the tissue, which could be the VvhA protein. It provides a basis for further research on immune pathogenesis and vaccine preparation.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
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本文编号：1895516