發(fā)布時間：2018-05-16 04:40

  本文選題：創(chuàng)傷弧菌 + vvhA　； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:創(chuàng)傷弧菌(V.vulfinicus)溶細(xì)胞素VvhA蛋白是由V.vulfinicus的vvhA基因所編碼的一種外分泌蛋白。它通過在靶細(xì)胞膜表面形成小孔發(fā)揮其生物作用,具有溶血活性和細(xì)胞毒性,既往研究認(rèn)為該基因的編碼產(chǎn)物在創(chuàng)傷弧菌感染人體中發(fā)揮了重要的作用。目前國內(nèi)外尚缺乏對vvhA基因及其編碼產(chǎn)物VvhA蛋白免疫應(yīng)答的研究報道。本文以V.vulfinicus臨床分離菌株Y066為研究對象,通過分子生物學(xué)、原核重組表達(dá)技術(shù)、生物信息學(xué)、動物模型和流式細(xì)胞術(shù)等手段對vvhA基因及其編碼產(chǎn)物的機體免疫應(yīng)答規(guī)律進(jìn)行了探究,這為深入研究其在V.vulfinicus致病機制中的地位及相關(guān)疫苗的制備奠定了基礎(chǔ)。方法:1.將V.vulfinicus Y066于哥倫比亞血瓊脂平板中培養(yǎng),采用普通生化反應(yīng)聯(lián)合16S rRNA序列分析法對其進(jìn)行鑒定。采用PCR手段,以V.vulfnicus Y066菌株基因組為模板,采用特異性引物擴增vvhA基因片段,采用原核重組表達(dá)技術(shù)構(gòu)建vvhA基因的原核表達(dá)載體,并送測序進(jìn)行驗證;2.原核表達(dá)載體經(jīng)測序后,推導(dǎo)其編碼產(chǎn)物的氨基酸序列。采用生物信息學(xué)分析方法獲得vvhA基因推導(dǎo)蛋白的基本理化性質(zhì)、細(xì)胞定位、信號肽及空間結(jié)構(gòu)等相關(guān)信息;3.以IPTG誘導(dǎo)E.coli Rosetta(DE3)宿主表達(dá)融合蛋白VvhA。用Ni2+-NTA親和層析柱對融合蛋白進(jìn)行純化和鑒定。以純化后的VvhA為抗原,通過免疫Balb/c小鼠制備抗-VvhA多克隆抗體,通過ELISA方法檢測抗體效價并檢測多克隆抗血清是否具有保護(hù)作用;4.采用同源重組的方法構(gòu)建vvhA基因敲除質(zhì)粒pBlueKM40-△vvhA::kan,采用電轉(zhuǎn)化法將其轉(zhuǎn)入V.vulfinicus Y066感受態(tài)細(xì)胞中以獲得vvhA基因缺失株;5.通過構(gòu)建動物感染模型,腹腔注射V.vulfinicus Y066野生株及vvhA基因缺失株,檢測小鼠脾臟Th1/Th2細(xì)胞亞群的變化,同時分離小鼠血清檢測IFN-γ的含量,以探討VvhA蛋白所引起的免疫應(yīng)答類型;同時取小鼠的肝臟、腎臟、小腸、肺臟、心臟和腦等組織,進(jìn)行固定及染色以檢測其組織病理學(xué)變化,分離小鼠血清檢測相關(guān)炎癥因子含量。結(jié)果:1.以V.vulfnicusY066為模板,成功構(gòu)建了 vvhA基因的原核表達(dá)載體pET28a-vvhA;2.生物信息學(xué)分析顯示,vvhA基因全長1416bp。該基因可編碼分子量為52.87kDa的由471個氨基組成的蛋白質(zhì)。該蛋白的等電點為7.55,屬于親水性的穩(wěn)定蛋白,該蛋白在1-20個氨基酸處存在信號肽,其細(xì)胞定位也預(yù)測該蛋白為外分泌蛋白;3.原核表達(dá)載體pET28a-vvhA經(jīng)誘導(dǎo)純化后獲得了高純度的VvhA蛋白。通過免疫小鼠獲得了效價為1:51200的鼠抗-VvhA多克隆抗體,并顯示出保護(hù)性作用;4.成功構(gòu)建了vvhA 基因的敲除載體pBlueKM40-△vvhA::kan,并獲得了 vvhA基因缺失株;5.通過動物感染模型、流式細(xì)胞術(shù)等免疫學(xué)手段檢測了小鼠脾臟Th1/Th2細(xì)胞亞群的變化,結(jié)果顯示,創(chuàng)傷弧菌野生株組小鼠脾臟Th1細(xì)胞亞群應(yīng)答明顯增強,Th2細(xì)胞亞群無明顯變化,其血清IFN-γ濃度也顯著高于缺失組。病理學(xué)檢測結(jié)果表明,小鼠肝組織受損最嚴(yán)重,且野生株肝臟損傷程度明顯重于缺失株,血清學(xué)檢測結(jié)果也與組織損傷結(jié)果一致(野生株:ALT 160U/L,AST 717 U/L;缺失株:ALT41U/L,AST147U/L)。結(jié)論:1.成功構(gòu)建了 vvhA基因的原核表達(dá)載體pET-28a-vvhA(1353bp)。2.VvhA蛋白是一種親水性高、穩(wěn)定性強的外分泌蛋白,有較強的抗原性,適合抗體的制備。該蛋白含有殺白細(xì)胞素和RICIN超家族,與該蛋白的細(xì)胞毒性作用密切相關(guān)。3.獲得了目的蛋白和高效價的鼠源VvhA多克隆抗體,動物實驗證實該多克隆抗體具有中和毒素的保護(hù)作用。4.成功構(gòu)建了vvhA基因缺失株。5.VvhA蛋白能引起小鼠Th1型細(xì)胞免疫應(yīng)答并對組織引起損傷,這可為VvhA蛋白免疫致病機制的深入研究和疫苗的制備提供了基礎(chǔ)。
[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.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R378
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本文編號：1895516