A型产气荚膜梭菌主要抗原蛋白筛选及其免疫保护作用

发布时间：2018-04-22 10:00

本文选题：产气荚膜梭菌 + 坏死性肠炎　；参考：《东北农业大学》2016年硕士论文

【摘要】：产气荚膜梭菌(Clostridium perfringens)最初是William Hery Welch发现的,因而又被命名为魏氏梭菌(Clostridium welchii),是一种G+厌氧芽孢杆菌。在繁殖过程中,C.perfringens能产生多种外毒素和酶,并且它们中的大部分都是具有致病作用的。其中Alpha毒素、Beta毒素、Epsilon毒素和Iota毒素是主要的致死毒素,根据这四种主要毒素可将C.perfringens分为A-E 5个类型。其中A型C.perfringens可引起鸡坏死性肠炎,该病对全球养殖业造成了重大经济损失,因而一直是研究的热点。从1891年C.perfringens被发现以来,对其致病机理和有效预防措施的研究从未间断,但仍缺乏细菌-宿主相互作用的信息和有效的疫苗来控制此菌的感染。本研究制备了C.perfringens标准株ATCC13124菌体蛋白样品,通过双向电泳对菌体蛋白进行分离,并利用全菌多抗进行双向电泳的Western-Blot检测。经过分析,本研究共选取了16个蛋白点,一式两份进行MALDI-TOF-MS和PMF分析,结果共有14种不同的蛋白被测出,除丙酮酸黄素氧还蛋白还原酶(PFO)与假定蛋白(HP)外,其他12种蛋白均为新鉴定出的免疫原性蛋白,大部分的免疫原性蛋白位于细胞质中,其多具有参与细胞生长与代谢的功能。本研究选取了半胱氨酸合成酶(cysteine synthase,Cys)、假定转酮酶(putative transketolase,PTC)、乙醇脱氢酶(alcohol dehydrogenase,ADH)以及嘌呤核苷磷酸化酶(purine nucleoside phosphorylase,PNP)四种免疫原性蛋白,构建其原核表达质粒并进行了蛋白表达,以纯化蛋白为抗原制备了多抗,Western-Blot分析表明所制备的四种多克隆抗体均能特异性结合C.perfringens菌体蛋白,具有很好的反应性。同时构建了四种蛋白的真核表达质粒,体外瞬时转染实验表明四种蛋白均有较好的表达,接着以真核表达质粒进行动物免疫保护实验。实验结果显示,所构建的四种重组真核表达质粒均能在不同程度上抵抗C.perfringens的攻击,降低了鸡只肠道病变程度,能刺激机体产生较好的免疫反应,其中Cys和PNP两种免疫抗原能产生较好的免疫保护作用。综上所述,本研究为C.perfringens的疫苗研发提供了物质基础,对鸡坏死性肠炎的临床控制具有重要意义。
[Abstract]:Clostridium perfringens (Clostridium perfringens) was first discovered by William Hery Welch, so it was named Clostridium welchiiana. C. perfringens can produce a variety of exotoxins and enzymes during reproduction, and most of them are pathogenic. Among them, Alpha toxin, Epsilon toxin and Iota toxin are the main lethal toxins. According to these four main toxins, C.perfringens can be classified into 5 types of A-E. Type A C.perfringens can cause necrotizing enteritis in chickens. Since the discovery of C.perfringens in 1891, the research on its pathogenic mechanism and effective preventive measures has been uninterrupted, but there is still a lack of information on bacteria-host interaction and effective vaccine to control the infection of this bacterium. In this study, we prepared the standard C.perfringens strain ATCC13124 cell protein sample, separated the bacterial protein by two-dimensional electrophoresis, and detected the Western-Blot by Western-Blot with whole bacterial polyclonal antibody. After analysis, a total of 16 protein spots were selected for MALDI-TOF-MS and PMF analysis in two copies. The results showed that 14 different proteins were detected, except pyruvate Flavin oxoredoxin reductase and hypothetical protein. The other 12 proteins are newly identified immunogenic proteins, most of which are located in the cytoplasm, and most of them are involved in cell growth and metabolism. In this study, four immunogenicity proteins, cysteine synthase (cysteine synthase), putative transketolase (PTC), ethanol dehydrogenase (ADH) and purine nucleoside phosphorylase (purine nucleoside phosphorylasePNPs), were selected, and their prokaryotic expression plasmids were constructed and expressed. Western-Blot analysis using purified protein as antigen showed that the four polyclonal antibodies could specifically bind to C.perfringens bacterial protein and had good reactivity. At the same time, the eukaryotic expression plasmids of four proteins were constructed. The transient transfection experiments in vitro showed that the four proteins were well expressed, and then the eukaryotic expression plasmids were used for animal immune protection experiments. The results showed that the four recombinant eukaryotic expression plasmids were able to resist the attack of C.perfringens to some extent, reduce the degree of intestinal lesions, and stimulate the body to produce a better immune response. Two immune antigens, Cys and PNP, can produce better immune protection. In conclusion, this study provides a material basis for the research and development of C.perfringens vaccine and is of great significance for the clinical control of necrotizing enteritis in chicken.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61

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