艰难梭菌tcdC基因的克隆表达与抗体制备

发布时间：2018-02-13 18:59

  本文关键词： 艰难梭菌 tcdC基因 原核表达 多克隆抗体制备　出处：《河北大学》2017年硕士论文　论文类型：学位论文

【摘要】：艰难梭菌(Clostridium difficile,CD)是一种厌氧生长的革兰氏阳性梭状芽孢杆菌,是人类肠道感染的主要致病菌。使用抗菌药物后可导致艰难梭菌过度生长,大量分泌毒素A(肠毒素)和毒素B(细胞毒素),产生一系列艰难梭菌感染(Clostridium difficile infection,CDI)症状。近年来,水环境及相关环境中艰难梭菌的检出率的不断增加,以及随着强毒株的出现,表明艰难梭菌感染在水生生物尤其是水产养殖生物中流行的可能性陡然增加。因此,深入开展艰难梭菌致病机理的研究在水生生物疾病防治方面显得尤为重要,对水产养殖及水环境健康具有积极的意义。研究表明,艰难梭菌TcdA和B毒素是机体致病的重要因子,其基因表达在致病区(PaLoc)中受多个因子调控。目前tcdC基因是否具有负调节功能尚存争论、在致病过程中的作用机制仍不明确。本研究以CD标准菌株(ATCC43255)作为研究对象,首先针对tcdC基因编码序列进行优化,将优化后的序列通过搭桥PCR方法重新合成,构建原核表达载体并转化到大肠杆菌中,诱导表达重组蛋白,用纯化的TcdC蛋白免疫动物,制备了高效价的多克隆抗体,并利用所制备的抗体对艰难梭菌TcdC蛋白的表达情况进行了初步研究。研究结果如下:(1)我们根据大肠杆菌同义密码子相对频率(RFSC)表对艰难梭菌tcdC基因的密码子进行了优化,并采用搭桥PCR的方法,成功扩增出优化后的tcdC基因全长序列。(2)采用分子克隆的方法,成功构建了艰难梭菌TcdC蛋白的原核表达质粒pBVGST-S(×2)、pBVGST-L、pBVIL1-S(×2)和pBVIL1-L,经过42℃水浴诱导后,结果诱导表达的各个重组蛋白与预期的大小相符,表达的蛋白主要以包涵体形式存在。采用亲和层析和离子交换层析对目的蛋白进行纯化,获得了高纯度的融合蛋白。(3)将上述分离纯化的目的蛋白GST-S(×2)、GST-L免疫兔子制备多克隆抗体,间接ELISA法检测到抗体效价均可达到1:6.4万以上。纯化抗体的电泳结果只出现50KD、25KD,与IgG的重链、轻链分子量吻合。经Western蛋白印记实验分析表明制备的多克隆抗体能够特异性识别相应的抗原,并且其中的TcdC-L多抗可特异性识别艰难梭菌TcdC蛋白。在对CD中TcdC蛋白的表达情况进行分析时发现,TcdC蛋白存在于胞内,也存在于培养上清中,是一种膜相关蛋白,并且在菌落生长的对数期大量表达,而在稳定期则表达量降低。综上所述,本研究成功制备了艰难梭菌TcdC重组蛋白和多克隆抗体,为后续研究tcdC基因在艰难梭菌致病过程中的作用及机制奠定了基础。
[Abstract]:Clostridium di traffic (CDCD) is an anaerobic gram-positive Clostridium aureus that is the leading cause of intestinal infections in humans. The use of antibiotics can lead to excessive growth of Clostridium diffuciatus. A large number of toxin A (enterotoxin) and toxin B (cytotoxin B) produce a series of symptoms of Clostridium difficile infection. In recent years, the detection rate of Clostridium difficile infection in water environment and related environment has been increasing, and with the emergence of virulent strains, The results indicate that the prevalence of Clostridium diffusa infection in aquatic organisms, especially in aquaculture, is increasing sharply. Therefore, it is very important to study the pathogenic mechanism of Clostridium davidii in the prevention and treatment of aquatic biological diseases. It has positive significance for aquaculture and water environment health. Studies show that TcdA and B toxin of Clostridium difficultii are important factors of organism pathogenicity. The expression of tcdC gene is regulated by many factors in the pathogenicity of Pa locus. At present, it is still controversial whether the tcdC gene has negative regulatory function, and the mechanism of its role in the pathogenesis is still unclear. In this study, CD standard strain ATCC 43255 was used as the research object. Firstly, the tcdC gene coding sequence was optimized, the optimized sequence was resynthesized by bypass PCR method, the prokaryotic expression vector was constructed and transformed into Escherichia coli, the recombinant protein was induced to express, and the purified TcdC protein was used to immunize animals. High titer polyclonal antibodies were prepared. The expression of TcdC protein in Clostridium diffracta was studied by using the prepared antibodies. The results are as follows: 1) We optimized the codon of tcdC gene of Clostridium difficulticus according to the relative frequency of codon synonymous codon of Escherichia coli. Using the method of bypass PCR, the optimized full-length tcdC gene sequence was successfully amplified. By molecular cloning, the prokaryotic expression plasmids pBVGST-S( 脳 2pBVGST-LBVVIL1-S-1) and pBVIL1-L were successfully constructed by molecular cloning method. After induced by water bath at 42 鈩，

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