本文關(guān)鍵詞： 艱難梭菌 tcdC基因 原核表達(dá) 多克隆抗體制備　出處：《河北大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：艱難梭菌(Clostridium difficile,CD)是一種厭氧生長的革蘭氏陽性梭狀芽孢桿菌,是人類腸道感染的主要致病菌。使用抗菌藥物后可導(dǎo)致艱難梭菌過度生長,大量分泌毒素A(腸毒素)和毒素B(細(xì)胞毒素),產(chǎn)生一系列艱難梭菌感染(Clostridium difficile infection,CDI)癥狀。近年來,水環(huán)境及相關(guān)環(huán)境中艱難梭菌的檢出率的不斷增加,以及隨著強(qiáng)毒株的出現(xiàn),表明艱難梭菌感染在水生生物尤其是水產(chǎn)養(yǎng)殖生物中流行的可能性陡然增加。因此,深入開展艱難梭菌致病機(jī)理的研究在水生生物疾病防治方面顯得尤為重要,對水產(chǎn)養(yǎng)殖及水環(huán)境健康具有積極的意義。研究表明,艱難梭菌TcdA和B毒素是機(jī)體致病的重要因子,其基因表達(dá)在致病區(qū)(PaLoc)中受多個因子調(diào)控。目前tcdC基因是否具有負(fù)調(diào)節(jié)功能尚存爭論、在致病過程中的作用機(jī)制仍不明確。本研究以CD標(biāo)準(zhǔn)菌株(ATCC43255)作為研究對象,首先針對tcdC基因編碼序列進(jìn)行優(yōu)化,將優(yōu)化后的序列通過搭橋PCR方法重新合成,構(gòu)建原核表達(dá)載體并轉(zhuǎn)化到大腸桿菌中,誘導(dǎo)表達(dá)重組蛋白,用純化的TcdC蛋白免疫動物,制備了高效價的多克隆抗體,并利用所制備的抗體對艱難梭菌TcdC蛋白的表達(dá)情況進(jìn)行了初步研究。研究結(jié)果如下:(1)我們根據(jù)大腸桿菌同義密碼子相對頻率(RFSC)表對艱難梭菌tcdC基因的密碼子進(jìn)行了優(yōu)化,并采用搭橋PCR的方法,成功擴(kuò)增出優(yōu)化后的tcdC基因全長序列。(2)采用分子克隆的方法,成功構(gòu)建了艱難梭菌TcdC蛋白的原核表達(dá)質(zhì)粒pBVGST-S(×2)、pBVGST-L、pBVIL1-S(×2)和pBVIL1-L,經(jīng)過42℃水浴誘導(dǎo)后,結(jié)果誘導(dǎo)表達(dá)的各個重組蛋白與預(yù)期的大小相符,表達(dá)的蛋白主要以包涵體形式存在。采用親和層析和離子交換層析對目的蛋白進(jìn)行純化,獲得了高純度的融合蛋白。(3)將上述分離純化的目的蛋白GST-S(×2)、GST-L免疫兔子制備多克隆抗體,間接ELISA法檢測到抗體效價均可達(dá)到1:6.4萬以上。純化抗體的電泳結(jié)果只出現(xiàn)50KD、25KD,與IgG的重鏈、輕鏈分子量吻合。經(jīng)Western蛋白印記實(shí)驗分析表明制備的多克隆抗體能夠特異性識別相應(yīng)的抗原,并且其中的TcdC-L多抗可特異性識別艱難梭菌TcdC蛋白。在對CD中TcdC蛋白的表達(dá)情況進(jìn)行分析時發(fā)現(xiàn),TcdC蛋白存在于胞內(nèi),也存在于培養(yǎng)上清中,是一種膜相關(guān)蛋白,并且在菌落生長的對數(shù)期大量表達(dá),而在穩(wěn)定期則表達(dá)量降低。綜上所述,本研究成功制備了艱難梭菌TcdC重組蛋白和多克隆抗體,為后續(xù)研究tcdC基因在艱難梭菌致病過程中的作用及機(jī)制奠定了基礎(chǔ)。
[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，

本文編號：1508871