本文關(guān)鍵詞： 糞腸球菌 毒力因子 溶血素 原核表達(dá)　出處：《福建醫(yī)科大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:檢測糞、屎腸球菌毒力基因分布的差別;構(gòu)建腸球菌溶血素cylLl,cylA重組子,體外表達(dá)cylLl,cylA蛋白。為研究腸球菌毒力機(jī)制以及疫苗的研究奠定基礎(chǔ)。 方法:根據(jù)Genebank上的腸球菌基因序列,合成cylA、cylLl、gelE、efaA引物,PCR檢測四種基因在臨床分離菌株中的分布情況。PCR擴(kuò)增cylLl,cylA基因片段,連接到pET42a載體上,轉(zhuǎn)化到Top10檢測陽性克隆,鑒定測序,重組質(zhì)粒轉(zhuǎn)化到BL21(DE)菌株,IPTG誘導(dǎo)表達(dá)腸球菌cylLl,cylA,SDS-PAGE分析cylLl, cylA蛋白,Western Blot進(jìn)行鑒定。 結(jié)果: 1.113株臨床分離腸球菌cylA,cylLl,gelE,efaA基因檢出率分別是53.98%、52.21%、63.72%、64.60%。其中92株糞腸球菌cylA,cylLl,gelE,efaA基因檢出率分別是58.7%、57.6%、70.7%、73.9%;21株屎腸球菌cylA,cylLl,gelE,efaA基因檢出率分別是33.3%、28.6%、33.3%、23.8%。糞腸球菌、屎腸球菌各毒力基因檢出率經(jīng)卡方檢驗(yàn),差別有顯著性。 2.構(gòu)建PET42a-cylLl,cylA重組子,體外表達(dá)cylLl,cylA蛋白,通過SDS-PAGE分析、Western Blot鑒定為cylLl,cylA融合蛋白。 結(jié)論: 1.糞腸球菌毒力基因檢出率比屎腸球菌高。2.在pET42a系統(tǒng)中成功表達(dá)了cylA,cylLl融合蛋白,為進(jìn)一步大規(guī)模表達(dá)和純化cylA,cylLl蛋白,研究其生物學(xué)活性及臨床診斷治療奠定基礎(chǔ)。
[Abstract]:Objective: to detect the distribution of virulence genes in feces and Enterococcus faecium. To construct the recombinant cylLllA recombinant of enterococcal hemolysin, and express cylLllA protein in vitro, which lays a foundation for the study of virulence mechanism of Enterococcus and the study of vaccine. Methods: according to the gene sequence of Enterococcus on Genebank, the primer Cyl Agna cyl Llngel EgelefaA was synthesized. PCR was used to detect the distribution of the four genes in the clinical isolates. PCR amplified the cylLlcylA gene fragment and ligated to the pET42a vector. The recombinant plasmid was transformed into BL21D) strain to induce the expression of CylLlcylA in Enterococcus. SDS-PAGE was used to identify cyl l and cylA protein by Western Blot. Results: 1. The detection rate of the gene of CylAcardia lllgel EefaA gene was 53.98% and 52.21%, 63.72%, respectively, in the clinical isolates of Enterococci. The positive rates of the gene were 53.98% and 52.21%, respectively. Among them, 92 strains of Enterococcus faecalis cyl Llngel Eguela A gene were 58.7% and 57.6%, 70.7% and 73.9%, respectively. The detection rate of EgelefaA gene in 21 strains of Enterococcus faecium was 33.328.6and 33.33.8. Enterococcus faecalis. The detection rate of virulence genes of Enterococcus faecium was significantly different by chi-square test. 2.Recombinant PET42a-cylLlcylA was constructed and expressed in vitro. The protein was analyzed by SDS-PAGE. Western Blot was identified as cylllA fusion protein. Conclusion: 1. The detection rate of virulence gene of Enterococcus faecalis was higher than that of Enterococcus faecium. 2. The fusion protein of cylAl l was successfully expressed in pET42a system, which was used to express and purify cylA on a large scale. CylLl protein, to study its biological activity and clinical diagnosis and treatment lay the foundation.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R378

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