肠球菌毒力因子的检测及溶血素cylLl和cylA基因的原核表达

发布时间：2018-01-22 05:19

本文关键词： 粪肠球菌毒力因子溶血素原核表达　出处：《福建医科大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的:检测粪、屎肠球菌毒力基因分布的差别;构建肠球菌溶血素cylLl,cylA重组子,体外表达cylLl,cylA蛋白。为研究肠球菌毒力机制以及疫苗的研究奠定基础。方法:根据Genebank上的肠球菌基因序列,合成cylA、cylLl、gelE、efaA引物,PCR检测四种基因在临床分离菌株中的分布情况。PCR扩增cylLl,cylA基因片段,连接到pET42a载体上,转化到Top10检测阳性克隆,鉴定测序,重组质粒转化到BL21(DE)菌株,IPTG诱导表达肠球菌cylLl,cylA,SDS-PAGE分析cylLl, cylA蛋白,Western Blot进行鉴定。结果: 1.113株临床分离肠球菌cylA,cylLl,gelE,efaA基因检出率分别是53.98%、52.21%、63.72%、64.60%。其中92株粪肠球菌cylA,cylLl,gelE,efaA基因检出率分别是58.7%、57.6%、70.7%、73.9%;21株屎肠球菌cylA,cylLl,gelE,efaA基因检出率分别是33.3%、28.6%、33.3%、23.8%。粪肠球菌、屎肠球菌各毒力基因检出率经卡方检验,差别有显著性。 2.构建PET42a-cylLl,cylA重组子,体外表达cylLl,cylA蛋白,通过SDS-PAGE分析、Western Blot鉴定为cylLl,cylA融合蛋白。结论: 1.粪肠球菌毒力基因检出率比屎肠球菌高。2.在pET42a系统中成功表达了cylA,cylLl融合蛋白,为进一步大规模表达和纯化cylA,cylLl蛋白,研究其生物学活性及临床诊断治疗奠定基础。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R378

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