屎肠球菌毒力基因hyl的鉴定和功能研究

发布时间：2018-01-03 14:17

本文关键词：屎肠球菌毒力基因hyl的鉴定和功能研究　出处：《重庆医科大学》2007年博士论文　论文类型：学位论文

【摘要】： 研究背景:肠球菌是人和动物肠道内的G+正常菌群。屎肠球菌毒力较弱,过去十多年内,屎肠球菌所引起的感染有所上升,由原来的不到10%,上升到30%～40%。而且临床上耐万古霉素肠球菌也绝大多数是屎肠球菌,在美国ICU达到了25%。由此可见,屎肠球菌感染的发病率上升可能与获得了新的毒力因子有关。然而屎肠球菌的致病机制和毒力因子研究甚少,加上多重天然耐药的存在,临床治疗非常困难。如何防治屎肠球菌的感染是近年来感染学界研究的热点。口服免疫治疗,可能是目前除药物治疗外的一种廉价、安全、实用性好、适合大规模高危人群免疫接种、发展前景非常好的新型治疗方法。透明质酸酶是致病性酿脓链球菌、金黄色葡萄球菌和肺炎链球菌等G+球菌的毒力因子之一。肠球菌原来属于D组链球菌,后来才独立分为一属,但其许多特性与链球菌相似。Rice等通过对屎肠球菌基因组进行类比分析,发现屎肠球菌染色体基因序列中有一开放读码框,全长1662bp,其DNA序列以及编码的氨基酸序列与酿脓链球菌透明质酸酶有高度同源性。并将这个基因命名为屎肠球菌透明质酸酶基因(Hyaluronidase, hylEfm)。为了研究hyl基因在屎肠球菌致病中的确切功能,我们通过构建屎肠球菌hyl基因突变菌株,研究hyl基因在屎肠球菌感染中的作用,并在此基础上对hyl基因进行克隆表达和纯化,探讨Hyl蛋白经口服免疫小鼠的免疫保护作用,为屎肠球菌的防治提供基础依据。目的:构建屎肠球菌hyl基因突变株,通过体外生长曲线和体内动物模型实验来研究hyl基因敲除前后屎肠球菌的毒力变化。在此基础上构建hyl基因重组表达质粒,在大肠埃希菌中诱导表达获得重组蛋白,探讨Hyl蛋白经口服免疫小鼠后,机体产生的免疫应答和体内抗菌保护作用。方法:用携带hyl基因插入序列的自杀质粒pTX4577通过体内同源重组,使hyl基因断裂而构建hyl基因突变株。突变株用PCR、脉冲场电泳和Southern blot进行鉴定。通过与野生株TX2466相比,观察突变株体外的生长能力以及小鼠腹膜炎模型和兔心内膜炎模型体内实验的毒力改变情况。然后用PCR扩增hyl基因片段,克隆至pQE-30质粒上,构建重组质粒pQE-hyl,转化E.coli DH5 ,经IPTG诱导表达融合蛋白Hyl;Western blot分析其免疫反应性。融合蛋白经镍离子柱纯化后,用Hyl蛋白和屎肠球菌全菌蛋白TX2466分别给小鼠灌胃进行免疫,ELISA检测小鼠血清IgG和IgA、小肠粘液sIgA和粪便sIgA水平。然后用TX2466腹腔攻击已免疫的小鼠,观察其体内抗菌保护性。结果:经同源重组,利用卡那霉素抗性筛选,PCR、脉冲场电泳和Southern blot进行鉴定获得基因突变株hyl mutant。突变株在体外的生长能力低于野生株。突变株在小鼠腹膜炎模型中的半数致死量(LD50)提高了7倍,在相同细菌感染浓度(3.7×109 cfu)下,hyl mutant组小鼠的存活率为50%,而野生株TX2466组小鼠的存活率为0,差异有显著性。6 h和12 h外周血白细胞变化低于野生株,6 h腹水中的TNF-α水平和腹膜活化的TNF-α蛋白低于野生株组。兔心内膜炎模型中,hyl mutant组则分别为(2.11±0.59)×105 cfu /g和(6.59±0.53)×105 cfu /g,而TX2466组主动脉瓣和壁性赘生物的菌落计数分别为(3.04±0.63)×106 cfu /g和(6.87±0.58)×106 cfu/g,差异同样有显著性。PCR扩增后,测序hyl基因全长1662 bp,为编码553个氨基酸残基的多肽。SDS-PAGE分析相对分子量约为60 000。表达量占全菌的38%以上,经亲和层析后可获得纯度为90%以上的重组蛋白。Western blot证实了其免疫反应性。灌胃免疫小鼠后,ELISA检测结果Hyl蛋白组血清IgA、IgG,粪sIgA,肠黏液sIgA分别为0.365±0.048,0.431±0.064,0.743±0.056和1.112±0.113 ,对照组则分别为0.051±0.013 ,0.098±0.019,0.102±0.032和0.187±0.051,差异有显著性。通过细菌攻击后,小鼠的平均存活时间也明显长于对照组。结论:hyl基因突变株hyl mutant构建成功,hyl基因在屎肠球菌致病中起着重要的作用,可能是屎肠球菌的毒力因子之一。hyl基因表达载体pQE-hyl构建成功,表达的融合蛋白Hyl经口服免疫可有效诱导粘膜免疫应答,产生高水平的sIgA,发挥局部粘膜免疫作用和抗菌保护作用。Hyl有可能作为预防屎肠球菌口服疫苗的候选抗原。
[Abstract]:Background: Enterococcus is human and animal intestinal G+ in normal flora. Enterococcus faecium is weak, in the past more than 10 years, enterococcus infection caused by increased by less than the original 10%, rising to 30% ~ 40%. vancomycin resistant enterococci faecium is most, reached 25%. the ICU in the United States, the incidence of excrement Enterococcus infection rate may be related to obtain new virulence factors. However, study on the pathogenesis and virulence factors of Enterococcus faecium have little multidrug resistance plus, clinical treatment is difficult. How to prevent the infection of Enterococcus faecium infection in recent years is the hotspot of research. Oral immunotherapy may be present in addition to drug treatment is a kind of cheap, safe, practical and suitable for mass vaccination in high-risk groups, the new treatment method of very good development prospects.
Hyaluronidase is Pathogenic Streptococcus pyogenes, virulence factor of Staphylococcus aureus and Streptococcus pneumoniae G+ aureus. Enterococcus belonged to group D Streptococcus, and later divided into a new genus, but many characteristics similar to Streptococcus.Rice by analogy analysis of Enterococcus faecium chromosome gene group, found that the gene sequence has an open reading frame, the full-length 1662bp DNA sequence and encoding the amino acid sequence of Streptococcus pyogenes hyaluronidase were highly homologous. And this gene is named Enterococcus faecium hyaluronidase gene (Hyaluronidase, hylEfm). For the exact function of hyl gene in the pathogenesis of Enterococcus faecium we, through the construction of hyl gene mutant strain of Enterococcus faecium, role of gene hyl in Enterococcus faecium infection, and on the basis of cloning hyl gene expression and purification. To discuss the protective effect of Hyl protein on mice immunized by oral immunization, and provide the basis for the prevention and control of Enterococcus faecium.
Objective: to construct the hyl gene mutant of Enterococcus faecium, to study hyl gene knock in virulence of Enterococcus faecium by before and after growth in vitro and in vivo animal experiment model curve. Based on the construction of hyl gene recombinant expression plasmid, induce the expression of recombinant protein in Escherichia coli, Hyl protein after oral immunization of mice after antimicrobial protection the role of the immune response and in vivo produced in the body.
Methods: Dutch act plasmid pTX4577 carrying hyl gene insertion sequences by homologous recombination, hyl gene breakage and construction of hyl mutant. The mutant was identified by PCR, pulsed field gel electrophoresis and Southern blot. Compared with the wild strain TX2466, growth ability and virulence in vitro observation mutant mouse peritonitis model and the rabbit endocarditis in vivo model changes. Then PCR amplification of hyl gene fragment was cloned into pQE-30 plasmid, the recombinant plasmid pQE-hyl was transformed into E.coli DH5, induced by IPTG Hyl fusion protein expression; analyze the immune reactivity of Western blot. The fusion protein was purified by Ni2 +, Hyl protein and the protein of Enterococcus faecium TX2466 mice were immunized with ELISA, serum IgG and IgA, sIgA and sIgA levels of small intestinal mucus feces. Then TX2466 abdominal attacks have immune mice, The antiseptic protection in vivo was observed.
Results: after homologous recombination, using kanamycin resistance screening, PCR, gene mutant hyl mutant. mutant in vitro growth ability than the wild strains were identified by pulsed field gel electrophoresis and Southern blot. The mutant mouse peritonitis models of the median lethal dose (LD50) was 7 times higher than that in the same bacterial infection concentration (3.7 * 109 CFU), hyl mutant group of mice and the survival rate was 50%, and the wild strain TX2466 mice survival rate was 0, the difference is the change of H and 12 h of peripheral white blood cells was significantly lower than that of wild strain.6, TNF- protein 6 h in ascites TNF- levels and peritoneal activation the lower than the wild strain group. The rabbit endocarditis model in hyl mutant group were (2.11 + 0.59) * 105 CFU and /g (6.59 + 0.53) * 105 CFU /g, and TX2466 group colony count of aortic and wall vegetations were (3.04 + 0.63) and /g (6.87 x 106 CFU + 0.58) * 106 cfu/g, Differences are also significant after.PCR amplification, sequencing the full-length of hyl gene was 1662 BP, the relative molecular weight of the polypeptide.SDS-PAGE encoding 553 amino acid residues of approximately 60000. protein accounted for more than 38% of the total bacteria, purity confirmed its immunoreactivity for more than 90% of the recombinant protein.Western blot by affinity chromatography. The mice were ELISA, Hyl protein detection results of serum IgA, IgG, dung sIgA, intestinal mucus sIgA were 0.365 + 0.048,0.431 + 0.064,0.743 + 0.056 and 1.112 + 0.113, the control group were 0.051 + 0.013, 0.098 + 0.019,0.102 + 0.032 and 0.187 + 0.051, the difference was significant. The bacterial attack later, the average survival time of mice was longer than the control group.
Conclusion: hyl gene mutant hyl mutant was successfully constructed, hyl gene plays an important role in the pathogenesis of Enterococcus, may be the expression vector pQE-hyl was successfully constructed one of the virulence factors of.Hyl gene of Enterococcus faecium, expression of fusion protein Hyl after oral immunization can induce effective mucosal immune response and produce higher levels of sIgA play local mucosa the immune effect of antibacterial and protective effect of.Hyl could be used as a candidate antigen for prevention of e.faecium oral vaccine.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378

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