本文關(guān)鍵詞：屎腸球菌毒力基因hyl的鑒定和功能研究　出處：《重慶醫(yī)科大學(xué)》2007年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 屎腸球菌 透明質(zhì)酸酶基因 鑒定 功能

【摘要】： 研究背景:腸球菌是人和動(dòng)物腸道內(nèi)的G+正常菌群。屎腸球菌毒力較弱,過(guò)去十多年內(nèi),屎腸球菌所引起的感染有所上升,由原來(lái)的不到10%,上升到30%～40%。而且臨床上耐萬(wàn)古霉素腸球菌也絕大多數(shù)是屎腸球菌,在美國(guó)ICU達(dá)到了25%。由此可見(jiàn),屎腸球菌感染的發(fā)病率上升可能與獲得了新的毒力因子有關(guān)。然而屎腸球菌的致病機(jī)制和毒力因子研究甚少,加上多重天然耐藥的存在,臨床治療非常困難。如何防治屎腸球菌的感染是近年來(lái)感染學(xué)界研究的熱點(diǎn)�？诜庖咧委�,可能是目前除藥物治療外的一種廉價(jià)、安全、實(shí)用性好、適合大規(guī)模高危人群免疫接種、發(fā)展前景非常好的新型治療方法。 透明質(zhì)酸酶是致病性釀膿鏈球菌、金黃色葡萄球菌和肺炎鏈球菌等G+球菌的毒力因子之一。腸球菌原來(lái)屬于D組鏈球菌,后來(lái)才獨(dú)立分為一屬,但其許多特性與鏈球菌相似。Rice等通過(guò)對(duì)屎腸球菌基因組進(jìn)行類(lèi)比分析,發(fā)現(xiàn)屎腸球菌染色體基因序列中有一開(kāi)放讀碼框,全長(zhǎng)1662bp,其DNA序列以及編碼的氨基酸序列與釀膿鏈球菌透明質(zhì)酸酶有高度同源性。并將這個(gè)基因命名為屎腸球菌透明質(zhì)酸酶基因(Hyaluronidase, hylEfm)。為了研究hyl基因在屎腸球菌致病中的確切功能,我們通過(guò)構(gòu)建屎腸球菌hyl基因突變菌株,研究hyl基因在屎腸球菌感染中的作用,并在此基礎(chǔ)上對(duì)hyl基因進(jìn)行克隆表達(dá)和純化,探討Hyl蛋白經(jīng)口服免疫小鼠的免疫保護(hù)作用,為屎腸球菌的防治提供基礎(chǔ)依據(jù)。 目的:構(gòu)建屎腸球菌hyl基因突變株,通過(guò)體外生長(zhǎng)曲線和體內(nèi)動(dòng)物模型實(shí)驗(yàn)來(lái)研究hyl基因敲除前后屎腸球菌的毒力變化。在此基礎(chǔ)上構(gòu)建hyl基因重組表達(dá)質(zhì)粒,在大腸埃希菌中誘導(dǎo)表達(dá)獲得重組蛋白,探討Hyl蛋白經(jīng)口服免疫小鼠后,機(jī)體產(chǎn)生的免疫應(yīng)答和體內(nèi)抗菌保護(hù)作用。 方法:用攜帶hyl基因插入序列的自殺質(zhì)粒pTX4577通過(guò)體內(nèi)同源重組,使hyl基因斷裂而構(gòu)建hyl基因突變株。突變株用PCR、脈沖場(chǎng)電泳和Southern blot進(jìn)行鑒定。通過(guò)與野生株TX2466相比,觀察突變株體外的生長(zhǎng)能力以及小鼠腹膜炎模型和兔心內(nèi)膜炎模型體內(nèi)實(shí)驗(yàn)的毒力改變情況。然后用PCR擴(kuò)增hyl基因片段,克隆至pQE-30質(zhì)粒上,構(gòu)建重組質(zhì)粒pQE-hyl,轉(zhuǎn)化E.coli DH5 ,經(jīng)IPTG誘導(dǎo)表達(dá)融合蛋白Hyl;Western blot分析其免疫反應(yīng)性。融合蛋白經(jīng)鎳離子柱純化后,用Hyl蛋白和屎腸球菌全菌蛋白TX2466分別給小鼠灌胃進(jìn)行免疫,ELISA檢測(cè)小鼠血清IgG和IgA、小腸粘液sIgA和糞便sIgA水平。然后用TX2466腹腔攻擊已免疫的小鼠,觀察其體內(nèi)抗菌保護(hù)性。 結(jié)果:經(jīng)同源重組,利用卡那霉素抗性篩選,PCR、脈沖場(chǎng)電泳和Southern blot進(jìn)行鑒定獲得基因突變株hyl mutant。突變株在體外的生長(zhǎng)能力低于野生株。突變株在小鼠腹膜炎模型中的半數(shù)致死量(LD50)提高了7倍,在相同細(xì)菌感染濃度(3.7×109 cfu)下,hyl mutant組小鼠的存活率為50%,而野生株TX2466組小鼠的存活率為0,差異有顯著性。6 h和12 h外周血白細(xì)胞變化低于野生株,6 h腹水中的TNF-α水平和腹膜活化的TNF-α蛋白低于野生株組。兔心內(nèi)膜炎模型中,hyl mutant組則分別為(2.11±0.59)×105 cfu /g和(6.59±0.53)×105 cfu /g,而TX2466組主動(dòng)脈瓣和壁性贅生物的菌落計(jì)數(shù)分別為(3.04±0.63)×106 cfu /g和(6.87±0.58)×106 cfu/g,差異同樣有顯著性。PCR擴(kuò)增后,測(cè)序hyl基因全長(zhǎng)1662 bp,為編碼553個(gè)氨基酸殘基的多肽。SDS-PAGE分析相對(duì)分子量約為60 000。表達(dá)量占全菌的38%以上,經(jīng)親和層析后可獲得純度為90%以上的重組蛋白。Western blot證實(shí)了其免疫反應(yīng)性。灌胃免疫小鼠后,ELISA檢測(cè)結(jié)果Hyl蛋白組血清IgA、IgG,糞sIgA,腸黏液sIgA分別為0.365±0.048,0.431±0.064,0.743±0.056和1.112±0.113 ,對(duì)照組則分別為0.051±0.013 ,0.098±0.019,0.102±0.032和0.187±0.051,差異有顯著性。通過(guò)細(xì)菌攻擊后,小鼠的平均存活時(shí)間也明顯長(zhǎng)于對(duì)照組。 結(jié)論:hyl基因突變株hyl mutant構(gòu)建成功,hyl基因在屎腸球菌致病中起著重要的作用,可能是屎腸球菌的毒力因子之一。hyl基因表達(dá)載體pQE-hyl構(gòu)建成功,表達(dá)的融合蛋白Hyl經(jīng)口服免疫可有效誘導(dǎo)粘膜免疫應(yīng)答,產(chǎn)生高水平的sIgA,發(fā)揮局部粘膜免疫作用和抗菌保護(hù)作用。Hyl有可能作為預(yù)防屎腸球菌口服疫苗的候選抗原。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R378

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相關(guān)期刊論文 前1條

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本文編號(hào)：1374244