本文選題：肺炎鏈球菌　切入點(diǎn)：D39Δspd1672　出處：《重慶醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 【背景】莢膜多糖是肺炎鏈球菌的主要毒力因子,它具有抗吞噬作用,可使其免受宿主免疫攻擊。莢膜丟失的肺炎鏈球菌很容易被吞噬細(xì)胞清除,毒力下降。本課題組前期工作從肺炎鏈球菌中篩選到一些體內(nèi)誘導(dǎo)基因,其中的基因spd1672經(jīng)過生物信息學(xué)分析,其表達(dá)產(chǎn)物含有脂質(zhì)A核心-O抗原連接酶結(jié)構(gòu)域,提示該基因可能與細(xì)菌莢膜形成相關(guān)。本課題擬對(duì)該基因與莢膜形成相關(guān)的功能進(jìn)行初步鑒定。 【方法】采用長臂同源多聚酶鏈?zhǔn)椒磻?yīng)(Long flanking homology polymerase chain reaction, LFH-PCR)構(gòu)建spd1672缺陷菌;用小鼠毒力體內(nèi)實(shí)驗(yàn)比較spd1672缺陷菌與野生菌的毒力;通過菌體連接實(shí)驗(yàn),將無莢膜的R6菌(含有SPD1672蛋白)和spd1672等位基因缺陷的R6菌株分別與D39Δspd1672缺陷菌的莢膜多糖(CPS)共同孵育,ELISA檢測(cè)菌體表面的CPS量,分析SPD1672蛋白的連接酶功能;利用透射電鏡技術(shù)、ELISA和Immunblotting技術(shù)分析spd1672缺陷后細(xì)菌表面莢膜多糖的改變;并通過FQ-PCR技術(shù)檢測(cè)cps基因簇cps2A、cps2D、cps2E、cps2H、cps2K、和RfbC的mRNA表達(dá)量,分析spd1672基因?qū)ηv膜多糖合成基因的調(diào)控作用。 【結(jié)果】成功構(gòu)建D39Δspd1672缺陷菌株;小鼠體內(nèi)毒力實(shí)驗(yàn)顯示注射D39Δspd1672缺陷菌的小鼠全部存活,毒力較野生菌株顯著減低(*P0.05),且注射到小鼠腹腔的缺陷菌全部被清除,腹腔液涂片未見細(xì)菌。透射電鏡觀察發(fā)現(xiàn)D39Δspd1672基因缺陷菌株莢膜結(jié)構(gòu)不復(fù)存在。菌體連接實(shí)驗(yàn)結(jié)果顯示,無莢膜的R6菌(含有SPD1672蛋白)與D39Δspd1672缺陷菌CPS共同孵育2小時(shí)后,R6菌體表面的CPS量增加,接近于野生菌的水平,spd1672等位基因缺陷R6菌株的CPS量未見增加;Immunoblotting分析結(jié)果顯示,D39Δspd1672缺陷菌的細(xì)胞壁檢測(cè)不到CPS,原生質(zhì)的CPS量較野生菌顯著減少,且缺乏大分子量的莢膜多糖成分;由于D39Δspd1672缺陷菌不能利用環(huán)境中的莢膜多糖,ELISA分析D39Δspd1672缺陷菌體中CPS量較野生菌顯著減少,但培養(yǎng)基上清的CPS量較野生菌多。熒光定量PCR測(cè)定結(jié)果顯示,除了cps2A無變化,D39Δspd1672缺陷菌株的cps2D、cps2E、cps2H、cps2K、RfbC基因表達(dá)都較野生菌的顯著下降(*P0.05)。 【結(jié)論】上述實(shí)驗(yàn)結(jié)果提示:spd1672基因表達(dá)產(chǎn)物能將莢膜多糖連接到細(xì)菌表面,是一種新的肺炎鏈球菌莢膜多糖連接酶,是否有聚合酶功能,尚需更多的實(shí)驗(yàn)證據(jù); spd1672基因還影響了多個(gè)莢膜多糖合成基因的表達(dá),在莢膜形成調(diào)控機(jī)制中起關(guān)鍵作用;小鼠體內(nèi)毒力實(shí)驗(yàn)提示spd1672基因是肺炎鏈球菌中的一個(gè)新的毒力因子。
[Abstract]:Background: capsule polysaccharide is the main virulence factor of Streptococcus pneumoniae.Streptococcus pneumoniae, which is lost in capsule, is easily cleared by phagocytes and its virulence decreases.Some induced genes were screened from Streptococcus pneumoniae in the early stage of our work. The gene spd1672 was analyzed by bioinformatics, and the expressed product contained the ligase domain of lipid A core antigen.These results suggest that this gene may be related to the formation of bacterial capsule.The purpose of this study is to identify the function of the gene associated with capsule formation.[methods] the long arm flanking homology polymerase chain reaction (LFH-PCR) was used to construct spd1672 deficient bacteria, the virulence of spd1672 deficiency bacteria and wild bacteria were compared with mice virulence in vivo.R6 strain without capsule (containing SPD1672 protein) and R6 strain with spd1672 allele deficiency were incubated with D39 螖 spd1672 deficient strain, respectively, to detect the CPS quantity on the cell surface and analyze the ligase function of SPD1672 protein.鍒╃敤閫忓皠鐢?shù)闀滄姡?

本文編號(hào)：1703822