本文選題：糞腸球菌 + Ace��； 參考：《南華大學(xué)》2007年碩士論文

【摘要】： 目的:構(gòu)建含糞腸球菌(Enterococcus faecalis,E.faecalis)ace基因的重組載體,在大腸桿菌M15中進(jìn)行誘導(dǎo)表達(dá),純化表達(dá)產(chǎn)物并進(jìn)行免疫活性分析,研究Ace蛋白在E.faecalis對膠原蛋白及HeLa細(xì)胞黏附過程中的作用,為探索Ace蛋白生物學(xué)功能及研發(fā)快速診斷試劑盒提供實(shí)驗依據(jù)。 方法:用Premier Primer 5.0引物設(shè)計軟件分析E.faecalis的ace基因序列,設(shè)計特異性引物,以E.faecalis標(biāo)準(zhǔn)株E.faecalis JH2-2為模板進(jìn)行聚合酶鏈反應(yīng)(PCR)擴(kuò)增ace基因,將其亞克隆入原核表達(dá)載體pQE30中,構(gòu)建重組質(zhì)粒pQE30/Ace,然后轉(zhuǎn)化至表達(dá)宿主菌M15中進(jìn)行誘導(dǎo)表達(dá),利用SDS-PAGE和Western blot進(jìn)行分析和鑒定表達(dá)結(jié)果,Ni-NTA親和層析柱純化重組蛋白,BCA法測定純化蛋白濃度。將純化的Ace重組蛋白(rAce)免疫新西蘭兔,用所得抗體對rAce的免疫活性進(jìn)行分析。用熒光素FITC標(biāo)記E.faecalis JH2-2進(jìn)行黏附實(shí)驗及黏附抑制實(shí)驗,免疫熒光顯微鏡觀察結(jié)果。 結(jié)果:PCR擴(kuò)增得到大小約930bp的目的片斷;構(gòu)建的重組質(zhì)粒經(jīng)PCR、雙酶切鑒定和測序鑒定證明其中插入片段為ace目的基因,測序結(jié)果與Genbank上登錄序列完全一致;SDS-PAGE分析顯示,在IPTG誘導(dǎo)下,重組工程菌表達(dá)了一相對分子質(zhì)量(Mr)約為37kDa的目的蛋白條帶,目的蛋白在菌體細(xì)胞內(nèi)主要以可溶性蛋白形式存在;經(jīng)Ni-NTA親和純化獲得了較高純度的重組蛋白;Western-blot檢測其能與6×His單克隆抗體發(fā)生特異性反應(yīng)。利用純化的rAce免疫新西蘭兔,間接ELISA法測定兔免疫血清特異性抗體效價在1:16 000以上;從E.faecalis細(xì)胞壁提取的Ace蛋白能與rAce免疫兔抗血清識別。黏附實(shí)驗顯示E.faecalis JH2-2能夠黏附于膠原蛋白Ⅰ及HeLa細(xì)胞表面;黏附抑制試驗顯示rAce多克隆抗體具有抑制46℃培養(yǎng)的E.faecalis JH2-2對膠原蛋白Ⅰ及HeLa細(xì)胞的黏附作用,這種抑制作用隨著血清稀釋度的增加而減弱。 結(jié)論: 1.成功構(gòu)建了pQE30/Ace原核表達(dá)載體,將其轉(zhuǎn)化至E.coliM15后表達(dá)出了一相對分子量(Mr)約為37kDa的重組蛋白; 2. Ace重組蛋白具有較好的免疫原性,能刺激新西蘭兔產(chǎn)生高效價的特異性抗體,并具有良好的免疫反應(yīng)性,有望應(yīng)用于E.faecalis快速診斷中; 3. Ace是一種具有黏附活性的蛋白,與E.faecalis致病性密切相關(guān)。
[Abstract]:Objective: to construct a recombinant vector containing Enterococcus faecalisus E. faecalisae gene and express it in Escherichia coli M15, purify the expression product and analyze its immunological activity, and study the role of Ace protein in the process of E.faecalis adhesion to collagen and HeLa cells. To explore the biological function of Ace protein and to develop a rapid diagnostic kit to provide experimental basis. Methods: Premier Primer 5.0 primer design software was used to analyze the ace gene sequence of E.faecalis, and specific primers were designed. The ace gene was amplified by polymerase chain reaction (PCR) using E.faecalis JH2-2 as template, and subcloned into the prokaryotic expression vector pQE30. The recombinant plasmid pQE30 / Acewas constructed, then transformed into the expression host strain M15 for induction expression. The expression results were analyzed and identified by SDS-PAGE and Western blot. The purified protein was purified by Ni-NTA affinity chromatography column. New Zealand rabbits were immunized with purified Ace recombinant protein rAce. the immunological activity of rAce was analyzed with the obtained antibodies. E.faecalis JH2-2 was labeled with fluorescein FITC for adhesion test and adhesion inhibition test. The results were observed by immunofluorescence microscope. Results the target fragment of 930bp was amplified by 1: PCR, and the recombinant plasmid was identified by PCR, double enzyme digestion and sequencing. The result of sequencing showed that the inserted fragment was ace target gene, and the result of sequencing was consistent with that of Genbank. Under the induction of IPTG, the recombinant engineering strain expressed a relative molecular weight (Mr) protein band of 37kDa, and the target protein existed mainly in the form of soluble protein in the cell. The recombinant protein with high purity was obtained by Ni-NTA affinity purification. Western-blot analysis showed that the recombinant protein could react specifically with 6 脳 His monoclonal antibody. The purified rAce was used to immunize New Zealand rabbits. The specific antibody titer of rabbit immunized by indirect ELISA was more than 1:16, and the Ace protein extracted from the cell wall of E.faecalis could be recognized with the antiserum of rAce. Adhesion assay showed that E.faecalis JH2-2 could adhere to the surface of collagen 鈪，

本文編號：1791817