本文選題：結(jié)核分支桿菌　切入點(diǎn)：卡介苗　出處：《華中科技大學(xué)》2011年碩士論文

【摘要】：一背景與目的: 使用BCG作為初免疫苗的異源性初免增強(qiáng)方案,可能將成為最有希望的新型TB免疫措施。ESAT-6和Ag85A是機(jī)體抗TB感染的兩個(gè)主要的保護(hù)性抗原。本研究中,我們克隆了ESAT-6和Ag85A的融合基因,分別構(gòu)建了表達(dá)ESAT-6和Ag85A融合蛋白的原核表達(dá)質(zhì)粒pPro685A和真核表達(dá)質(zhì)粒pcD685A,研究了pcD685A增強(qiáng)BCG初免誘導(dǎo)的免疫反應(yīng),同時(shí)探討pcD685A增強(qiáng)BCG初免對(duì)小鼠抗TB感染的保護(hù)性。 二方法: 1.利用PCR技術(shù)從M.tb H37Rv株中擴(kuò)增Ag85A編碼基因fbpA和ESAT-6編碼基因esxA,利用基因拼接(GeneSOEing)方法,合成esxA-fbpA融合基因,并分別克隆入原核表達(dá)載體pProEXHTb和真核表達(dá)載體PcDNA3.1(+)中,重組質(zhì)粒命名為pPro685A和pcD685A。 2.將pPro685A重組原核表達(dá)質(zhì)粒轉(zhuǎn)化入感受態(tài)大腸桿菌BL21,誘導(dǎo)并純化出r685A蛋白。蛋白的表達(dá)和純化的過(guò)程,分別用SDS-PAGE驗(yàn)證,并用anti-ESAT-6和anti-Ag85A Ab經(jīng)Western blotting驗(yàn)證。純化r685A蛋白的純度進(jìn)一步用SDS-PAGE證實(shí),并經(jīng)BCA法定量。 3.將pcD685A重組質(zhì)粒轉(zhuǎn)化入感受態(tài)E.coli DH5α,規(guī)模化提取、純化pcD685A重組質(zhì)粒,并經(jīng)分光光度法測(cè)定質(zhì)粒濃度。 4.利用BCG(中國(guó)株)初免pcD685A增強(qiáng)的方式免疫C57BL/6小鼠。通過(guò)ELISA法檢測(cè)外周血抗r685A蛋白IgG抗體,qRT-PCR檢測(cè)肺臟表達(dá)的IFN-γ和IL-10的手段,來(lái)評(píng)價(jià)在小鼠模型中的免疫原性。 5.對(duì)免疫后小鼠用M.tb H37Rv毒株攻擊實(shí)驗(yàn)進(jìn)行攻擊,測(cè)定組織荷菌量、組織病理學(xué)改變等指標(biāo)評(píng)價(jià)疫苗的保護(hù)性。 三結(jié)果: 1.成功的構(gòu)建了esxA-fbpA融合基因的真核表達(dá)載體和原核表達(dá)載體。 2. SDS-PAGE和Western blotting證實(shí)r685A原核表達(dá)和純化成功。純化的r685A經(jīng)SDS-PAGE證實(shí)。 3. pcD685A誘導(dǎo)表達(dá)了特異性IgG的抗體反應(yīng),BCG初免pcD685A增強(qiáng)免疫組明顯高于其它組。qRT-PCR檢測(cè)肺臟細(xì)胞因子,除了空載體組,其他各組的IFN-γ反應(yīng)水平均增加。pcD685A增強(qiáng)BCG初免組誘導(dǎo)小鼠肺臟產(chǎn)生了最高劑量的IFN-γ,并比單獨(dú)BCG組或者pcD685A組IFN-γ有顯著提高。 4.更重要的是pcD685A增強(qiáng)免疫BCG初免與單獨(dú)BCG或pcD685A組相比,能明顯的減少肺臟和脾臟的荷菌量。 5.小鼠用M.tb H37Rv毒株攻擊后,肺臟組織病理學(xué)檢查結(jié)果顯示BCG初免pcD685A增強(qiáng)組的肺臟病理變化最輕微。 四結(jié)論: 因此,我們的研究顯示pcD685A可能將是一種有效地增強(qiáng)BCG免疫的抗TB疫苗。
[Abstract]:Background and purpose:The use of BCG as a primary immune enhancement regimen may be the most promising new TB immunization method. ESAT-6 and Ag85A are the two main protective antigens against TB infection.In this study, we cloned the fusion gene of ESAT-6 and Ag85A, constructed the prokaryotic expression plasmid pPro685A and eukaryotic expression plasmid pcD685A expressing ESAT-6 and Ag85A fusion protein respectively, and studied the enhancement of the immune response induced by BCG by pcD685A.To explore the protective effect of pcD685A on anti-TB infection in mice.Two methods:1.Ag85A encoding gene fbpA and ESAT-6 coding gene esxA were amplified from M.tb H37Rv strain by PCR technique, and esxA-fbpA fusion gene was synthesized by gene splicing method. EsxA-fbpA fusion gene was cloned into prokaryotic expression vector pProEXHTb and eukaryotic expression vector PcDNA3.1 (). The recombinant plasmids were named pPro685A and pcD685A respectively.2.The recombinant prokaryotic expression plasmid of pPro685A was transformed into the competent Escherichia coli BL21 to induce and purify r685A protein.The process of protein expression and purification was verified by SDS-PAGE, anti-ESAT-6 and anti-Ag85A Ab by Western blotting.The purity of purified r685A protein was further confirmed by SDS-PAGE and measured by BCA.3.The recombinant plasmid of pcD685A was transformed into the competent E.coli DH5 偽. The recombinant plasmid of pcD685A was extracted and purified on a large scale. The concentration of the recombinant plasmid was determined by spectrophotometry.4.C57BL/6 mice were immunized with BCG (Chinese strain) with the method of pcD685A enhancement.5.M.tb H37Rv strain was used to attack the immunized mice. The tissue count and histopathological changes were measured to evaluate the protection of the vaccine.Three outcomes:1.The eukaryotic expression vector and prokaryotic expression vector of esxA-fbpA fusion gene were successfully constructed.2.SDS-PAGE and Western blotting confirmed the prokaryotic expression and purification of r685A.The purified r685A was confirmed by SDS-PAGE.3. The antibody response induced by pcD685A to express specific IgG was significantly higher in the pcD685A immunized group than in the other groups, except for the empty vector group.The level of IFN- 緯 reaction in other groups was increased. PcD685A enhanced the production of IFN- 緯 in lung of mice in BCG group, and it was significantly higher than that in BCG group or pcD685A group.4.More importantly, pcD685A enhanced immunized BCG could significantly reduce the amount of bacteria in the lungs and spleen compared with the group of BCG or pcD685A alone.5.The lung histopathological examination of mice with M.tb H37Rv strain showed that the lung histopathological changes were the most slight in the group of pcD685A enhanced by BCG.IV. Conclusion:Therefore, our study suggests that pcD685A may be an anti-TB vaccine that enhances BCG immunization effectively.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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相關(guān)期刊論文 前1條

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本文編號(hào)：1699829