ESAT-6-Ag85A融合基因DNA疫苗增强卡介苗初免的免疫原性和保护性

发布时间：2018-04-02 09:48

  本文选题：结核分支杆菌　切入点：卡介苗　出处：《华中科技大学》2011年硕士论文

【摘要】：一背景与目的: 使用BCG作为初免疫苗的异源性初免增强方案,可能将成为最有希望的新型TB免疫措施。ESAT-6和Ag85A是机体抗TB感染的两个主要的保护性抗原。本研究中,我们克隆了ESAT-6和Ag85A的融合基因,分别构建了表达ESAT-6和Ag85A融合蛋白的原核表达质粒pPro685A和真核表达质粒pcD685A,研究了pcD685A增强BCG初免诱导的免疫反应,同时探讨pcD685A增强BCG初免对小鼠抗TB感染的保护性。 二方法: 1.利用PCR技术从M.tb H37Rv株中扩增Ag85A编码基因fbpA和ESAT-6编码基因esxA,利用基因拼接(GeneSOEing)方法,合成esxA-fbpA融合基因,并分别克隆入原核表达载体pProEXHTb和真核表达载体PcDNA3.1(+)中,重组质粒命名为pPro685A和pcD685A。 2.将pPro685A重组原核表达质粒转化入感受态大肠杆菌BL21,诱导并纯化出r685A蛋白。蛋白的表达和纯化的过程,分别用SDS-PAGE验证,并用anti-ESAT-6和anti-Ag85A Ab经Western blotting验证。纯化r685A蛋白的纯度进一步用SDS-PAGE证实,并经BCA法定量。 3.将pcD685A重组质粒转化入感受态E.coli DH5α,规模化提取、纯化pcD685A重组质粒,并经分光光度法测定质粒浓度。 4.利用BCG(中国株)初免pcD685A增强的方式免疫C57BL/6小鼠。通过ELISA法检测外周血抗r685A蛋白IgG抗体,qRT-PCR检测肺脏表达的IFN-γ和IL-10的手段,来评价在小鼠模型中的免疫原性。 5.对免疫后小鼠用M.tb H37Rv毒株攻击实验进行攻击,测定组织荷菌量、组织病理学改变等指标评价疫苗的保护性。 三结果: 1.成功的构建了esxA-fbpA融合基因的真核表达载体和原核表达载体。 2. SDS-PAGE和Western blotting证实r685A原核表达和纯化成功。纯化的r685A经SDS-PAGE证实。 3. pcD685A诱导表达了特异性IgG的抗体反应,BCG初免pcD685A增强免疫组明显高于其它组。qRT-PCR检测肺脏细胞因子,除了空载体组,其他各组的IFN-γ反应水平均增加。pcD685A增强BCG初免组诱导小鼠肺脏产生了最高剂量的IFN-γ,并比单独BCG组或者pcD685A组IFN-γ有显著提高。 4.更重要的是pcD685A增强免疫BCG初免与单独BCG或pcD685A组相比,能明显的减少肺脏和脾脏的荷菌量。 5.小鼠用M.tb H37Rv毒株攻击后,肺脏组织病理学检查结果显示BCG初免pcD685A增强组的肺脏病理变化最轻微。 四结论: 因此,我们的研究显示pcD685A可能将是一种有效地增强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 伪. 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- 纬 reaction in other groups was increased. PcD685A enhanced the production of IFN- 纬 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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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