甲型流感病毒M2基因真核表达载体的构建及其免疫保护性的初步研究
发布时间：2018-11-09 17:31
【摘要】：甲型流感病毒M2蛋白是一种具有高保守性，可形成离子通道并影响流感病毒血凝素(hemagglutinin，HA)天然构象的形成，有利于病毒脱壳，具有型特异性，，被认为是具有交叉免疫保护性的结构蛋白。DNA疫苗能够同时刺激机体B细胞，辅助性T细胞(helper T cell，Th)和细胞毒性T细胞(cytotoxic T lymphocytes，CTL)，产生特异性体液免疫和细胞免疫，易于操作，经济有效，目前已成为疫苗研究的热点。美国国立卫生研究院(National Institutes of Health，NIH)和世界卫生组织(World Health Organization，WHO)都强调流感DNA疫苗是流感疫苗研究的一个重要方向。 目的 本研究的目的在于通过构建甲型流感病毒M2蛋白的真核表达载体，用M2蛋白的真核表达载体核酸免疫实验动物后再用流感病毒攻击，观察重组质粒pcDNA3.1(+)／M2的免疫保护作用，为进一步研究M2蛋白的交叉保护作用奠定基础。 方法 我们首先将人流感病毒A／PR／8／34(H1N1)经鸡胚尿囊液接种培养，用红细胞吸附释放法(absorption and release-RBC，AR-RBC)纯化病毒。在甲型流感病毒M2基因的克隆方面，先提取人流感病毒株A／PR／8／34(H1N1)的RNA，用特异引物进行RT-PCR扩增获得M2基因。将扩增的M2基因片段克隆入真核表达质粒载体pcDNA3.1(+)，转化感受态E．coli JM109并筛选阳性克隆。双酶切、多聚合酶链反应(polymerase chain reaction，PCR)鉴定重组质粒
[Abstract]:The M2 protein of influenza A virus is a highly conserved protein that can form ion channels and affect the formation of natural conformation of influenza virus hemagglutinin (hemagglutinin,HA). DNA vaccine can stimulate both B cell, helper T cell (helper T cell,Th and cytotoxic T cell (cytotoxic T lymphocytes,CTL. To produce specific humoral and cellular immunity, easy to operate, economical and effective, has become a hot spot in vaccine research. The National Institutes of Health (National Institutes of Health,NIH) and the World Health Organization (World Health Organization,WHO) have emphasized that influenza DNA vaccine is an important direction of influenza vaccine research. Objective to construct the eukaryotic expression vector of M2 protein of influenza A virus and immunize experimental animals with the eukaryotic expression vector of M2 protein. The immunoprotective effect of recombinant plasmid pcDNA3.1 () / M2 was observed, which laid a foundation for further study on the cross protection of M2 protein. Methods Human influenza virus A/PR/8/34 (H1N1) was cultured in chicken embryo allantoic fluid and purified by erythrocyte adsorption release method (absorption and release-RBC,AR-RBC). In the cloning of M2 gene of influenza A virus, the M2 gene was obtained by RT-PCR amplification from the RNA, of human influenza virus strain A/PR/8/34 (H1N1) with specific primers. The amplified M2 gene fragment was cloned into eukaryotic expression plasmid pcDNA3.1 (), and transformed into receptive E.coli JM109 and positive clones were screened. Identification of Recombinant plasmid by double enzyme digestion and Polymerase chain reaction (polymerase chain reaction,PCR)
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373
本文编号：2321092
[Abstract]:The M2 protein of influenza A virus is a highly conserved protein that can form ion channels and affect the formation of natural conformation of influenza virus hemagglutinin (hemagglutinin,HA). DNA vaccine can stimulate both B cell, helper T cell (helper T cell,Th and cytotoxic T cell (cytotoxic T lymphocytes,CTL. To produce specific humoral and cellular immunity, easy to operate, economical and effective, has become a hot spot in vaccine research. The National Institutes of Health (National Institutes of Health,NIH) and the World Health Organization (World Health Organization,WHO) have emphasized that influenza DNA vaccine is an important direction of influenza vaccine research. Objective to construct the eukaryotic expression vector of M2 protein of influenza A virus and immunize experimental animals with the eukaryotic expression vector of M2 protein. The immunoprotective effect of recombinant plasmid pcDNA3.1 () / M2 was observed, which laid a foundation for further study on the cross protection of M2 protein. Methods Human influenza virus A/PR/8/34 (H1N1) was cultured in chicken embryo allantoic fluid and purified by erythrocyte adsorption release method (absorption and release-RBC,AR-RBC). In the cloning of M2 gene of influenza A virus, the M2 gene was obtained by RT-PCR amplification from the RNA, of human influenza virus strain A/PR/8/34 (H1N1) with specific primers. The amplified M2 gene fragment was cloned into eukaryotic expression plasmid pcDNA3.1 (), and transformed into receptive E.coli JM109 and positive clones were screened. Identification of Recombinant plasmid by double enzyme digestion and Polymerase chain reaction (polymerase chain reaction,PCR)
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373
【引证文献】
相关博士学位论文 前1条
1 张博;H1N1亚型猪流感病毒分离鉴定、鉴别诊断及M2e重组质粒免疫效力研究[D];四川农业大学;2011年
本文编号：2321092
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