發(fā)布時間：2018-11-09 17:31

【摘要】：甲型流感病毒M2蛋白是一種具有高保守性，可形成離子通道并影響流感病毒血凝素(hemagglutinin，HA)天然構(gòu)象的形成，有利于病毒脫殼，具有型特異性，，被認(rèn)為是具有交叉免疫保護(hù)性的結(jié)構(gòu)蛋白。DNA疫苗能夠同時刺激機體B細(xì)胞，輔助性T細(xì)胞(helper T cell，Th)和細(xì)胞毒性T細(xì)胞(cytotoxic T lymphocytes，CTL)，產(chǎn)生特異性體液免疫和細(xì)胞免疫，易于操作，經(jīng)濟(jì)有效，目前已成為疫苗研究的熱點。美國國立衛(wèi)生研究院(National Institutes of Health，NIH)和世界衛(wèi)生組織(World Health Organization，WHO)都強調(diào)流感DNA疫苗是流感疫苗研究的一個重要方向。 目的 本研究的目的在于通過構(gòu)建甲型流感病毒M2蛋白的真核表達(dá)載體，用M2蛋白的真核表達(dá)載體核酸免疫實驗動物后再用流感病毒攻擊，觀察重組質(zhì)粒pcDNA3.1(+)／M2的免疫保護(hù)作用，為進(jìn)一步研究M2蛋白的交叉保護(hù)作用奠定基礎(chǔ)。 方法 我們首先將人流感病毒A／PR／8／34(H1N1)經(jīng)雞胚尿囊液接種培養(yǎng)，用紅細(xì)胞吸附釋放法(absorption and release-RBC，AR-RBC)純化病毒。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，用特異引物進(jìn)行RT-PCR擴增獲得M2基因。將擴增的M2基因片段克隆入真核表達(dá)質(zhì)粒載體pcDNA3.1(+)，轉(zhuǎn)化感受態(tài)E．coli JM109并篩選陽性克隆。雙酶切、多聚合酶鏈反應(yīng)(polymerase chain reaction，PCR)鑒定重組質(zhì)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)
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R373

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 張博;H1N1亞型豬流感病毒分離鑒定、鑒別診斷及M2e重組質(zhì)粒免疫效力研究[D];四川農(nóng)業(yè)大學(xué);2011年

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