【摘要】：登革病毒為黃病毒屬的主要成員，是一類有包膜的單股正鏈RNA病毒。自然界存在的登革病毒有四個血清型，每型均可使人發(fā)病，癥狀從較輕微的DF，到較嚴重的DHF／DSS．自二十世紀中葉以來，登革病毒的傳播已成為熱帶和亞熱帶地區(qū)嚴重的公共衛(wèi)生問題，隨著全球變暖和國際交往的日益頻繁，DF／DHF發(fā)病率迅速上升，每年約有1億人感染，威脅著世界上近三分之一人口的健康，對此WHO、有關(guān)國家防疫部門及病毒學(xué)家始終予以高度重視。但目前仍無安全有效的疫苗批準上市。 疫苗研究的起始階段著重于傳統(tǒng)的減毒活疫苗和滅活疫苗，但多年的臨床試驗數(shù)據(jù)表明，其四價疫苗不足以同時提供針對四個血清型的保護性免疫應(yīng)答。近年，許多病毒學(xué)家致力于用能引起長期免疫保護反應(yīng)的病毒亞單位抗原做疫苗。為避免ADE，，人們將研究焦點從最初的E蛋白轉(zhuǎn)向非結(jié)構(gòu)蛋白，主要是NS1。近年多采用真核表達系統(tǒng)如桿狀病毒和痘苗病毒等作為亞單位疫苗的載體，自上世紀80年代以來，痘病毒真核表達載體己大量用于構(gòu)建JEV、HIV、流感、乙型肝炎和HSV等的疫苗候選株。 對NS1蛋白的抗原性和保護性研究的很多，已試驗動物證明能保護動物免受致死劑量病毒的攻擊，針對NS3蛋白近年研究也日益增多，因為該蛋白有很好的免疫原性，有多個T細胞識別位點，盡管就其細胞免疫所產(chǎn)生的免疫保護作用在國內(nèi)外的研究都很少�；谝陨腺Y料，本研究將登革病毒的NS1和NS3分別重組到痘病毒載體上，通過痘病毒復(fù)制，穩(wěn)定的表達有活性的目的蛋白，將重組活病毒進行動物試驗，檢測其抗體滴度，并做致死劑量病毒攻擊，初步探索NS1／NS3的免疫保護性。 首先，分別構(gòu)建了含登革2和登革4的NS1、NS3的重組質(zhì)粒NS1／NS3—pGS20。利用Trizol試劑從感染DEN2和DEN4的乳鼠腦中提取病毒RNA，然后分別進行RT—PCR，得到DEN2和DEN4的NS1、NS3基因。先后分別插入克隆載體pMD18—T中測序檢查序列的正確性，再通過目的片斷和穿梭載體上
[Abstract]:Dengue virus is a major member of the genus flavivirus, and is a type of enveloped single-strand RNA virus. The dengue virus in nature has four serotypes, each of which can cause a person to develop, and the symptoms can be reduced from a minor DF to a more severe DHF/ DSS. Since the middle of the twentieth century, the transmission of dengue virus has become a serious public health problem in the tropical and subtropical regions. With the increasing frequency of global warming and international exchanges, the incidence of DF/ DHF has increased rapidly, and about 100 million people are infected every year. The health of nearly a third of the world's population is a threat to the world, and the WHO, the national epidemic prevention department and the virologist have always attached great importance to it. But there are still no safe and effective vaccines for marketing. The start-up phase of the vaccine study is focused on traditional attenuated live vaccines and inactivated vaccines, but for many years clinical trials have shown that its tetravalent vaccine is not sufficient to provide for four blood at the same time In recent years, a number of virologists have been working to respond to long-term immune protection. The virus subunit antigen is a vaccine. In order to avoid ADE, the focus is shifted from the initial E protein to the non-junction In recent years, the eukaryotic expression vector of the poxvirus has been used in the construction of JEV, HIV, influenza, hepatitis B and the carrier of the subunit vaccine by using the eukaryotic expression system such as baculovirus and vaccinia virus in recent years. A vaccine candidate for HSV and the like. Many of the antigenic and protective studies of the NS1 protein have been shown to be able to protect the animals from the attack of the lethal dose virus, which has also been increasing in recent years for NS3 proteins, as it The protein has good immunogenicity, and has a plurality of T-cell recognition sites, Based on the above data, the NS1 and NS3 of the dengue virus are respectively recombined onto the poxvirus vector, and the active protein is expressed by the poxvirus replication and the stable expression of the active protein, and the recombinant live virus is carried out for animals. Test, detect the antibody titer, and make a fatal dose of disease In this paper, the immune protection of NS1/ NS3 was first explored. The recombinant plasmid NS1/ NS3-pGS20 of the NS1, NS3 of the leather 2 and the dengue 4 was extracted with the Trizol reagent from the rat brain infected with DEN2 and DEN4, and then The NS1 and NS3 gene of DEN2 and DEN4 were obtained by RT-PCR respectively.
【學(xué)位授予單位】：中國藥品生物制品檢定所
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前3條

1 王樹聲;劉明團;梁富雄;陳錦華;;用異羥基洋地黃毒甙配基(Digoxigenin)標記cDNA-RNA雜交試驗檢測登革熱病毒的研究[J];廣西醫(yī)學(xué);1991年01期

2 秦鄂德;黃病毒抗原結(jié)構(gòu)及其抗原性的研究進展[J];軍事醫(yī)學(xué)科學(xué)院院刊;1993年03期

3 徐偉文;登革出血熱致病機理研究進展[J];中國人獸共患病雜志;1998年02期

本文編號：2494420