表达登革病毒非结构蛋白的重组痘苗病毒的构建和表达

发布时间：2019-06-06 15:49

【摘要】：登革病毒为黄病毒属的主要成员，是一类有包膜的单股正链RNA病毒。自然界存在的登革病毒有四个血清型，每型均可使人发病，症状从较轻微的DF，到较严重的DHF／DSS．自二十世纪中叶以来，登革病毒的传播已成为热带和亚热带地区严重的公共卫生问题，随着全球变暖和国际交往的日益频繁，DF／DHF发病率迅速上升，每年约有1亿人感染，威胁着世界上近三分之一人口的健康，对此WHO、有关国家防疫部门及病毒学家始终予以高度重视。但目前仍无安全有效的疫苗批准上市。疫苗研究的起始阶段着重于传统的减毒活疫苗和灭活疫苗，但多年的临床试验数据表明，其四价疫苗不足以同时提供针对四个血清型的保护性免疫应答。近年，许多病毒学家致力于用能引起长期免疫保护反应的病毒亚单位抗原做疫苗。为避免ADE，，人们将研究焦点从最初的E蛋白转向非结构蛋白，主要是NS1。近年多采用真核表达系统如杆状病毒和痘苗病毒等作为亚单位疫苗的载体，自上世纪80年代以来，痘病毒真核表达载体己大量用于构建JEV、HIV、流感、乙型肝炎和HSV等的疫苗候选株。对NS1蛋白的抗原性和保护性研究的很多，已试验动物证明能保护动物免受致死剂量病毒的攻击，针对NS3蛋白近年研究也日益增多，因为该蛋白有很好的免疫原性，有多个T细胞识别位点，尽管就其细胞免疫所产生的免疫保护作用在国内外的研究都很少。基于以上资料，本研究将登革病毒的NS1和NS3分别重组到痘病毒载体上，通过痘病毒复制，稳定的表达有活性的目的蛋白，将重组活病毒进行动物试验，检测其抗体滴度，并做致死剂量病毒攻击，初步探索NS1／NS3的免疫保护性。首先，分别构建了含登革2和登革4的NS1、NS3的重组质粒NS1／NS3—pGS20。利用Trizol试剂从感染DEN2和DEN4的乳鼠脑中提取病毒RNA，然后分别进行RT—PCR，得到DEN2和DEN4的NS1、NS3基因。先后分别插入克隆载体pMD18—T中测序检查序列的正确性，再通过目的片断和穿梭载体上
[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.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

【参考文献】