基于痘苗病毒天坛株的改良载体的研究及HIV-1疫苗的构建

发布时间：2018-08-26 16:11

【摘要】： 1981年以前,痘苗病毒天坛株作为天花疫苗在中国被广泛应用并为消灭天花发挥了重要作用。之前我们已经鉴定了野生型天坛株在体内体外的生物学特性,并且已经通过检测鼻腔途径导致的体重降低和直接颅腔接种鉴定了其毒力。为了开发天坛株作为活载体来构建疫苗,我们通过同源重组的方法将绿色荧光蛋白基因GFP导入到其基因组的不同位置,构建了三种改良的痘苗病毒天坛株。在Vero细胞上,基于绿色荧光蛋白的表达筛选得到了三种改良的痘苗病毒天坛株并利用Vero进行了增殖。对纯化的改良痘苗病毒天坛株在体内体外进行了遗传型和表型的鉴定。测序结果表明确实是在试验预期设计的位点发生了重组。我们发现在Vero细胞上进行多次传代以后GFP依然十分稳定,这表明三个位点都能够耐受外源基因并能够应用于将来的重组疫苗构建。我们对三种改良病毒在14种细胞上的生长特性进行了测试,跟野生型的天坛株相比,他们表现出了不同程度降低的致细胞病变病变效应和缩小的宿主范围,但表达GFP的水平相当。进一步的,我们发现在小鼠内导致体重降低的毒力跟野生型天坛株相比下降了250到1000倍以上而且在老鼠的中枢神经系统不能进行复制。与此同时,三种改良病毒都能在小鼠体内刺激产生较强的针对外源基因GFP的体液和细胞免疫反应。结合以上几点,改良病毒的复制能力能在体内产生比较高的免疫反应。在减小源自父本野生天坛株的毒力的同时,我们希望能同时保留其复制能力,以获得理想的活病毒载体应用于包括艾滋病在内的传染病的疫苗的研发。作为最初的尝试,我们还构建了融合表达华中地区艾滋病毒流行株的nef,tat和rev基因的改良痘苗病毒天坛株。通过PCR的方法,我们去除了nef的终止密码,tat的起始和终止密码以及rev的起始密码以在一个启动子控制之下进行融合表达。可以检测到融合表达的蛋白质。已经用融合表达nef,tat和rev基因的改良痘苗病毒天坛株对小鼠进行了免疫并收集了第0,3和5周的血清,在第5周时收集了小鼠脾细胞。通过Elisa检测了针对外源基因的抗体反应,现在正在进行ELISpot试验检测免疫反应。
[Abstract]:Before 1981, Temple of Heaven strain of vaccinia virus was widely used as smallpox vaccine in China and played an important role in smallpox eradication. We have previously identified the biological characteristics of the wild Temple of Heaven strain in vitro and in vivo, and its virulence has been evaluated by detecting the weight loss caused by the nasal pathway and direct cranial inoculation. In order to develop Temple of Heaven strain as a live vector to construct vaccine, we constructed three improved vaccinia virus Temple of Heaven strain by homologous recombination of green fluorescent protein gene GFP into different positions of its genome. Three improved vaccinia virus Temple of Heaven strains were obtained based on the expression of green fluorescent protein in Vero cells and were proliferated by Vero. The genetic type and phenotype of the purified Temple of Heaven strain of vaccinia virus were identified in vitro and in vivo. The sequencing results showed that the recombination did take place at the intended site of the experiment. We found that GFP remained stable after multiple passages on Vero cells, which indicated that all three loci could tolerate foreign genes and be used to construct recombinant vaccines in the future. We tested the growth characteristics of three modified viruses on 14 kinds of cells. Compared with the wild type Temple of Heaven strain, they showed different degrees of reduced cytopathic effect and reduced host range. But the level of GFP expression was similar. Further, we found that the virulence that led to weight loss in mice was 250 to 1000 times lower than that of the wild Temple of Heaven strain and could not be replicated in the central nervous system of mice. At the same time, all of the three modified viruses could produce strong humoral and cellular immune responses to exogenous gene GFP in mice. Combined with the above, the modified virus replication ability in vivo can produce a relatively high immune response. While reducing the virulence derived from the paternal wild Temple of Heaven strain, we hope to retain its replicability at the same time so as to obtain the ideal live virus vector for the development of vaccines for infectious diseases, including AIDS. As an initial attempt, we also constructed an improved vaccinia virus Temple of Heaven strain that fused the nef,tat and rev genes expressing HIV epidemic strains in Central China. By using the method of PCR, we remove the start and end cipher of nef and the start password of rev for fusion expression under the control of a promoter. Fusion proteins can be detected. The modified vaccinia virus Temple of Heaven strain, which expressed nef,tat and rev genes, was used to immunize the mice and collect the sera of the 3rd and 5th weeks, and the spleen cells of the mice were collected at the 5th week. Antibody responses to foreign genes were detected by Elisa, and ELISpot tests are being conducted to detect immune responses.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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