【摘要】： 1981年以前,痘苗病毒天壇株作為天花疫苗在中國(guó)被廣泛應(yīng)用并為消滅天花發(fā)揮了重要作用。之前我們已經(jīng)鑒定了野生型天壇株在體內(nèi)體外的生物學(xué)特性,并且已經(jīng)通過(guò)檢測(cè)鼻腔途徑導(dǎo)致的體重降低和直接顱腔接種鑒定了其毒力。為了開(kāi)發(fā)天壇株作為活載體來(lái)構(gòu)建疫苗,我們通過(guò)同源重組的方法將綠色熒光蛋白基因GFP導(dǎo)入到其基因組的不同位置,構(gòu)建了三種改良的痘苗病毒天壇株。在Vero細(xì)胞上,基于綠色熒光蛋白的表達(dá)篩選得到了三種改良的痘苗病毒天壇株并利用Vero進(jìn)行了增殖。對(duì)純化的改良痘苗病毒天壇株在體內(nèi)體外進(jìn)行了遺傳型和表型的鑒定。測(cè)序結(jié)果表明確實(shí)是在試驗(yàn)預(yù)期設(shè)計(jì)的位點(diǎn)發(fā)生了重組。我們發(fā)現(xiàn)在Vero細(xì)胞上進(jìn)行多次傳代以后GFP依然十分穩(wěn)定,這表明三個(gè)位點(diǎn)都能夠耐受外源基因并能夠應(yīng)用于將來(lái)的重組疫苗構(gòu)建。我們對(duì)三種改良病毒在14種細(xì)胞上的生長(zhǎng)特性進(jìn)行了測(cè)試,跟野生型的天壇株相比,他們表現(xiàn)出了不同程度降低的致細(xì)胞病變病變效應(yīng)和縮小的宿主范圍,但表達(dá)GFP的水平相當(dāng)。進(jìn)一步的,我們發(fā)現(xiàn)在小鼠內(nèi)導(dǎo)致體重降低的毒力跟野生型天壇株相比下降了250到1000倍以上而且在老鼠的中樞神經(jīng)系統(tǒng)不能進(jìn)行復(fù)制。與此同時(shí),三種改良病毒都能在小鼠體內(nèi)刺激產(chǎn)生較強(qiáng)的針對(duì)外源基因GFP的體液和細(xì)胞免疫反應(yīng)。結(jié)合以上幾點(diǎn),改良病毒的復(fù)制能力能在體內(nèi)產(chǎn)生比較高的免疫反應(yīng)。在減小源自父本野生天壇株的毒力的同時(shí),我們希望能同時(shí)保留其復(fù)制能力,以獲得理想的活病毒載體應(yīng)用于包括艾滋病在內(nèi)的傳染病的疫苗的研發(fā)。 作為最初的嘗試,我們還構(gòu)建了融合表達(dá)華中地區(qū)艾滋病毒流行株的nef,tat和rev基因的改良痘苗病毒天壇株。通過(guò)PCR的方法,我們?nèi)コ薾ef的終止密碼,tat的起始和終止密碼以及rev的起始密碼以在一個(gè)啟動(dòng)子控制之下進(jìn)行融合表達(dá)�？梢詸z測(cè)到融合表達(dá)的蛋白質(zhì)。已經(jīng)用融合表達(dá)nef,tat和rev基因的改良痘苗病毒天壇株對(duì)小鼠進(jìn)行了免疫并收集了第0,3和5周的血清,在第5周時(shí)收集了小鼠脾細(xì)胞。通過(guò)Elisa檢測(cè)了針對(duì)外源基因的抗體反應(yīng),現(xiàn)在正在進(jìn)行ELISpot試驗(yàn)檢測(cè)免疫反應(yīng)。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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本文編號(hào)：2205446