含HIV-1 gag基因的重组腺病毒5型与35型嵌合病毒免疫效果研究

发布时间：2018-05-20 08:59

本文选题：HIV-1 + 艾滋病疫苗　；参考：《北京工业大学》2007年硕士论文

【摘要】： 近年来艾滋病毒,即人类免疫缺陷病毒(Human Immunodeficiency Virus,HIV)在世界范围内迅速传播,尤其是在非洲,已经成为死亡的首要病因。联合国艾滋病规划署和世界卫生组织2006年11月21日共同发布的《2006年世界艾滋病报告》显示,2006年全球新增艾滋病病毒感染者430万,使艾滋病病毒感染者总数达3,950万,同时全球又有290万人死于艾滋病。截至2006年10月31日,我国历年累计报告HIV感染者183,733例,其中艾滋病病人40,667例,死亡12,464例。国际医学界至今尚无治愈艾滋病的有效药物和疗法。因而研制针对我国HIV流行株的预防性和治疗性疫苗迫在眉睫。目前研制的预防性疫苗还不能产生针对HIV的持久免疫力,本研究的目的是构建有效抑制HIV复制的治疗性疫苗,有效后再用于预防。在经过抗逆转录病毒药物治疗、病人血浆HIV病毒载量下降后,应用该疫苗替代或减少化学药物的应用,从而达到抑制病毒复制、减轻病情的作用。本研究首先比较HIV-1 gag基因野生型(wt.gag)与密码子优化型(mod.gag)在体外细胞水平表达量,经Western Blot证实mod.gag基因的表达水平明显高于wt.gag基因。因此后续实验均采用mod.gag构建的重组病毒株。经PCR,Western Blot,间接免疫荧光鉴定了密码子优化过的HIV-1 gag基因在重组腺病毒(rAd5/F35-mod.gag)的正确插入和体外细胞水平的表达;用rAd5/F35-mod.gag以不同的方式单独或与DNA,rAAV2/1联合免疫BALB/c小鼠,ELISA检测小鼠血清中的P24特异性抗体,细胞内细胞因子染色法检测小鼠特异性细胞毒性T淋巴细胞(CTL)应答。结果重组腺病毒rAd5/F35-mod.gag中gag正确插入并且在体外细胞水平可以很好的表达目的基因gag;除PBS对照组外,在小鼠体内rAd5/F35-mod.gag单独或与DNA联合免疫均可诱导特异性的CTL应答和血清IgG抗体反应,单独免疫rAd5/F35-mod.gag两次分泌IFN-γ的CD8+ T细胞占总CD8+T细胞的8.828±1.554%,该组CTL反应的效果优于DNA初免,rAd5/F35-mod.gag加强的联合免疫组(3.62±2.03%)。单独免疫rAd5/F35-mod.gag组抗HIV-1 P24 IgG抗体滴度为1:12800,高于其他各组,反应结果与细胞免疫反应结果一致。腺病毒5型(Ad5)在人群中的感染率较高,预先存在的抗体可能会降低腺病毒载体携带的目的基因在体内的表达水平从而减弱其免疫原性。本研究通过改变Ad5型的纤突蛋白Fiber构建了含有gag基因的rAd5/F35-mod.gag载体,期望可以避免Ad5特异性免疫反应的抑制作用。首先用Ad5-GFP免疫小鼠来诱导针对Ad5的特异性免疫反应,再用rAd5/F35-mod.gag和rAd5-mod.gag免疫小鼠,检测HIV-1 Gag特异性的细胞免疫反应。Ad5-GFP初次感染4周后,rAd5/F35-mod.gag免疫2次后分泌IFN-γ的CD8+ T细胞占总CD8+T细胞的1.71±0.05%,低于rAd5/F35-mod.gag单独免疫2次的特异性细胞免疫水平(4.34±3.27%);而rAd5 -mod.gag在预先存在Ad5特异性免疫反应影响下,HIV特异性细胞免疫水平0.75±0.06%,远低于rAd5-mod.gag单独免疫2次的细胞免疫水平(4.70±0.73%)。因此rAd35/F35受到预先存在Ad5特异性免疫反应的抑制作用相对较小。因此重组腺病毒rAd5/F35-mod.gag只改变Ad5纤突蛋白Fiber,可以部分避免Ad5特异性免疫反应的抑制作用。上述结果表明rAd5/F35-mod.gag载体用做疫苗可诱导强而持久的HIV-1特异性细胞及体液免疫反应,是一种有希望的HIV候选疫苗。
[Abstract]:In recent years, HIV, the human immunodeficiency virus (Human Immunodeficiency Virus, HIV), has spread rapidly worldwide, especially in Africa, which has become the leading cause of death. The United Nations Programme on AIDS and the World Health Organization (WHO) and the World Health Organization (WHO), jointly issued by the United Nations Programme on HIV / AIDS and the World Health Organization (WHO) November 21, 2006 World AIDS report, show that 2006 Global The total number of HIV infected people was 4 million 300 thousand, the total number of HIV infected people was 39 million 500 thousand, and 2 million 900 thousand people died of AIDS in the world. Up to October 31, 2006, there were 183733 cases of HIV infection in China, including 40667 cases of AIDS patients and 12464 deaths. There is no effective drug to cure AIDS in the international medical field. Therefore, it is urgent to develop preventive and therapeutic vaccines targeting HIV strains in China.
The present preventive vaccine can not produce persistent immunity against HIV. The aim of this study is to construct a therapeutic vaccine that effectively inhibits HIV replication and to be used for prevention. After antiretroviral therapy, the vaccine should be used to replace or reduce the use of chemical drugs after the decrease of HIV viral load in the plasma of the patients. It can inhibit the replication of the virus and reduce the effect of the disease.
In this study, the level of expression of HIV-1 gag gene wild type (wt.gag) and codon optimization (mod.gag) in vitro was compared. The expression level of mod.gag gene was obviously higher than that of wt.gag gene by Western Blot. Therefore, the recombinant virus strain constructed by mod.gag was used in the follow-up experiment. The indirect immunofluorescence was identified by PCR, Western Blot, and indirect immunofluorescence. The HIV-1 gag gene has been optimized by the symbol in the correct insertion of recombinant adenovirus (rAd5/F35-mod.gag) and the expression of the cell level in vitro; the specific anti body in the serum of the mice was detected by rAd5/F35-mod.gag in a different way or with DNA, rAAV2/1 combined with BALB/c mice, and ELISA was used to detect the specificity of the mouse serum. The intracellular cytokine staining method was used to detect the specificity of the mice. Cytotoxic T lymphocyte (CTL) response. Results gag was correctly inserted in the recombinant adenovirus rAd5/F35-mod.gag and expressed the target gene gag well at the cell level in vitro. Except for the PBS control group, the rAd5/F35-mod.gag alone or combined with DNA in mice could induce specific CTL response and serum IgG antibody response, alone. The CD8+ T cells that immunized rAd5/F35-mod.gag two times IFN- gamma accounted for 8.828 + 1.554% of the total CD8+T cells. The effect of CTL reaction in this group was better than that of the primary DNA and the combined immune group (3.62 + 2.03%) strengthened with rAd5/F35-mod.gag. The anti HIV-1 P24 IgG antibody titer of the rAd5/F35-mod.gag group was 12800, which was higher than that of the other groups, and the reaction results and cellular immunity were higher than those in the other groups. The results of the reaction are consistent.
The infection rate of adenovirus type 5 (Ad5) is higher in the population. The pre existing antibody may reduce the expression level of the target gene in the adenovirus vector in vivo to weaken its immunogenicity. This study constructs the rAd5/F35-mod.gag vector containing the gag gene by changing the Ad5 type of the fibrinoid protein Fiber, hoping to avoid the specific Ad5 specificity. The inhibitory effect of sexual immune response. First, the specific immune response to Ad5 was induced by Ad5-GFP immunization in mice. The mice were immunized with rAd5/F35-mod.gag and rAd5-mod.gag to detect the HIV-1 Gag specific cellular immune response for 4 weeks after primary infection, and CD8+ T cells secreting IFN- gamma after rAd5/F35-mod.gag immunization for 2 times accounted for the total CD8+T cells. The 1.71 + 0.05%, lower than the specific immunization level of rAd5/F35-mod.gag alone (4.34 + 3.27%), and rAd5 -mod.gag in the pre existence of Ad5 specific immune response, the HIV specific cell immune level was 0.75 + 0.06%, far below the cellular immune level of 2 times alone (4.70 + 0.73%). Therefore, rAd35/F35 received. The inhibition of Ad5 specific immunoreaction in advance is relatively small. Therefore, the recombinant adenovirus rAd5/F35-mod.gag only changes the Ad5 fibrin protein Fiber, which can partly avoid the inhibitory effect of Ad5 specific immune response.
The above results show that rAd5/F35-mod.gag vector can induce strong and persistent HIV-1 specific cell and humoral immune response, which is a promising candidate for HIV vaccine.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373;R392

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