结核分枝杆菌MPT83重组腺病毒载体的构建及表达

发布时间：2018-01-17 13:28

本文关键词：结核分枝杆菌MPT83重组腺病毒载体的构建及表达　出处：《西南大学》2008年硕士论文　论文类型：学位论文

【摘要】：结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染引起的慢性传染病,MTB可能侵入人体全身各种器官,但主要侵犯肺,称为肺结核。据世界卫生组织(WHO)统计,全世界每年有1000万新发病例,150万人死于结核病。我国高居全球结核病高负担国家的第二位,现有MTB感染人数已达4亿,传染性TB患者达到200万。卡介苗(BCG)作为目前唯一用于预防结核病的疫苗,对于儿童重症结核病如粟粒型结核和结核性脑膜炎具有预防作用,但是对于预防成年人结核病效果不理想,保护率为0-80%不等。目前,很多国家都在研究结核新疫苗,已有200多种的候选疫苗,其中有的正在进行动物模型实验,有的已经进入临床实验。近几十年,随着耐药MTB增多及HIV共感染等问题,加重了结核病的严重性。所以开发新型有效的结核病疫苗迫在眉睫,希望尽快开发出更安全、高效、低成本、并可广泛用于各类人群的新型结核病疫苗。本文以广泛使用的Ad-Easy腺病毒包装系统为基础,构建结核分枝杆菌MPT83重组腺病毒疫苗。首先PCR获取结核分枝杆菌MPT83基因,然后再将MPT83基因重组于穿梭载体pAdTrack-CMV;通过化学转化法将线性化的重组穿梭质粒(pAdTrack-CMV-MPT83)和病毒骨架质粒(pAdEasy-1)共转入大肠杆菌BJ5183,重组穿梭质粒和病毒骨架质粒在BJ5183发生同源重组形成重组腺病毒质粒(rAd-GFP-MPT83);线性化的重组腺病毒质粒转染HEK293细胞,并于HEK293细胞中包装成病毒;mRNA和蛋白水平上检测目的基因的表达;大量扩增腺病毒,体外通过MTT实验初步验证重组腺病毒的免疫原性;体内通过重组腺病毒免疫小鼠,整体荧光成像观察报告基因绿色荧光蛋白(GFP)在小鼠体内的表达与分布,间接跟踪目的基因在小鼠体内的表达情况。结果表明: (1)成功构建结核分枝杆菌MPT83重组腺病毒载体,构建的重组腺病毒载体于HEK293细胞中成功包装腺病毒。通过RT-PCR和Western-Blotting实验,分别于mRNA和蛋白水平上检测均表明结核分枝杆菌MPT83基因在HEK293细胞中表达。 (2)MTT实验表明:vAd-GFP-MPT83病毒液比vAd-GFP病毒液具有更强的刺激体外淋巴细胞增殖的能力,证实了目的蛋白具有很强的刺激淋巴细胞增殖的能力,即其免疫原性。 (3)小鼠活体实验表明:vAd-GFP-MPT83病毒免疫小鼠后,报告基因GFP于免疫后第4d达到高峰,一直到半个月之后还有很强的表达,同时表达的范围也由原来注射的部位向周边扩散。
[Abstract]:Tuberculosis tuberculosis (TB) is caused by Mycobacterium tuberculosis. MTB) the chronic infectious disease caused by MTB infection may invade various organs of the human body, but mainly invades the lungs, known as tuberculosis. According to the World Health Organization (WHO) statistics. There are 10 million new cases and 1.5 million deaths from tuberculosis in the world every year. China ranks second among the high-burden countries in the world, and the current number of MTB infection has reached 400 million. BCG, the only vaccine used to prevent tuberculosis, has a preventive effect on children with severe tuberculosis, such as miliary tuberculosis and tuberculous meningitis. At present, many countries are studying new TB vaccine, and there are more than 200 candidate vaccines. In recent decades, with the increase of drug-resistant MTB and HIV co-infection and other problems. Therefore, it is urgent to develop new and effective TB vaccine. We hope to develop a more safe, efficient, low-cost and widely used TB vaccine for all kinds of people as soon as possible. Based on the widely used Ad-Easy adenovirus packaging system, the recombinant adenovirus vaccine of Mycobacterium tuberculosis MPT83 was constructed. Firstly, the MPT83 gene of Mycobacterium tuberculosis was obtained by PCR. Then the MPT83 gene was recombined into the shuttle vector pAdTrack-CMV. The linearized recombinant shuttle plasmid pAdTrack-CMV-MPT83) and the viral skeleton plasmid pAdEasy-1) were co-transfected into Escherichia coli BJ5183 by chemical transformation. The recombinant shuttle plasmid and viral skeleton plasmid were homologous recombined in BJ5183 to form recombinant adenovirus plasmid rAd-GFP-MPT83; The linearized recombinant adenovirus plasmid was transfected into HEK293 cells and packaged into HEK293 cells. The expression of the target gene was detected at the level of mRNA and protein. A large number of adenovirus was amplified and the immunogenicity of recombinant adenovirus was preliminarily verified by MTT in vitro. Mice were immunized with recombinant adenovirus in vivo, and the expression and distribution of report gene GFP in mice were observed by whole fluorescence imaging. Indirect tracking of the expression of the target gene in mice. The results showed that: The recombinant adenovirus vector of Mycobacterium tuberculosis MPT83 was constructed successfully. The recombinant adenovirus vector was successfully packaged in HEK293 cells by RT-PCR and Western-Blotting experiments. Both mRNA and protein levels showed that the MPT83 gene of Mycobacterium tuberculosis was expressed in HEK293 cells. The results of MTT assay showed that the vAd-GFP virus solution had a stronger ability to stimulate lymphocyte proliferation in vitro than that of vAd-GFP virus solution. It is confirmed that the target protein has a strong ability to stimulate lymphocyte proliferation, that is, its immunogenicity. (3) in vivo mouse experiment showed that the reporter gene GFP reached its peak on the 4th day after inoculation with the virus, and was still strongly expressed half a month later. At the same time, the range of expression from the original injection site to the peripheral diffusion.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346;Q789

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