恶性疟多表位疫苗M.RCAg-1在大肠杆菌中的表达纯化及稳定溶液体系的筛选

发布时间：2018-01-30 01:31

本文关键词： 恶性疟原虫多表位疫苗表达纯化稳定溶液体系　出处：《北京协和医学院》2012年硕士论文　论文类型：学位论文

【摘要】：疟疾是当今世界上对人类危害最严重的三大传染性疾病之一,主要流行于热带和亚热带地区。根据WHO最新统计数据估计,仅2010年全世界就有2.16亿疟疾临床病例,大约65.5万人因其而死亡,2010年全球有33亿人面临感染疟疾的风险,而且大多数为非洲撒哈拉以南地区5岁以下儿童和孕妇。在感染人的5种疟原虫中,恶性疟原虫是危害最严重、致死率最高的一种。近年来,尽管在疟疾控制方面取得了显著成效,但由于抗药性恶性疟原虫虫株和抗杀虫剂蚊媒的产生及蔓延,使得传统疟疾防治方法受到严峻挑战,这就加剧了人们对于预防性和治疗性疟疾疫苗的需求。由于疟原虫生活史复杂,抗原表达具有期特异性、虫株间差异性、虫体间的高度变异性和逃避宿主免疫攻击的机制等特点,基于单个抗原或表位的疫苗在相关实验中的免疫效果并不理想。因此,多期多价人工疫苗成为目前抗疟疫苗的研究热点。本课题组对恶性疟原虫多表位疫苗的研制进行了长期的探索,前期利用表位改组技术构建了多表位基因库,并用库免疫血清筛选出了免疫原性最佳、稳定性最好的随机组合多表位人工抗原M.RCAg-1,最终获得了原核表达的高效工程菌。M.RCAg-1与佐剂乳化后免疫小鼠和新西兰大白兔,结果显示其具有较强的免疫原性,特异性抗体在体外可抑制疟原虫生长(Growth Inhibition Assay, GIA)。表明M.RCAg-1可作为恶性疟原虫红内期候选疫苗,进一步优化、开发。但是,由于初步放大生产所获得的重组蛋白量较低,而且其在-80℃保存几周后会出现降解或聚集,给后续研究带来了困难。因此,获得高纯度稳定的M.RCAg-1重组蛋白是临床前研究的关键步骤。本研究在前期工作的基础上,根据多表位嵌合抗原疫苗M.RCAg-1的理化性质,利用镍亲和层析、阴离子交换层析和凝胶过滤层析等不同组合方式的5种纯化方案对其进行纯化,根据最终纯度和回收率选择最佳纯化方案,摸索出了多表位嵌合抗原疫苗M.RCAg-1的纯化步骤。与此同时,选用了蛋白结晶试剂盒Crystal Screen HT(HR2-130)中的溶液与M.RCAg-1蛋白液混合,通过倒置显微镜观察和12%SDS-PAGE电泳图谱,筛选出了最适合M.RCAg-1的溶液体系。此外,鉴于二硫键在蛋白质的正确折叠、高级结构的形成以及保持其生物活性等方面都起着重要作用,本文也对M.RCAg-1的二硫键定位进行了初步分析。本研究主要取得以下进展： 1.在大肠杆菌中可溶性表达M.RCAg-1,表达量达到20%以上。 2.摸索出纯化多表位嵌合抗原疫苗M.RCAg-1的最佳步骤,为下游放大生产和进一步研究M.RCAg-1的免疫效果奠定了基础。在1L培养基中平均获得206.68mg菌体总蛋白,目的蛋白M.RCAg-1约占菌体总蛋白的20.92%,最终纯化结果显示目的蛋白纯度大于99%,回收率大于15%。 3.利用蛋白结晶试剂盒Crystal Screen HT (HR2-130),筛选出了使M.RCAg-1稳定的溶液体系。经研究证实M.RCAg-1溶解于PBS后可在-80℃保存六个月以上不发生降解。 4.对M.RCAg-1重组蛋白进行二硫键定位分析。初步分析结果显示,M.RCAg-1分子中Cys215-Cys329形成二硫键,Cys215-Cys215形成链间二硫键。
[Abstract]:Malaria is one of the world's harm to human and one of the three most severe infectious diseases, mainly in tropical and subtropical regions. According to the latest statistics WHO estimates, only in 2010 the whole world has 216 million clinical cases of malaria, about 655 thousand deaths in 2010, 3 billion 300 million people worldwide are at risk of infection. And most of sub Saharan Africa under the age of 5 children and pregnant women. In 5 kinds of human Plasmodium infection, Plasmodium falciparum is the most serious hazard, one of the highest death rate. In recent years, although has made remarkable achievements in malaria control, but the emergence and spread of drug-resistant falciparum malaria strains and insecticide resistant mosquitoes, which makes the traditional method of malaria control under severe challenge, which exacerbated the people for the prevention and treatment of malaria vaccine needs. Because the life cycle of Plasmodium antigen complex. As with stage specific differences among plants, insects, worms of the height between the variability and evade the host immune attack mechanism, based on the immune effect of single antigen epitope vaccine in the experiments is not ideal. Therefore, many vaccines have become a hot artificial multivalent vaccine against malaria.
The research group of the Plasmodium falciparum multi epitope vaccine development for a long period of exploration, early use of epitope shuffling technique to construct a multi epitope gene library and library immune serum screened the immunogenicity of the best, the best combination of stochastic stability of polyepitope artificial antigen M.RCAg-1, finally achieved high efficiency prokaryotic engineering the expression of strain.M.RCAg-1 and adjuvant immunized mice and rabbits. The results show that it has strong immunogenicity and specificity of the antibody could inhibit parasite growth in vitro (Growth Inhibition Assay, GIA). The results indicated that M.RCAg-1 can be used as a candidate vaccine against Plasmodium falciparum, further optimize the development. However, due to the initial amplification the amount of recombinant protein production is low, and in the -80 stored at a few weeks will appear after degradation or aggregation, bring difficulties to subsequent research. Therefore, to obtain high purity and stable M.RC Ag-1 recombinant protein is a key step in preclinical research.
This study on the basis of previous work, according to the physicochemical properties of multi epitope chimeric antigen M.RCAg-1 vaccine, using nickel affinity chromatography, 5 purification methods of anion exchange chromatography and gel filtration chromatography for the purification of different combinations of the final, according to the purity and recovery rate to choose the best purification scheme, worked out a multi table the purification steps a chimeric antigen vaccine M.RCAg-1. At the same time, the protein crystallization kit of Crystal Screen HT (HR2-130) and M.RCAg-1 protein in mixed liquid solution, observed by inverted microscope and 12%SDS-PAGE electrophoresis, screened out the most suitable solution system of M.RCAg-1. In addition, in view of the two disulfide bonds in the correct protein folding, formation advanced structure and maintain the biological activity and plays an important role, the two disulfide bonds of M.RCAg-1 positioning are analyzed. This research mainly takes The following progress is made:
1. in Escherichia coli, the expression of M.RCAg-1 was more than 20% in soluble expression.
2. to find out the optimum purification steps of polyepitope chimeric vaccine M.RCAg-1, which laid the foundation for the further study of M.RCAg-1 amplification production and downstream immunity effect. The average 206.68mg of total bacterial protein 1L in culture medium, the target protein accounted for about M.RCAg-1 of the total bacterial protein 20.92%, final purification results showed that target protein purity is higher than 99%, recovery the rate of more than 15%.
3., we used the protein crystallization kit Crystal Screen HT (HR2-130) to screen out the solution system that made M.RCAg-1 stable. It was proved that M.RCAg-1 can be stored at -80 temperature for six months without degradation after being dissolved in PBS.
4., we carried out two sulfur bond location analysis for M.RCAg-1 recombinant protein. Preliminary analysis showed that Cys215-Cys329 formed two sulfur bonds in M.RCAg-1 molecule and Cys215-Cys215 formed two sulfur bonds between chains.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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