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  本文關鍵詞： 惡性瘧原蟲 多表位疫苗 表達純化 穩(wěn)定溶液體系　出處：《北京協(xié)和醫(yī)學院》2012年碩士論文　論文類型：學位論文

【摘要】：瘧疾是當今世界上對人類危害最嚴重的三大傳染性疾病之一,主要流行于熱帶和亞熱帶地區(qū)。根據(jù)WHO最新統(tǒng)計數(shù)據(jù)估計,僅2010年全世界就有2.16億瘧疾臨床病例,大約65.5萬人因其而死亡,2010年全球有33億人面臨感染瘧疾的風險,而且大多數(shù)為非洲撒哈拉以南地區(qū)5歲以下兒童和孕婦。在感染人的5種瘧原蟲中,惡性瘧原蟲是危害最嚴重、致死率最高的一種。近年來,盡管在瘧疾控制方面取得了顯著成效,但由于抗藥性惡性瘧原蟲蟲株和抗殺蟲劑蚊媒的產(chǎn)生及蔓延,使得傳統(tǒng)瘧疾防治方法受到嚴峻挑戰(zhàn),這就加劇了人們對于預防性和治療性瘧疾疫苗的需求。由于瘧原蟲生活史復雜,抗原表達具有期特異性、蟲株間差異性、蟲體間的高度變異性和逃避宿主免疫攻擊的機制等特點,基于單個抗原或表位的疫苗在相關實驗中的免疫效果并不理想。因此,多期多價人工疫苗成為目前抗瘧疫苗的研究熱點。 本課題組對惡性瘧原蟲多表位疫苗的研制進行了長期的探索,前期利用表位改組技術構建了多表位基因庫,并用庫免疫血清篩選出了免疫原性最佳、穩(wěn)定性最好的隨機組合多表位人工抗原M.RCAg-1,最終獲得了原核表達的高效工程菌。M.RCAg-1與佐劑乳化后免疫小鼠和新西蘭大白兔,結果顯示其具有較強的免疫原性,特異性抗體在體外可抑制瘧原蟲生長(Growth Inhibition Assay, GIA)。表明M.RCAg-1可作為惡性瘧原蟲紅內(nèi)期候選疫苗,進一步優(yōu)化、開發(fā)。但是,由于初步放大生產(chǎn)所獲得的重組蛋白量較低,而且其在-80℃保存幾周后會出現(xiàn)降解或聚集,給后續(xù)研究帶來了困難。因此,獲得高純度穩(wěn)定的M.RCAg-1重組蛋白是臨床前研究的關鍵步驟。 本研究在前期工作的基礎上,根據(jù)多表位嵌合抗原疫苗M.RCAg-1的理化性質(zhì),利用鎳親和層析、陰離子交換層析和凝膠過濾層析等不同組合方式的5種純化方案對其進行純化,根據(jù)最終純度和回收率選擇最佳純化方案,摸索出了多表位嵌合抗原疫苗M.RCAg-1的純化步驟。與此同時,選用了蛋白結晶試劑盒Crystal Screen HT(HR2-130)中的溶液與M.RCAg-1蛋白液混合,通過倒置顯微鏡觀察和12%SDS-PAGE電泳圖譜,篩選出了最適合M.RCAg-1的溶液體系。此外,鑒于二硫鍵在蛋白質(zhì)的正確折疊、高級結構的形成以及保持其生物活性等方面都起著重要作用,本文也對M.RCAg-1的二硫鍵定位進行了初步分析。本研究主要取得以下進展： 1.在大腸桿菌中可溶性表達M.RCAg-1,表達量達到20%以上。 2.摸索出純化多表位嵌合抗原疫苗M.RCAg-1的最佳步驟,為下游放大生產(chǎn)和進一步研究M.RCAg-1的免疫效果奠定了基礎。在1L培養(yǎng)基中平均獲得206.68mg菌體總蛋白,目的蛋白M.RCAg-1約占菌體總蛋白的20.92%,最終純化結果顯示目的蛋白純度大于99%,回收率大于15%。 3.利用蛋白結晶試劑盒Crystal Screen HT (HR2-130),篩選出了使M.RCAg-1穩(wěn)定的溶液體系。經(jīng)研究證實M.RCAg-1溶解于PBS后可在-80℃保存六個月以上不發(fā)生降解。 4.對M.RCAg-1重組蛋白進行二硫鍵定位分析。初步分析結果顯示,M.RCAg-1分子中Cys215-Cys329形成二硫鍵,Cys215-Cys215形成鏈間二硫鍵。
[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.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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本文編號：1474919