多价血吸虫蛋白疫苗的优选与原核表达研究

发布时间：2018-01-11 22:27

本文关键词：多价血吸虫蛋白疫苗的优选与原核表达研究　出处：《华中科技大学》2012年硕士论文　论文类型：学位论文

【摘要】：血吸虫病是一种世界性的传染性疾病，，它严重危害着人类的生命健康，阻碍社会发展，常规的预防和治疗手段难以遏制形势日益严峻的血吸虫疫情，研制血吸虫疫苗是预防血吸虫病最有效的方法之一。目前血吸虫蛋白疫苗和血吸虫核酸疫苗是主要的两种疫苗形式。血吸虫核酸疫苗能够为实验动物提供一定的免疫保护力，然而其安全性在短时间内还无法得到确认。与核酸疫苗相比，蛋白疫苗的技术较为成熟，且其安全性得到验证，因此血吸虫蛋白疫苗有可能首先实行产业化。血吸虫蛋白疫苗有单价蛋白疫苗和多价蛋白疫苗形式，生产多价蛋白疫苗可以减少生产成本和简化生产工艺。然而，利用基因工程技术生产多价蛋白疫苗时，是否因为蛋白融合之后片段过大而影响其合成是一个需要研究的问题。为此，本研究建立了血吸虫蛋白疫苗原核表达系统，对单价蛋白疫苗和多价蛋白疫苗的原核表达进行了深入的研究，取得了以下实验结果： (1)根据WHO提供的血吸虫疫苗候选抗原以及利用生物信息学方法分析抗原的疏水性和高级结构，筛选出合适的血吸虫抗原基因（Sj26, SjGAPDH,Sj26.GAPDH）来进行研究。 (2)克隆到了日本血吸虫单价抗原基因Sj26和SjGAPDH以及融合抗原基因Sj26.GAPDH，利用分子生物学方法将抗原基因连接到原核表达载体上，构建成原核表达系统，经酶切验证和测序验证，表明构建成功。通过镍离子层析柱的亲和层析来对目的蛋白进行分离纯化，并且摸索出镍离子层析柱的最佳洗脱条件。 (3)利用酶联免疫吸附测定(ELISA)对分离得到的单价日本血吸虫抗原蛋白和融合的抗原蛋白进行定量分析。统计学分析表明，融合抗原蛋白的原核表达量与单价抗原蛋白的原核表达量没有明显差异，从而对血吸虫蛋白疫苗的优选作出了初步判断。
[Abstract]:Schistosomiasis is a worldwide infectious disease, which seriously endangers human life and health, hinders the development of society, and it is difficult to contain the increasingly severe situation of schistosomiasis by conventional prevention and treatment. Development of schistosomiasis vaccine is one of the most effective methods to prevent schistosomiasis. At present, Schistosoma protein vaccine and schistosomiasis nucleic acid vaccine are two main forms of vaccine. Schistosoma nucleic acid vaccine can provide certain amount of vaccine for laboratory animals. Immune protection. However, its safety can not be confirmed in a short time. Compared with nucleic acid vaccine, protein vaccine technology is more mature, and its safety has been verified. Therefore, it is possible that the protein vaccine of Schistosoma japonicum can be industrialized first. The protein vaccine of Schistosoma japonicum can be produced in the form of monovalent protein vaccine and polyvalent protein vaccine. The production of polyvalent protein vaccines can reduce production costs and simplify production processes. However, when using genetic engineering techniques to produce multivalent protein vaccines. It is a problem that whether the protein fusion fragment is too large to affect its synthesis. Therefore, the prokaryotic expression system of Schistosoma japonicum protein vaccine has been established in this study. The prokaryotic expression of monovalent protein vaccine and polyvalent protein vaccine was studied. The following results were obtained: 1) according to the candidate antigen of Schistosoma japonicum vaccine provided by WHO and the analysis of hydrophobicity and advanced structure of antigen by bioinformatics, the suitable gene of Schistosoma japonicum antigen (Sj26) was selected. SjGAPDH Sj26. GAPDH). The monovalent antigen genes Sj26 and SjGAPDH of Schistosoma japonicum and the fusion antigen gene Sj26.GAPDH were cloned. The prokaryotic expression system was constructed by ligating the antigen gene to the prokaryotic expression vector by molecular biological method. The expression system was confirmed by restriction endonuclease digestion and sequencing. The results showed that the target protein was separated and purified by affinity chromatography of nickel ion chromatography column, and the optimum elution conditions were found out. (3) the monovalent Schistosoma japonicum antigen protein and fusion antigen protein were quantitatively analyzed by enzyme linked immunosorbent assay (Elisa). There was no significant difference between the prokaryotic expression of fusion antigen protein and that of monovalent antigen protein, so the preliminary judgement was made on the optimal selection of Schistosoma japonicum protein vaccine.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R532.21;R392.11

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