本文關(guān)鍵詞：多價血吸蟲蛋白疫苗的優(yōu)選與原核表達(dá)研究　出處：《華中科技大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 日本血吸蟲 原核表達(dá) 蛋白疫苗 生物信息學(xué)分析

【摘要】：血吸蟲病是一種世界性的傳染性疾病，，它嚴(yán)重危害著人類的生命健康，阻礙社會發(fā)展，常規(guī)的預(yù)防和治療手段難以遏制形勢日益嚴(yán)峻的血吸蟲疫情，研制血吸蟲疫苗是預(yù)防血吸蟲病最有效的方法之一。目前血吸蟲蛋白疫苗和血吸蟲核酸疫苗是主要的兩種疫苗形式。血吸蟲核酸疫苗能夠為實驗動物提供一定的免疫保護(hù)力，然而其安全性在短時間內(nèi)還無法得到確認(rèn)。與核酸疫苗相比，蛋白疫苗的技術(shù)較為成熟，且其安全性得到驗證，因此血吸蟲蛋白疫苗有可能首先實行產(chǎn)業(yè)化。血吸蟲蛋白疫苗有單價蛋白疫苗和多價蛋白疫苗形式，生產(chǎn)多價蛋白疫苗可以減少生產(chǎn)成本和簡化生產(chǎn)工藝。然而，利用基因工程技術(shù)生產(chǎn)多價蛋白疫苗時，是否因為蛋白融合之后片段過大而影響其合成是一個需要研究的問題。為此，本研究建立了血吸蟲蛋白疫苗原核表達(dá)系統(tǒng)，對單價蛋白疫苗和多價蛋白疫苗的原核表達(dá)進(jìn)行了深入的研究，取得了以下實驗結(jié)果： (1)根據(jù)WHO提供的血吸蟲疫苗候選抗原以及利用生物信息學(xué)方法分析抗原的疏水性和高級結(jié)構(gòu)，篩選出合適的血吸蟲抗原基因（Sj26, SjGAPDH,Sj26.GAPDH）來進(jìn)行研究。 (2)克隆到了日本血吸蟲單價抗原基因Sj26和SjGAPDH以及融合抗原基因Sj26.GAPDH，利用分子生物學(xué)方法將抗原基因連接到原核表達(dá)載體上，構(gòu)建成原核表達(dá)系統(tǒng)，經(jīng)酶切驗證和測序驗證，表明構(gòu)建成功。通過鎳離子層析柱的親和層析來對目的蛋白進(jìn)行分離純化，并且摸索出鎳離子層析柱的最佳洗脫條件。 (3)利用酶聯(lián)免疫吸附測定(ELISA)對分離得到的單價日本血吸蟲抗原蛋白和融合的抗原蛋白進(jìn)行定量分析。統(tǒng)計學(xué)分析表明，融合抗原蛋白的原核表達(dá)量與單價抗原蛋白的原核表達(dá)量沒有明顯差異，從而對血吸蟲蛋白疫苗的優(yōu)選作出了初步判斷。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R532.21;R392.11

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