本文關鍵詞：日本血吸蟲重組多價疫苗的研制與免疫保護功能評估　出處：《上海師范大學》2006年碩士論文　論文類型：學位論文

  更多相關文章： 日本血吸蟲 多價 核酸疫苗 重組抗原

【摘要】：目的：血吸蟲病是一種重要的人獸共患寄生蟲病，其疫苗的研究是防治該病的重要措施之一。迄今所研制出的一些日本血吸蟲亞單位基因工程疫苗保護力效果還不夠理想，加強多價疫苗的研究也是探索提高疫苗保護效果的有效途徑之一。本研究應用基因工程技術研制由多個不同抗原表位富集區(qū)組成的抗日本血吸蟲多價核酸疫苗和多價重組抗原疫苗，通過對小鼠進行的免疫保護實驗比較不同的疫苗所誘導的免疫保護效果，以期尋找到具有更高保護力的抗日本血吸蟲疫苗。 方法：本研究通過生物信息學的方法預測和篩選了日本血吸蟲抱雌溝蛋白(SjGCP)的抗原表位，應用PCR技術擴增了日本血吸蟲23KDa表膜蛋白(Sj23)，日本血吸蟲谷胱甘肽-S-轉移酶(Sj28 GST)和日本血吸蟲抱雌溝蛋白(SjGCP)的各一段富含表位的肽段所對應的編碼核苷酸片段，運用基因工程重組技術將此三種核苷酸片段以不同的組合方式分別插入載體pCMV-手寫體的和pGEX-2T的中，構建成了pCMV-BSj23-BSj28的，pCMV-BSjGCP-BSj23，pCMV-BSjGCP-BSj23-BSj28三種多價核酸疫苗及pGEX-BSj23-BSj28，pGEX-BSjGCP-BSj23，pGEX-BSjGCP-BSj23-BSj28三種原核表達質粒。三種真核質粒pCMV-BSj23-BSj28的，pCMV-BSjGCP-BSj23，pCMV-BSjGCP-BSj23-BSj28的經純化后直接以肌肉注射的方法免疫昆明系小鼠，，將三種重組原核表達質粒pGEX-BSj23-BSj28，pGEX-BSjGCP-BSj23，pGEX-BSjGCP-BSj23-BSj28的轉化BL21后進行了誘導表達和純化，結合福氏佐劑以皮下注射的方式免疫BalB／c小鼠。三免后，用日本血吸蟲尾蚴分別攻擊感染小鼠，42天后剖殺沖蟲并計數(shù)。 結果：成功構建了三種重組的真核表達質粒pCMV-BSj23-BSj28，pCMV-BSjGCP-BSj23，pCMV-BSjGCP-BSj23-BSj28，并在免疫保護試驗中分別獲得了6.30％，14.76％和64.95％的減蟲率：成功制備了三種重組抗原疫苗pGEX-BSj23-BSj28的，pGEX-BSjGCP-BSj23，pGEX-BSjGCP-BSj23-BSj28的，并在免疫保護實驗中分別獲得了13.65％，15.7％和57.99％的減蟲率。 結論：重組的多價核酸疫苗pCMV-BSjGCP-BSj23-BSj28的和重組的多價抗原pGEX-BSjGCP-BSj23-BSj28的在動物免疫實驗中獲得了較好的免疫保護效果，表明該種構建彩價疫苗的方法具有潛在的應用價值。
[Abstract]:Objective: schistosomiasis is an important parasitic disease, the vaccine research is one of the important measures for the prevention and treatment of disease. The effect of genetic engineering subunit vaccine of Schistosoma japonicum protection so far developed is not ideal, strengthen the research of polyvalent vaccine is also exploring the effective way to improve the protective effect of this vaccine. Study on the application of genetic engineering technology development is composed of a plurality of different of the immunodominant region of Schistosoma japonicum multivalent DNA vaccine and multivalent recombinant antigen vaccine, immune protective effect induced by immune protection test were conducted for the comparison of different vaccines, in order to find the anti Schistosoma vaccine has higher protection.
Methods: This study through the method of bioinformatics prediction and screening of sjgcp protein (SjGCP) epitopes was amplified by PCR of Schistosoma japonicum 23KDa membrane protein of Schistosoma japonicum (Sj23), glutathione -S- transferase (Sj28 GST) and Schistosoma japonicum GYNECOPHORAL canal protein (SjGCP) the corresponding nucleotide fragment encoding a peptide rich in table position, using gene engineering the three nucleotide fragments in different combinations were inserted into the vector pCMV- script and pGEX-2T, constructed by pCMV-BSj23-BSj28, pCMV-BSjGCP-BSj23, pCMV-BSjGCP-BSj23-BSj28 three kinds of nucleic acid vaccine and pGEX-BSj23-BSj28, pGEX-BSjGCP-BSj23. PGEX-BSjGCP-BSj23-BSj28 three prokaryotic expression plasmid. Three eukaryotic plasmid pCMV-BSj23-BSj28, pCMV-BSjGCP-BSj23, pCMV-BSjGCP-BSj23-BSj28 after purification Direct intramuscular injection of immune to Kunming mice, three Recombinant Prokaryotic expression plasmid pGEX-BSj23-BSj28, pGEX-BSjGCP-BSj23, pGEX-BSjGCP-BSj23-BSj28 after the conversion of BL21 was expressed and purified, combined with Freund's adjuvant by subcutaneous injection of immune BalB / c mice. Three after immunization, with cercariae of Schistosoma japonicum infected mice were 42. Days later killed insects and counting.
Results: we successfully constructed the eukaryotic expression plasmid pCMV-BSj23-BSj28, three recombinant pCMV-BSjGCP-BSj23, pCMV-BSjGCP-BSj23-BSj28, and the immune protection test were obtained respectively 6.30%, 14.76% and 64.95% of worm reduction rate: the successful preparation of three kinds of recombinant vaccines of pGEX-BSj23-BSj28, pGEX-BSjGCP-BSj23, pGEX-BSjGCP-BSj23-BSj28, and the immune protection test were obtained 13.65%, 15.7% and 57.99% of worm reduction rate.
Conclusion: recombinant polyvalent nucleic acid vaccine pCMV-BSjGCP-BSj23-BSj28 and recombinant polyvalent antigen pGEX-BSjGCP-BSj23-BSj28 have good immune protection effect in animal immune experiment, indicating that this method of constructing color value vaccine has potential application value.

【學位授予單位】：上海師范大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

【引證文獻】

相關碩士學位論文 前2條

1 陳實;重組抗原pGEX-BSjGCP-BSj23診斷家畜日本血吸蟲病研究[D];南京農業(yè)大學;2010年

2 徐妮為;抗日本血吸蟲病雙價共表達DNA疫苗pVAX1/SjCB·hIL-18的構建及其動物保護性研究[D];中南大學;2010年

本文編號：1417006