日本血吸虫重组多价疫苗的研制与免疫保护功能评估

发布时间：2018-01-13 02:15

本文关键词：日本血吸虫重组多价疫苗的研制与免疫保护功能评估　出处：《上海师范大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：血吸虫病是一种重要的人兽共患寄生虫病，其疫苗的研究是防治该病的重要措施之一。迄今所研制出的一些日本血吸虫亚单位基因工程疫苗保护力效果还不够理想，加强多价疫苗的研究也是探索提高疫苗保护效果的有效途径之一。本研究应用基因工程技术研制由多个不同抗原表位富集区组成的抗日本血吸虫多价核酸疫苗和多价重组抗原疫苗，通过对小鼠进行的免疫保护实验比较不同的疫苗所诱导的免疫保护效果，以期寻找到具有更高保护力的抗日本血吸虫疫苗。方法：本研究通过生物信息学的方法预测和筛选了日本血吸虫抱雌沟蛋白(SjGCP)的抗原表位，应用PCR技术扩增了日本血吸虫23KDa表膜蛋白(Sj23)，日本血吸虫谷胱甘肽-S-转移酶(Sj28 GST)和日本血吸虫抱雌沟蛋白(SjGCP)的各一段富含表位的肽段所对应的编码核苷酸片段，运用基因工程重组技术将此三种核苷酸片段以不同的组合方式分别插入载体pCMV-手写体的和pGEX-2T的中，构建成了pCMV-BSj23-BSj28的，pCMV-BSjGCP-BSj23，pCMV-BSjGCP-BSj23-BSj28三种多价核酸疫苗及pGEX-BSj23-BSj28，pGEX-BSjGCP-BSj23，pGEX-BSjGCP-BSj23-BSj28三种原核表达质粒。三种真核质粒pCMV-BSj23-BSj28的，pCMV-BSjGCP-BSj23，pCMV-BSjGCP-BSj23-BSj28的经纯化后直接以肌肉注射的方法免疫昆明系小鼠，，将三种重组原核表达质粒pGEX-BSj23-BSj28，pGEX-BSjGCP-BSj23，pGEX-BSjGCP-BSj23-BSj28的转化BL21后进行了诱导表达和纯化，结合福氏佐剂以皮下注射的方式免疫BalB／c小鼠。三免后，用日本血吸虫尾蚴分别攻击感染小鼠，42天后剖杀冲虫并计数。结果：成功构建了三种重组的真核表达质粒pCMV-BSj23-BSj28，pCMV-BSjGCP-BSj23，pCMV-BSjGCP-BSj23-BSj28，并在免疫保护试验中分别获得了6.30％，14.76％和64.95％的减虫率：成功制备了三种重组抗原疫苗pGEX-BSj23-BSj28的，pGEX-BSjGCP-BSj23，pGEX-BSjGCP-BSj23-BSj28的，并在免疫保护实验中分别获得了13.65％，15.7％和57.99％的减虫率。结论：重组的多价核酸疫苗pCMV-BSjGCP-BSj23-BSj28的和重组的多价抗原pGEX-BSjGCP-BSj23-BSj28的在动物免疫实验中获得了较好的免疫保护效果，表明该种构建彩价疫苗的方法具有潜在的应用价值。
[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.

【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

【引证文献】