弓形虫多价和多表位核酸疫苗黏膜免疫的实验研究

发布时间：2018-06-30 05:07

本文选题：弓形虫 + 核酸疫苗　；参考：《山东大学》2006年博士论文

【摘要】：目前，在经历了减毒或弱毒疫苗后，针对弓形虫病疫苗的研究主要集中在以保护性抗原为基础成分的蛋白疫苗和核酸疫苗上。作为继病原体疫苗、亚单位疫苗之后的第三代疫苗，核酸疫苗使得弓形虫疫苗的研究进入到一个新的阶段。但动物和人体实验均表明，单价疫苗的免疫效果不太理想，发展多种抗原组合、针对不同生活史发育阶段的复合多价疫苗是弓形虫疫苗研究的一个发展方向与共识。另外，在复合多价疫苗的研究当中，选取某些强保护性抗原中含T淋巴细胞和(或)B淋巴细胞表位构成的多表位疫苗，是新近发展起来的的疫苗研制趋势。本课题首次将弓形虫多价和多表位基因与霍乱毒素CTXA_2/B的基因融合构建弓形虫的多价和多表位DNA疫苗，采用重组减毒胞内菌(鼠伤寒减毒沙门氏菌)通过粘膜自然感染途径运送DNA疫苗，对DNA疫苗在体内外诱导的免疫应答进行检测，对重组减毒沙门氏菌DNA菌苗的粘膜免疫效果和霍乱毒素在粘膜免疫中的佐剂作用作初步探讨。本研究分两个部分：第一部分：观察弓形虫主要表面抗原SAG1、SAG2与霍乱毒素A2/B亚基复合基因真核质粒免疫小鼠所诱导的免疫反应。将SAG1基因、SAG2基因及CTXA2/B基因定向连接插入真核表达质粒pcDNA3.1，经酶切及测序，获得pcDNA3.1-SAG1，，pcDNA3.1-SAG1-SAG2及pcDNA3.1-SAG1-SAG2-CTXA2/B的重组子；将重组真核质粒在体外进行初步诱导，脂质体Cellfectamine~(TM)2000介导重组真核表达质粒转染人宫颈癌细胞系Hela细胞，RT-PCR从转染细胞的RNA中扩增得到单价和多价基因片断，证实经质粒载体携带的单价和多价基因均已
[Abstract]:At present, after the attenuated or attenuated vaccine, the research on toxoplasmosis vaccine is mainly focused on protein vaccine and nucleic acid vaccine based on protective antigen. As the third generation vaccine after pathogen vaccine, subunit vaccine, nucleic acid vaccine makes the research of Toxoplasma gondii vaccine into a new stage. However, animal and human experiments show that the immunological effect of univalent vaccine is not ideal, and the development of multiple antigen combinations is a development direction and consensus in the research of Toxoplasma gondii vaccine. In addition, in the study of multivalent vaccine, it is a new developing trend to select the polyepitope vaccine containing T lymphocytes and / or B lymphocyte epitopes in some strong protective antigens. In this study, the multivalent and multiepitope DNA vaccine of Toxoplasma gondii was constructed by fusion of the multivalent and multiepitope genes of Toxoplasma gondii with the cholera toxin CTXA 2 / B gene for the first time. DNA vaccine was transported by recombinant attenuated intracellular bacteria (Salmonella typhimurium) through natural mucosal infection to detect the immune response induced by DNA vaccine in vivo and in vitro. The mucosal immunity of recombinant attenuated Salmonella DNA vaccine and the adjuvant effect of cholera toxin in mucosal immunity were studied. This study is divided into two parts: the first part: to observe the eukaryotic plasmid immunization of Toxoplasma gondii main surface antigen SAG1-SAG2 and cholera toxin A2 / B subunit compound gene. Immune response induced by mice. The recombinant plasmid pcDNA3.1-SAG1-SAG1-SAG1-SAG2 and pcDNA3.1-SAG2-CTXA2-CTXA2 / B were obtained by restriction endonuclease digestion and sequencing, and the recombinant eukaryotic plasmids were preliminarily induced in vitro, and the recombinant plasmid was inserted into the eukaryotic expression plasmid pcDNA3.1-SAG1-SAG2-CTXA2-CTXA2.The recombinant plasmid was inserted into eukaryotic expression plasmid pcDNA3.1-SAG1-SAG1-SAG1-SAG2-CTXA2 / B by enzyme digestion and sequencing. The recombinant eukaryotic expression plasmid Cellfectamine ~ (TM) 2000 was transfected into human cervical cancer cell line Hela cell line by RT-PCR. The monovalent and multivalent gene fragments were amplified from the RNA of the transfected cells. It was confirmed that both the univalent and multivalent genes carried by the plasmid vector had been obtained.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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