弓形虫P30单基因疫苗与P30-ROP2复合基因疫苗免疫小鼠的实验研究

发布时间：2018-01-19 11:30

本文关键词： 弓形虫 P3O基因 ROP2基因复合基因核酸疫苗　出处：《山东大学》2005年硕士论文　论文类型：学位论文

【摘要】：随着分子生物学技术在寄生虫学研究领域的应用和发展,弓形虫疫苗从最早的全虫疫苗到新近迅速发展起来的核酸疫苗均取得了很大进展。但是,弓形虫灭活疫苗免疫保护力低;减毒活疫苗易恢复毒力,不能用于人体;亚单位疫苗生产过程复杂,且抗原蛋白不能形成正确的天然构象因此免疫活性不足,使得弓形虫核酸疫苗成为一个新的研究方向。同时,越来越多的证据表明,弓形虫阶段特异性抗原分子只能激发机体的阶段特异性保护免疫。因此,使用来自不同阶段的多抗原基因的复合基因疫苗可能刺激机体产生较全面的免疫保护性。 P30(SAG1)作为弓形虫速殖子期特异的主要表面抗原之一,具有高度的免疫原性和免疫保护性,是弓形虫感染免疫诊断和疫苗开发的的主要候选抗原。ROP2(Rhoptry2)蛋白是弓形虫棒状体分泌的一种蛋白,主要协助虫体入侵宿主细胞,在弓形虫生活史的速殖子期、缓殖子期和子孢子期中均有表达,具有高度的保守性和免疫原性,能充分刺激机体的免疫系统,引发保护性免疫反应,其作为疫苗候选分子具有巨大的潜力。本研究应用基因工程方法构建弓形虫单基因真核表达质粒pcDNA3.1-P30;并将之与pcDNA3.1-P30-ROP2复合基因真核表达质粒分别瞬时转染人宫颈癌细胞系(Hela)细胞,转染48小时后用Trizol提取细胞总RNA,以Oligo(dT)18为引物逆转录生成cDNA第一条链;对管家基因β—actin进行PCR验证逆转录;然后分别对P30、P30-ROP2基因片段进行PCR,证实真核表达载体pcDNA3.1能够携带弓形虫抗原基因P30及P30-ROP2在哺乳动物细胞中转录表达。此后,用碱裂解法大量制备质粒pcDNA3.1-P30及pcDNA3.1-P30-ROP2,以肌注方式接种BalB/c小鼠(100μg/只),分别于2周、4周后加强免疫一次,并设立PBS组、pcDNA3.1空载体组为对照,观察分析核酸疫苗所诱导的小鼠的免疫效应以及抵
[Abstract]:With the application and development of molecular biology technology in parasitology research, Toxoplasma gondii vaccine has made great progress from the earliest whole worm vaccine to the newly developed nucleic acid vaccine. Toxoplasma gondii inactivated vaccine has low immune protection; Attenuated live vaccine is easy to restore virulence, can not be used in human body; The production process of subunit vaccine is complicated, and the antigen protein can not form the correct natural conformation. Therefore, the immune activity is insufficient, which makes Toxoplasma nucleic acid vaccine a new research direction. At the same time, more and more evidence shows that. Toxoplasma gondii stage specific antigen molecules can only stimulate the body stage specific protection of immunity. The use of multiple gene vaccines from different stages of polyantigen genes may stimulate a more comprehensive immune protection. As one of the major surface antigens of Toxoplasma gondii Tachyzoites, P30 Sag1 has high immunogenicity and immunogenicity. Toxoplasma gondii (Toxoplasma gondii) is the main candidate antigen for immunological diagnosis and vaccine development of Toxoplasma gondii. It is a protein secreted by Toxoplasma gondii Rhoptry2, which mainly assists in invading host cells of Toxoplasma gondii. Toxoplasma gondii has been expressed in the tachyzoites, bradyzoites and sporozoites. It is highly conservative and immunogenicity, which can stimulate the immune system and induce protective immune response. It has great potential as a vaccine candidate. In this study, the eukaryotic expression plasmid pcDNA3.1-P30 of Toxoplasma gondii was constructed by genetic engineering. Human cervical cancer cell line Hela was transfected with the eukaryotic expression plasmid of pcDNA3.1-P30-ROP2 gene. After 48 hours of transfection, the total RNAs were extracted by Trizol, and the first strand of cDNA was generated by reverse transcription with Oligo(dT)18 as the primer. The housekeeper gene 尾 -actin was reverse transcripted by PCR. The P30 P30-ROP2 gene fragment was then analyzed by PCR. It was confirmed that the eukaryotic expression vector pcDNA3.1 could carry Toxoplasma gondii antigen gene P30 and P30-ROP2 into mammalian cells for transcriptional expression. The plasmids pcDNA3.1-P30 and pcDNA3.1-P30-ROP2were prepared by alkaline lysis method and inoculated intramuscularly into BalB/c mice (100 渭 g / mouse). The immune response of mice induced by nucleic acid vaccine was observed and compared with that of PBS group with pcDNA3.1 empty vector as control group. The immune response of mice induced by nucleic acid vaccine was observed and analyzed.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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