【作者】 杨婷婷；

【导师】 何深一；

【作者基本信息】 山东大学 ， 病原生物学， 2005， 硕士

【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更多还原

【Abstract】 Toxoplasma gondii is the aetiological agent of toxoplasmosis and is the most frequent and best known of the parasitic diseases. Recently, the development of vaccines against toxoplasmosis has progressed considerably due to the application of the technology of molecular biology.Research showed that the inactivated vaccine was insufficient to induce proper immune responses. A vaccine based on the live attenuated S48 strain was developed for veterinary uses^[1]. However, it is not suitable for human use for the possibility of reverting to a pathogenic strain. Subunit vaccine using nature or recombinant parasite antigens is expensive, difficult to purified and has a short shelf life therefore it is not appropriate for wide-ranging application. Under the present scenario, development of DNA vaccine against toxoplasmosis is an attractive alternative.P30, or SAG1, is the main surface antigen of the Toxoplasma tachyzoite covered 3 to 5 percent of the gross proteins of T. gondii. Being highly immunogenic and immuno-protective, it was considered as an important candidate for effective diagnostic reagents and vaccine antigen^[4,5]. More recent reports showed that ROP2, a p54 kDa protein component of the rhoptries expressed in three stages of T. gondii life cycle ,participated in the parasite invasion into host cells by mediating host organelle association with the parasitophorous vacuole membrane （PVM）, and had also been proposed as a vaccine candidate against toxoplasmosis.Meanwhile, accumulating evidence indicates that vaccination with stage-specific antigens leads to stage-limited protection12’31. Based on this background, we studied the efficacy of immunization with P30-R0P2-encoding compound DNA vaccine and induced resistance against challenge with the virulent RH T. gondii strain, at the same time compared with those of a single gene vaccine encoding P30.In this study, gene engineering technology was exploited to construct the monogene eukaryotic expression plasmid pcDNA3. 1-P30. Then it was transiently introduced into Hela cells by liposomes with the recombinant eukaryotic expression plasmid pcDNA3. 1-P3O-ROP2 respectively. The total RNA of the cell culture was extracted by use of Trizol 48 hs after transfection. The eukaryotic expression vector pcDNA3. 1 was verified to have the capability to transcript and translate the P30 and the P30-R0P2 genes in mammal cells through RT-PCR methods. House-keeping gene P — actin was used as an inner control. After that abundant plasmids pcDNA3. 1-P30 and pcDNA3. 1-P3O-ROP2 were extracted by the means of alkaline lysis. BALB/c mice were intramuscularly immunized with the compound DNA vaccine encoding P30 and ROP2 genes （100 y g per animal） or equal dose of monovalent DNA vaccine encoding P30 single gene. Two booster injections were carried out at 2-week intervals. As negative controls, the empty vector pcDNA3.1 and PBS buffer were injected in the other two groups respectively. Mice were bled at day 13 （a day before the second dose）, day 27（a day before the last dose）, day 42（14 days after the last dose） and day 56（28 days after the last dose）. All sera was analysed individually. Challenge or removal of spleens took place at day 56 unless otherwise stated. Each mice inoculated with DNA constructions or PBSbuffer were challenged intraperitoneally with 105 tachyzoite forms of T. gondii RH strain. The proliferation activity of T cells and the activity of NK cells were determined by means of MTT assay. The total IgG and cytokines of IFN-y^ IL-4 were determined by ELISA. Flow cytometry was applied to detect the subclones of T cells. Results: Both immunized groups （group injected with pcDNA3.1-P30 or pcDNA3. 1-P30-ROP2） gained the highest IgG at day 56, while a more transparent production of antigen-specific IgG was raised in the compound vaccination group compared to the single vaccination one （P<0. 01） . The pcDNA3.1-P30-ROP2 group raised higher IFN-y （P<0. 01） , CD8+ T cell proportion （P<0.01） . The proliferation activity of T cells of group pcDNA3.1-P30-ROP2 increased evidently （P<0. 01） .while neither group showed significant NKC killing activity. No IL-4 was detected in all the groups. Although no mice survived a lethal challenge with highly virulent Toxoplasma gondii RH strain, compound DNA vaccination induced a long-lasting protection.Our results showed that DNA vaccination could induced specific immune response against T. gondii, including the specific antibody and a protective Thl response, and activated the non-specific immune system such as the NK cells as well. Compound DNA vaccine of genes from different livestages of T. gondii elicit better immunoprotectivity in mice than vaccine of a single gene.更多还原