弓形虫ADF基因亚单位疫苗与核酸疫苗的实验免疫研究

发布时间：2018-04-10 13:09

本文选题：弓形虫 + 肌动蛋白解聚因子　；参考：《吉林大学》2009年硕士论文

【摘要】： 弓形虫(Toxoplasma gondii)是一种重要的机会性致病原虫,广泛寄生于人体及动物的组织细胞内,引起严重的人兽共患病。据估计全世界1/3的人群感染本病,我国人群的感染率为5%～10%。目前尚未找到理想的防治该病的药物,且由于研制新药成本费用高、药物残留以及减毒疫苗毒力返强等问题的存在,使弓形虫基因工程疫苗的研究成为新的趋势。有学者通过对弓形虫抗原及其诱导的免疫反应进行研究后认为有效的疫苗对该病的防治有一定的作用。为探讨ADF基因对弓形虫感染的免疫保护作用,本研究在已知ADF基因序列的基础上设计引物,扩增ADF开放阅读框,将其克隆入原核表达载体并表达出重组ADF蛋白,并对ADF基因在弓形虫速殖子内的分布进行定位;同时用DNA重组技术将弓形虫ADF基因与真核表达载体pVAX1相连,构建真核表达载体,并将重组真核表达质粒转染Hela细胞,验证其在真核细胞中的表达;将重组ADF蛋白与真核表达质粒分别免疫小鼠检测其免疫保护力。结果显示,扩增出365bp的ADF开放阅读框,经Blast比对显示同源性为98%,并表达出17KDa的重组融合蛋白,应用免疫荧光抗体技术将ADF基因定位于速殖子胞质内及质膜上;用间接免疫荧光方法在重组质粒转染后的Hela细胞中检测到特异蛋白,通过Western blot试验证实所得蛋白具有反应原性;重组ADF蛋白免疫保护结果显示,随着免疫次数的增加抗体滴度逐渐增加,试验组与对照组相比差异极显著(P0.01)。CD4+与CD8+T淋巴细胞数与对照组相比差异显著(P0.01),CD4+/CD8+ T淋巴细胞数的值差异不显著(P0.05)。试验小鼠均未能抵抗弓形虫强毒株攻虫感染,但试验组生存时间延长,与对照组差异不显著(P0.05),脑组织中包囊数减少,减虫率达30%;核酸疫苗免疫保护结果表明,试验组与对照组相比,特异性抗体滴度、CD4+与CD8+T淋巴细胞数均差异极显著(P0.01),CD4+/CD8+ T淋巴细胞数的值差异不显著(P0.05)。攻虫后小鼠生存时间得到延长,与PBS对照组差异显著(P0.05),与pVAX1对照组差异不显著(P0.05),包囊减少可达到42.8%。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is an important opportunistic protozoa, widely parasitic in human and animal tissue cells, causing serious zoonosis.It is estimated that a third of the world's population is infected with the disease.At present, no ideal drug has been found to prevent and cure the disease. Due to the high cost of new drugs, the existence of drug residues and the strong virulence of attenuated vaccine, the research of Toxoplasma gondii genetic engineering vaccine has become a new trend.Through the study of Toxoplasma gondii antigen and its induced immune response, some scholars think that the effective vaccine has certain effect on the prevention and treatment of the disease.In order to investigate the immune protection of ADF gene against Toxoplasma gondii infection, primer was designed based on known ADF gene sequence, ADF open reading frame was amplified, cloned into prokaryotic expression vector and expressed recombinant ADF protein.The distribution of ADF gene in Toxoplasma gondii tachyzoites was located, and the ADF gene of Toxoplasma gondii was linked to the eukaryotic expression vector pVAX1 by DNA recombination technique, and the eukaryotic expression vector was constructed, and the recombinant eukaryotic expression plasmid was transfected into Hela cells.The recombinant ADF protein and the eukaryotic expression plasmid were immunized to test the immune protection of mice.The results showed that the ADF open reading frame of 365bp was amplified and the homology was 98 by Blast comparison, and the recombinant fusion protein of 17KDa was expressed. The ADF gene was located in the cytoplasm and plasma membrane of tachyzoites by immunofluorescence antibody technique.The specific protein was detected by indirect immunofluorescence assay in Hela cells transfected with recombinant plasmid, and the reactivity of the protein was confirmed by Western blot assay.With the increase of immunization times, the titer of antibody increased gradually, the difference between the test group and the control group was extremely significant (P 0.01). The number of CD8 T lymphocytes was significantly different from that of the control group. There was no significant difference in the number of CD 4 / CD 8 T lymphocytes between the test group and the control group (P 0.05).The experimental mice could not resist the infection of Toxoplasma gondii virulent strain, but the survival time of the test group was prolonged, the difference was not significant compared with the control group (P0.05), the number of the cyst in the brain tissue was decreased, and the worm reduction rate was 30%.Compared with the control group, the specific antibody titer (CD _ 4) and the number of CD8 T lymphocytes were significantly different between the test group and the control group. There was no significant difference in the number of CD _ 4 / CD _ 8 T lymphocytes between the test group and the control group (P 0.05).The survival time of the mice after attack was prolonged, which was significantly different from that of PBS control group (P 0.05) and pVAX1 control group (P 0.05), and the cysts decreased to 42.8%.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【引证文献】