TgMIC 5和16基因序列分析及其免疫保护性研究

发布时间：2018-02-10 03:36

本文关键词： 弓形虫微线体蛋白5 微线体蛋白16 序列分析 DNA疫苗　出处：《安徽农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：弓形虫作为一种呈世界性分布的专性细胞内寄生的顶覆门原虫,严重威胁着人类的健康,影响畜牧业的发展。孕妇感染弓形虫,会造成流产、产死胎、胎儿畸形。在畜牧行业,尤其是对山羊、绵羊以及猪等家畜,弓形虫感染会导致家畜的流产、产死胎、木乃伊胎等,给畜牧行业带来巨大的经济损失。本研究首次研究不同宿主、地理来源以及基因型的刚地弓形虫虫株的TgMIC5和TgMIC16基因的序列变异情况,并验证其是否能够作为刚地弓形虫种群分析和基因分型的遗传标记。序列分析揭示,TgMIC5基因序列的遗传变异率仅为0.2-1.3%,无法有效区分不同基因型的弓形虫虫株,不能作为基因分型的标记分子用于弓形虫种群遗传学研究;TgMIC16基因序列的遗传变异率为0.1-0.9%,系统发育树分析表明TgMIC16基因能够区分不同基因型弓形虫,而PCR-RFLP方法进一步验证了TgMIC16基因能够区分不同基因型弓形虫。因此,TgMIC16基因是一个潜在的用于种群研究和基因分型的标记基因。本研究还对TgMIC5、TgMIC16以及TgGRA16单独和混合免疫的保护性进行了评价。结果表明:免疫组小鼠血清中的抗体IgG以及抗体亚类IgG1和IgG2a的含量均有明显的升高;CD3+CD4+CD8-辅助性T细胞以及CD3+CD8+CD4-T细胞的含量明显增高。说明pVAX-MIC5和pVAX-MIC16能诱导小鼠机体产生高水平的Th 1型细胞免疫和体液免疫应答反应。且经弓形虫RH株致死性和PRU株慢性攻击感染后,单基因免疫组和联合免疫组小鼠都能有效延长小鼠存活时间(空白对照组:4.1±0.31d、p VAX-MIC5:8.8±0.55d、pVAX-MIC16:7.8±0.33d、pVAX-MIC5+MIC16:10±0.63d、pVAX-MIC5+MIC16+GRA16:12.5±1.06d),减少脑包囊荷虫率(pVAX-MIC5:28.00%、pVAX-MIC16:39.44%、pVAX-MIC5+MIC16:48.06%、pVAX-MIC5+MIC16+GRA16:58.44%),且联合免疫的免疫效果显著高于单基因免疫。本研究说明这两个抗原具有良好的免疫原性,能诱导小鼠产生高水平的免疫应答以抵抗弓形虫的感染,且二者的联合适合于混合疫苗的研究。
[Abstract]:Toxoplasma gondii, as a worldwide specialized cell parasitic protozoa, is a serious threat to human health and affects the development of animal husbandry. Pregnant women infected with Toxoplasma gondii can cause miscarriage, stillbirth and fetal malformation. Especially for domestic animals such as goats, sheep and pigs, Toxoplasma gondii infection can lead to abortion, stillbirth, mummy fetus and so on. Sequence variations of TgMIC5 and TgMIC16 genes of Toxoplasma gondii strains from geographic sources and genotypes, The genetic variation rate of TgMIC5 gene was only 0.2-1.3, which could not be used to distinguish Toxoplasma gondii strains with different genotypes, the results showed that TgMIC5 gene could not be used as a genetic marker for Toxoplasma gondii population analysis and genotyping, and the genetic variation rate of TgMIC5 gene sequence was only 0.2-1.3. Genetic study of Toxoplasma gondii TgMIC16 gene sequence genetic variation rate of TgMIC16 gene was 0.1-0.90.The phylogenetic tree analysis showed that TgMIC16 gene could distinguish different genotypes of Toxoplasma gondii. The PCR-RFLP method further verified that the TgMIC16 gene can distinguish different genotypes of Toxoplasma gondii. Therefore, the TgMIC16 gene is a potential marker gene for population research and genotyping. TgMIC5, TgMIC16 and TgGRA16 are also immunized separately and mixed. The results showed that the levels of antibody IgG, antibody subclass IgG1 and IgG2a in serum of immunized mice were significantly increased, and the contents of CD3 CD4 CD8-helper T cells and CD3 CD8 CD4-T cells were significantly increased. The results showed that pVAX-MIC5 and pVAX-MIC16 could induce high level of Th 1 cellular and humoral immune responses in mice, and were infected by Toxoplasma gondii RH strain and PRU strain by chronic attack. The survival time of mice in single gene immunization group and combined immunization group was significantly prolonged (blank control group: 4.1 卤0.31 d VAX-MIC5:8.8 卤0.55 d pVAX-MIC167.8 卤0.33 d pVAX-MIC5 MIC16:10 卤0.63 d pVAX-MIC5 MIC16 GRA16:12.5 卤1.06 d), and the rate of pVAX-MIC5: 28.00% pVAX-MIC5: pVAX-MIC1639.44 DX mice was significantly higher than that of pVAX-MICX-5 MIC16 GRA1658.440.The immune effect of the combined immunization was significantly higher than that of the single gene immunized mice with pVAX-MIC5, pVAX-MIC5, pVAX-MIC5, pVAX-MIC5, pVAX-MIC5, pVAX-MIC5, pVAX-MIC5, pVAX-MIC5, pVAX-MIC5, pVAX-MIC5, pVAX-MIC5 and pVAX-MIC5. Studies have shown that these two antigens have good immunogenicity. It can induce a high level of immune response to Toxoplasma gondii infection in mice, and the combination of the two is suitable for the study of mixed vaccine.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.4

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