细粒棘球绦虫重组蛋白P29诱导绵羊的免疫保护力及其免疫机制研究

发布时间：2018-07-07 23:12

本文选题：细粒棘球蚴 + r　；参考：《宁夏医科大学》2015年硕士论文

【摘要】：目的:通过细粒棘球绦虫重组蛋白P29(r Eg.P29)对绵羊免疫保护作用及其免疫机制的研究,为包虫病的免疫预防及疫苗的研制奠定理论基础,并积累有用的资料。方法:利用基因工程重组技术制备的细粒棘球绦虫重组蛋白P29免疫绵羊(免疫2次),并在第二次免疫后用制备好的的细粒棘球绦虫的虫卵感染动物,通过与对照组的比较来观察和计算重组蛋白是否能够诱导动物产生对病原体的免疫保护力(检测并统计动物肝脏包囊荷数,用减囊率公式计算出重组蛋白所引起的免疫保护力);同时,在重组蛋白免疫和虫卵感染动物前后,分别采集各时间段的动物血液、收集血清,用ELISA法检测动物血清的抗体(Ig G、Ig A、Ig M、Ig G1、Ig G2)变化水平及细胞因子(IL-2、IL-4、IL-10、IFN-γ)的变化水平,以此来观察重组蛋白免疫及虫卵感染动物前后所引的体液免疫和细胞免疫变化水平。结果:1.r Eg.P29免疫绵羊获得了94.8%的免疫保护力。2.r Eg.P29在诱导绵羊产生免疫保护力的同时,引起动物体液免疫指标的变化:r Eg.P29免疫动物2周后,引起总Ig G、Ig G1、Ig G2水平的增高,与免疫前和对照组的抗体水平相比,差异具有统计学意义(P0.01)。在虫卵感染后,免疫组Ig G、Ig G1、Ig G2始终保持较高的水平,与对照组相比具有明显的统计学差异(P0.01)。Ig M的水平在免疫组的早期即出现明显的上升期,随后下降,在虫卵感染后也出现了一个明显的上升期。Ig A在实验组中免疫前、后相比,有一定的上升,上升的速度不及Ig G、Ig G1、Ig G2,但上升水平与免疫前比较,差异有统计学意义(P0.01),在虫卵感染后,与对照组相比较,Ig A有了明显的上升(P0.01),并能持续较长时间。r Eg.P29免疫及虫卵感染动物后Ig G、Ig G1、Ig G2能长期保持较高的水平,提示重组蛋白能通过诱导动物产生高水平的Ig G来抵抗病原体的感染。Ig M在重组蛋白免疫的早期及虫卵感染的早期出现一个明显的升高,随后水平下降,说明其具有早期体液免疫效应。Ig A在免疫后能够出现增高,但不如Ig G水平变化显著,在虫卵感染后,其水平与对照组相比有了明显的上升。3.r Eg.P29在诱导绵羊产生免疫保护力的同时,引起动物发生细胞免疫指标的变化:在免疫组中,感染前、后相比较,IFN-γ水平显著升高(P0.05),IL-2、IL-4、IL-10水平变化不显著。免疫组与对照组相比,感染前、后的IFN-γ、IL-2水平均有显著的升高(P0.01),IL-4只在感染前较对照组有升高(P0.01),感染后与对照组相比无明显差异,IL-10感染前、后差异均无统计学意义。而在对照组中,感染前、后相比,IL-4水平上升显著(P0.05),IFN-γ、IL-2及IL-10在感染前后的变化不明显。由于IL-2、IFN-γ主要是由Th1型细胞所分泌,IL-4、IL-10主要是由Th2型细胞所分泌。r Eg.P29免疫动物组(免疫组)与对照相比,免疫组能够明显的引起IFN-γ和IL-2水平的增高,且在虫卵感染后IFN-γ水平进一步增高,提示r Eg.P29能介导Th1型细胞为主的细胞免疫应答。对照组在动物感染前后IFN-γ和IL-2和IL-10的变化不明显,但IL-4在感染后水平有明显的升高,提示未获得免疫保护力的对照组可能介导的是以Th2型细胞为主的细胞免疫应答。结论:1.r Eg.P29免疫绵羊获得了94.8%的免疫保护力,是较好的抗包虫病的疫苗候选分子。2.r Eg.P29免疫绵羊后引起以Ig G、Ig G1、Ig G2、Ig A和Ig M水平增高为主的体液免疫应答。3.r Eg.P29免疫绵羊引起以IL-2与IFN-γ水平增高为主的Th1型细胞免疫应答。
[Abstract]:Aim: To study the immune protection and immune mechanism of Echinococcus granulosus recombinant protein P29 (R Eg.P29), and to lay a theoretical foundation for the immunization prevention and vaccine development of echinococcosis, and accumulate useful information. Method: the recombinant protein P29 of Echinococcus granulosus was prepared by gene engineering recombinant technology to immunize sheep (2 After second times of immunization, the eggs infected with Echinococcus granulosus were prepared. By comparing with the control group, it was observed and calculated whether the recombinant protein could induce the immune protection of the animal to the pathogen (detection and statistics of the number of hepatic cysts in animal liver, and the expression of the recombinant protein was calculated by the formula of reducing the cyst rate. " At the same time, before and after the immunization of the recombinant protein and the infected animals, the blood of animals in each time period was collected, the serum was collected, and the change level of the antibody of animal serum (Ig G, Ig A, Ig M, Ig G1, Ig G2) and the change level of the cytokine (IL-2, Ig G2) were detected by ELISA to observe the immunization of the recombinant protein and the eggs. Results: 1.r Eg.P29 immune sheep obtained 94.8% immune protective power.2.r Eg.P29, which induced the immune protective ability of sheep, and caused the changes of animal humoral immunity index. After 2 weeks, R Eg.P29 immune animals caused the increase of total Ig G, Ig G1, Ig G2 level. The difference in the level of antibody before and in the control group was statistically significant (P0.01). After the infection of the eggs, the immune group Ig G, Ig G1, Ig G2 kept a high level, and there was a significant difference between the control group and the control group (P0.01) the level of.Ig M in the early stage of the immune group, then decreased, after the infection of the eggs. There was also an obvious rise period of.Ig A before immunization in the experimental group, and then compared to a certain increase, the rising speed was less than Ig G, Ig G1, Ig G2, but the rise level was statistically significant (P0.01) compared with the pre immunization (P0.01). Compared with the control group, the Ig A had a significant rise (P0.01) and could continue to continue for a long time after the infection of the eggs. Ig G, Ig G1, Ig G2 can maintain a high level for a long time after Eg.P29 immunization and egg infection, suggesting that the recombinant protein can resist pathogen infection by inducing a high level of Ig G in animals..Ig M in the early stage of recombinant protein immunization and early infection of eggs appears an obvious increase, then the level decline, indicating that it has early stage. The humoral immune effect.Ig A can increase after immunization, but not as significant as Ig G. After the infection of the eggs, the level of.3.r Eg.P29 is significantly higher than that of the control group, and.3.r Eg.P29 can induce the immune protection of the sheep and cause the changes of the immune index of the animals: in the immune group, before infection, and in the posterior comparison, IFN- The level of gamma (P0.05), IL-2, IL-4 and IL-10 had no significant changes. Compared with the control group, the level of IFN- and IL-2 increased significantly before infection (P0.01), and IL-4 only increased before infection (P0.01) before infection (P0.01), and there was no significant difference between the control group and the control group. The difference was not statistically significant before the infection, and there was no significant difference between the IFN- and the control group. In the control group, the level of IL-4 increased significantly before and after infection (P0.05), and the changes of IFN- gamma, IL-2 and IL-10 were not obvious before and after infection. As IL-2, IFN- gamma was mainly secreted by Th1 type cells, IL-4, IL-10 was mainly secreted by Th2 type cells. And the level of IL-2 increased, and the level of IFN- gamma increased further after the infection of the eggs, suggesting that R Eg.P29 could mediate the cellular immune response of Th1 cells. The changes of IFN- gamma and IL-2 and IL-10 in the control group were not obvious before and after the infection, but the level of IL-4 at the post infection level was obviously elevated, suggesting that the control group that had not acquired immune protection might be possible. The immune response was mediated by Th2 type cells. Conclusion: 1.r Eg.P29 immune sheep obtained 94.8% immune protection. It is a good vaccine candidate for anti echinococcosis,.2.r Eg.P29 immune sheep caused by Ig G, Ig G1, Ig G2, Ig, and higher level of immune response. 2 Th1 cell immune response was mainly associated with increased IFN- gamma level.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R532.32

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