本文選題：細(xì)粒棘球蚴 + r　； 參考：《寧夏醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:通過細(xì)粒棘球絳蟲重組蛋白P29(r Eg.P29)對(duì)綿羊免疫保護(hù)作用及其免疫機(jī)制的研究,為包蟲病的免疫預(yù)防及疫苗的研制奠定理論基礎(chǔ),并積累有用的資料。方法:利用基因工程重組技術(shù)制備的細(xì)粒棘球絳蟲重組蛋白P29免疫綿羊(免疫2次),并在第二次免疫后用制備好的的細(xì)粒棘球絳蟲的蟲卵感染動(dòng)物,通過與對(duì)照組的比較來觀察和計(jì)算重組蛋白是否能夠誘導(dǎo)動(dòng)物產(chǎn)生對(duì)病原體的免疫保護(hù)力(檢測(cè)并統(tǒng)計(jì)動(dòng)物肝臟包囊荷數(shù),用減囊率公式計(jì)算出重組蛋白所引起的免疫保護(hù)力);同時(shí),在重組蛋白免疫和蟲卵感染動(dòng)物前后,分別采集各時(shí)間段的動(dòng)物血液、收集血清,用ELISA法檢測(cè)動(dòng)物血清的抗體(Ig G、Ig A、Ig M、Ig G1、Ig G2)變化水平及細(xì)胞因子(IL-2、IL-4、IL-10、IFN-γ)的變化水平,以此來觀察重組蛋白免疫及蟲卵感染動(dòng)物前后所引的體液免疫和細(xì)胞免疫變化水平。結(jié)果:1.r Eg.P29免疫綿羊獲得了94.8%的免疫保護(hù)力。2.r Eg.P29在誘導(dǎo)綿羊產(chǎn)生免疫保護(hù)力的同時(shí),引起動(dòng)物體液免疫指標(biāo)的變化:r Eg.P29免疫動(dòng)物2周后,引起總Ig G、Ig G1、Ig G2水平的增高,與免疫前和對(duì)照組的抗體水平相比,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。在蟲卵感染后,免疫組Ig G、Ig G1、Ig G2始終保持較高的水平,與對(duì)照組相比具有明顯的統(tǒng)計(jì)學(xué)差異(P0.01)。Ig M的水平在免疫組的早期即出現(xiàn)明顯的上升期,隨后下降,在蟲卵感染后也出現(xiàn)了一個(gè)明顯的上升期。Ig A在實(shí)驗(yàn)組中免疫前、后相比,有一定的上升,上升的速度不及Ig G、Ig G1、Ig G2,但上升水平與免疫前比較,差異有統(tǒng)計(jì)學(xué)意義(P0.01),在蟲卵感染后,與對(duì)照組相比較,Ig A有了明顯的上升(P0.01),并能持續(xù)較長(zhǎng)時(shí)間。r Eg.P29免疫及蟲卵感染動(dòng)物后Ig G、Ig G1、Ig G2能長(zhǎng)期保持較高的水平,提示重組蛋白能通過誘導(dǎo)動(dòng)物產(chǎn)生高水平的Ig G來抵抗病原體的感染。Ig M在重組蛋白免疫的早期及蟲卵感染的早期出現(xiàn)一個(gè)明顯的升高,隨后水平下降,說明其具有早期體液免疫效應(yīng)。Ig A在免疫后能夠出現(xiàn)增高,但不如Ig G水平變化顯著,在蟲卵感染后,其水平與對(duì)照組相比有了明顯的上升。3.r Eg.P29在誘導(dǎo)綿羊產(chǎn)生免疫保護(hù)力的同時(shí),引起動(dòng)物發(fā)生細(xì)胞免疫指標(biāo)的變化:在免疫組中,感染前、后相比較,IFN-γ水平顯著升高(P0.05),IL-2、IL-4、IL-10水平變化不顯著。免疫組與對(duì)照組相比,感染前、后的IFN-γ、IL-2水平均有顯著的升高(P0.01),IL-4只在感染前較對(duì)照組有升高(P0.01),感染后與對(duì)照組相比無明顯差異,IL-10感染前、后差異均無統(tǒng)計(jì)學(xué)意義。而在對(duì)照組中,感染前、后相比,IL-4水平上升顯著(P0.05),IFN-γ、IL-2及IL-10在感染前后的變化不明顯。由于IL-2、IFN-γ主要是由Th1型細(xì)胞所分泌,IL-4、IL-10主要是由Th2型細(xì)胞所分泌。r Eg.P29免疫動(dòng)物組(免疫組)與對(duì)照相比,免疫組能夠明顯的引起IFN-γ和IL-2水平的增高,且在蟲卵感染后IFN-γ水平進(jìn)一步增高,提示r Eg.P29能介導(dǎo)Th1型細(xì)胞為主的細(xì)胞免疫應(yīng)答。對(duì)照組在動(dòng)物感染前后IFN-γ和IL-2和IL-10的變化不明顯,但I(xiàn)L-4在感染后水平有明顯的升高,提示未獲得免疫保護(hù)力的對(duì)照組可能介導(dǎo)的是以Th2型細(xì)胞為主的細(xì)胞免疫應(yīng)答。結(jié)論:1.r Eg.P29免疫綿羊獲得了94.8%的免疫保護(hù)力,是較好的抗包蟲病的疫苗候選分子。2.r Eg.P29免疫綿羊后引起以Ig G、Ig G1、Ig G2、Ig A和Ig M水平增高為主的體液免疫應(yīng)答。3.r Eg.P29免疫綿羊引起以IL-2與IFN-γ水平增高為主的Th1型細(xì)胞免疫應(yīng)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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R532.32

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉晶,張國慶,鄭世民;寄生蟲疫苗的研究進(jìn)展[J];遼寧畜牧獸醫(yī);2003年06期

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本文編號(hào)：2106590