口蹄疫灭活疫苗免疫猪抗体“灰色区”细胞免疫水平差异分析

发布时间：2018-06-27 00:29

本文选题：口蹄疫病毒 + 灭活疫苗　；参考：《中国农业科学院》2016年硕士论文

【摘要】：有研究发现动物免疫口蹄疫灭活疫苗后,机体内抗口蹄疫病毒抗体水平与攻毒后动物的保护力之间存在密切联系。根据攻毒前机体内抗体水平与攻毒后动物保护力之间的关系可将抗口蹄疫病毒抗体水平进行分类,分别为:抗体白色区,即完全保护区,体内抗体水平很高,动物感染口蹄疫病毒后处于完全保护状态;黑色区,即完全不保护区,体内抗体水平很低,动物感染口蹄疫病毒后全部发病;灰色区,即部分保护区,体内抗体水平处于白色区和黑色区之间,动物感染口蹄疫病毒后,有些动物处于保护状态,而有些动物会发病。在灰色区内还会出现抗体水平高的动物会发病,而抗体水平较低的动物得到保护的现象。本研究的主要目的是探究抗体灰色区内动物保护力不同的机制。为探索口蹄疫免疫抗体灰色区形成的机制,本实验选取33头健康长白猪免疫自行制备的O型口蹄疫灭活疫苗,于免疫后28天进行同源强毒攻毒,随后连续观察十天确定动物发病情况。于免疫前及免疫后第28天采集血液样品进行试验,使用液相阻断ELISA测定血清中抗体水平,流式细胞术测定外周血中淋巴细胞亚群含量,抗体芯片技术测定血清中细胞因子含量。根据免疫后第28天时血清中抗体水平与攻毒后动物发病情况的关系,确定免疫抗体灰色区的范围。结果表明猪免疫口蹄疫灭活疫苗后,灰色区抗体效价的范围为1:6-1:45,灰色区动物在免疫前后CTL细胞含量及TNF-α、IFN-γ、IL-12、IL-15、IL-18、MIG、GM-CSF、IL-1α、TGF-α、TWEAK-R等细胞因子的含量均发生变化。灰色区内保护组动物免疫后CTL细胞含量与免疫前相比出现升高,而发病动物则出现显著性降低。对于IFN-γ、IL-12、GM-CSF、IL-1α、IL-15和TNF-α这几种细胞因子,其含量在灰色区保护组动物中免疫后与免疫前相比出现升高,而发病动物则没有发生变化或出现不同程度的降低。MIG和TWEAK R在保护组动物中免疫前后没有明显变化,但是发病动物其含量出现下降。而对于IL-18和TGF-α,保护组动物在免疫后其含量低于免疫前,不保护组动物IL-18的含量在免疫后反而出现上升,TGF-α的含量没有明显变化。实验结果显示,负责细胞免疫应答的CTL细胞及TNF-α、IFN-γ、IL-12和IL-15等Th1型细胞因子能够提高灰色区内动物抗口蹄疫病毒感染的能力,由此可得出免疫后诱导的细胞免疫应答强弱与口蹄疫抗体灰色区内动物保护力的高低有关。同时选用另外33头健康长白猪免疫口蹄疫灭活疫苗,观察整个免疫阶段外周血淋巴细胞亚群动态变化。分别于免疫前,免疫后第1、3、5、7、10、14、21、35天采集抗凝血,进行流式细胞术实验检测血液中细胞亚群的分布情况,同时测定血清中抗体水平。结果显示,在整个免疫期Th细胞含量变化与B细胞含量变化存在相关性,同时也与抗体水平的变化相关,这表明了Th细胞与B细胞之间存在相互作用的关系,共同介导体液免疫应答。DCs含量在免疫后快速升高,说明其在短期内即可被活化发挥功能。结果表明,疫苗免疫后DCs能快速发挥功能,使固有免疫应答朝着适应性免疫应答的方向发展。B细胞和Th细胞含量的升高说明体液免疫应答被激活,产生的高水平和高度特异性的抗体可对机体起到保护作用,同时CTL细胞含量也出现升高,说明细胞免疫应答在该过程中也发挥了至关重要的作用。
[Abstract]:There is a close relationship between the level of anti FMDV antibody and the protection ability of the animals after inoculation of the inactivated vaccine against FMDV. The anti body level of FMD virus can be classified according to the relationship between the level of antibody in the body of the virus and the protective ability of the animal after the attack, which is the white area of the antibody, respectively. In the complete protection area, the level of antibody in the body is very high, the animal infected with FMDV is completely protected, the black area, that is, the completely non protected area, the level of antibody in the body is very low, the animal infected with the foot and mouth disease virus, and the grey area, that is, part of the protective area, the body anti body level is between the white area and the black area, and the animal infect the mouth of the mouth. After pestilence virus, some animals are in protective condition, and some animals will occur. In the gray area, there will be a high level of antibody in animals, and the animals with low antibody level are protected. The main purpose of this study is to explore the mechanism of different animal protection ability in the gray area of the antibody. The mechanism of color region formation was selected by 33 healthy long white pigs immunized with O inactivated vaccine of foot and mouth disease (FMDV). After 28 days of immunization, it was observed for ten days for ten days. The blood samples were collected before and twenty-eighth days after immunization, and the serum resistance was measured by liquid phase blocking ELISA. The content of lymphocyte subsets in peripheral blood was measured by flow cytometry, and the content of cytokines in serum was determined by antibody chip technique. The range of grey area of immune antibody was determined according to the relationship between the serum antibody level and the incidence of attack after twenty-eighth days after immunization. The range of antibody titer was 1:6-1:45, and the content of CTL cells in the gray area and the content of TNF- alpha, IFN- gamma, IL-12, IL-15, IL-18, MIG, GM-CSF, IL-1 a, TGF- alpha, TWEAK-R and other cytokines were all changed. For IFN- gamma, IL-12, GM-CSF, IL-1 a, IL-15 and TNF- alpha, the content of these cytokines increased in the grey area protection group after immunization, but the incidence of.MIG and TWEAK R in the animals did not change significantly, but there was no obvious change in the immune system in the animals of the protective group. The content of the sick animals decreased. For IL-18 and TGF- alpha, the contents of the animals in the protection group were lower than those before the immune system. The content of IL-18 in the non protective group rose but the content of TGF- alpha did not change obviously. The experimental results showed that the CTL cells and TNF- alpha, IFN- gamma, IL-12 and IL-15, which were responsible for the cellular immune response. Cytokines can improve the ability of animals to resist foot and mouth disease virus infection in the gray area, thus it can be concluded that the immune response induced by immune response is related to the level of animal protection in the grey area of FMD antibody. At the same time, 33 other healthy long white pigs immunized foot-and-mouth vaccine was selected to observe the peripheral blood lymph nodes in the whole immune phase. Dynamic changes in cell subgroups. Anticoagulants were collected on day 1,3,5,7,10,14,21,35 before immunization, and the distribution of cell subgroups in blood was detected by flow cytometry and serum antibody levels were measured at the same time by flow cytometry. The results showed that there was a correlation between the changes of Th cells in the whole immune period and the changes of B cells in the whole immune period, and also in the same time. The change of antibody level is associated with the interaction between Th cells and B cells. The.DCs content of the common dielectric liquid immune response is rapidly increased after immunization, indicating that it can be activated in the short term. The result shows that the immune response of the vaccine to the immune response is fast and that the intrinsic immune response will be adapted to the adaptive immune response. The increase in the content of.B and Th cells indicates that the humoral immune response is activated. The high level and highly specific antibodies can protect the body, and the content of CTL cells also increases, indicating that the cellular immune response also plays a vital role in this process.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

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