牛分枝杆菌与非结核分枝杆菌Ag85b蛋白免疫交叉反应的研究

发布时间：2018-05-12 01:43

本文选题：牛结核分枝杆菌 + 非结核分枝杆菌　；参考：《中国农业科学院》2012年硕士论文

【摘要】：牛结核病（Bovine Tuberculosis）是由牛结核分枝杆菌(Mycobacterium bovis, M.bovis)引起的一种人畜共患的慢性消耗性传染病，严重阻碍畜牧业的发展和威胁人类的健康。此外，环境中还存在许多非结核分枝杆菌(Nontuberculous Mycobacteriua, NTM)，这些NTM引起的疾病也都呈现结核样症状，对结核分枝杆菌的诊断和治疗造成一定的影响，Ag85b蛋白是分枝杆菌培养物中的一种分泌蛋白，是结核分枝杆菌的一种重要的保护性抗原。Ag85b蛋白主要存在于牛分枝杆菌（Mycobacterium bovis，M.bovis）、结核分枝杆菌(Mycobacterium tuberculosis，M.tuberculosis)和NTM（Nontuberculous Mycobacterium,NTM）中，所以研究M.bovis与NTM Ag85b蛋白免疫交叉反应，对牛结核病的诊断及防治具有重要的意义。本研究分别以M.bovis、副结核分枝杆菌（Mycobacterium avium subsp.paratuberculosis，M.paratuberculosis）海分枝杆菌(Mycobacterium marinum, M.marinum)、耻垢分枝杆菌(Mycobacterium smegmatis, M.smegmatis)、鸟分枝杆菌(Mycobacterium avium, M.avium)2型扩增Ag85b基因，分别构建了五种分枝杆菌的原核和真核表达重组质粒。诱导表达五种重组Ag85b（rAg85b）蛋白，用Ni-His-resin纯化目的蛋白，Western-blot鉴定后免疫小鼠，制备多克隆抗体。构建的五种真核表达重组质粒分别免疫3月龄豚鼠，分离豚鼠的抗血清，以五种rAg85b蛋白作为包被抗原，间接ELISA检测五种抗血清之间的交叉反应情况。分离免疫豚鼠的脾淋巴细胞，分别用五种rAg85b蛋白进行刺激，测定淋巴细胞增殖情况及IFN-γ的分泌情况。本研究获得了具有较好免疫原性的五种rAg85b蛋白，间接ELISA结果表明，，五种分枝杆菌Ag85b蛋白之间均存在血清学交叉反应，并且每一种分枝杆菌的rAg85b蛋白均能够刺激其他各实验组的淋巴细胞增殖和诱导IFN-γ分泌水平的增加，显示M.paratuberculosis、M.smegmatis、M.marinum、M.bovis、M.avium的Ag85b蛋白之间存在很严重的交叉反应，这将会给Ag85b作为牛结核病的候选疫苗的免疫监测造成很大的障碍。因此，如果将Ag85b作为牛结核病新型疫苗的组分，应该加上可以鉴别疫苗免疫与自然感染的标记物，这对于疫苗的监测及牛群的净化将具有重要的意义。同时本研究还提示了Ag85b对结核分枝杆菌和非结核分枝杆菌还存在交叉保护作用，使得Ag85b可以作为一种良好的辅助物，在增强牛结核病疫苗的免疫应答的同时对NTM的感染提供免疫交叉保护效应。
[Abstract]:Bovine tuberculosis (TB) is a chronic and expendable infectious disease caused by Mycobacterium bovisis (M. bovisis), which seriously hinders the development of animal husbandry and threatens human health. In addition, there are also many non-tuberculous Mycobacterium mycobacterium, NTMN in the environment, and the diseases caused by these NTM also show tuberculous symptoms. Ag85b protein is a secretory protein in the culture of Mycobacterium tuberculosis. It is an important protective antigen of Mycobacterium tuberculosis. Ag85b protein mainly exists in Mycobacterium bovisus M. bovisus, Mycobacterium tuberculosism (M. tuberculosism) and NTM(Nontuberculous Mycobacterium tuberculiensis (NTM). Therefore, the immune cross reaction between M.bovis and NTM Ag85b protein was studied. It is of great significance for the diagnosis, prevention and treatment of bovine tuberculosis. In this study, the prokaryotic and eukaryotic expression recombinant plasmids of Mycobacterium marinum, M. marinumum, Mycobacterium smeatis, M. smegmatis, Mycobacterium avium, M.avium)2 were constructed by M. bovisis, Mycobacterium avium subsp. paratuberculosism M. paratuberculosism, M. marinumum, M. smegmatisme, Mycobacterium avium, M.avium)2 respectively. Five recombinant Ag85b rAg85b) proteins were induced and purified by Ni-His-resin and identified by Western-blot to immunize mice to prepare polyclonal antibodies. Five recombinant eukaryotic expression plasmids were constructed to immunize 3 month old guinea pigs. The antiserum of guinea pigs was isolated. Five kinds of rAg85b proteins were used as coated antigens. The cross reaction between the five antisera was detected by indirect ELISA. The spleen lymphocytes of guinea pigs were isolated and stimulated with five kinds of rAg85b proteins. The proliferation of lymphocytes and the secretion of IFN- 纬 were measured. In this study, five rAg85b proteins with good immunogenicity were obtained. Indirect ELISA results showed that there were serological cross reactions among the five Ag85b proteins of Mycobacterium. Moreover, the rAg85b protein of each type of Mycobacterium can stimulate the proliferation of lymphocytes and induce the increase of IFN- 纬 secretion in other experimental groups, indicating that there is a very serious cross reaction between the Ag85b protein of M. gmatissimus M. bovisum M. avium. This will pose a major obstacle to the immunological monitoring of Ag85b as a vaccine candidate for bovine tuberculosis. Therefore, if Ag85b is used as a component of new bovine tuberculosis vaccine, it should be added with markers that can distinguish vaccine immunity from natural infection, which will be of great significance for vaccine monitoring and cattle purification. At the same time, this study also indicated that Ag85b has cross-protection effect on Mycobacterium tuberculosis and non-tuberculosis mycobacterium, so that Ag85b can be used as a good adjuvant. In addition to enhancing the immune response of bovine tuberculosis vaccine, the immune cross protection against NTM infection is provided.
【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】：2012
【分类号】：R392.1

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