本文選題：牛結核分枝桿菌 + 非結核分枝桿菌��； 參考：《中國農業(yè)科學院》2012年碩士論文

【摘要】：牛結核病（Bovine Tuberculosis）是由牛結核分枝桿菌(Mycobacterium bovis, M.bovis)引起的一種人畜共患的慢性消耗性傳染病，嚴重阻礙畜牧業(yè)的發(fā)展和威脅人類的健康。此外，環(huán)境中還存在許多非結核分枝桿菌(Nontuberculous Mycobacteriua, NTM)，這些NTM引起的疾病也都呈現(xiàn)結核樣癥狀，對結核分枝桿菌的診斷和治療造成一定的影響，Ag85b蛋白是分枝桿菌培養(yǎng)物中的一種分泌蛋白，是結核分枝桿菌的一種重要的保護性抗原。Ag85b蛋白主要存在于牛分枝桿菌（Mycobacterium bovis，M.bovis）、結核分枝桿菌(Mycobacterium tuberculosis，M.tuberculosis)和NTM（Nontuberculous Mycobacterium,NTM）中，所以研究M.bovis與NTM Ag85b蛋白免疫交叉反應，對牛結核病的診斷及防治具有重要的意義。 本研究分別以M.bovis、副結核分枝桿菌（Mycobacterium avium subsp.paratuberculosis，M.paratuberculosis）海分枝桿菌(Mycobacterium marinum, M.marinum)、恥垢分枝桿菌(Mycobacterium smegmatis, M.smegmatis)、鳥分枝桿菌(Mycobacterium avium, M.avium)2型擴增Ag85b基因，分別構建了五種分枝桿菌的原核和真核表達重組質粒。誘導表達五種重組Ag85b（rAg85b）蛋白，用Ni-His-resin純化目的蛋白，Western-blot鑒定后免疫小鼠，制備多克隆抗體。構建的五種真核表達重組質粒分別免疫3月齡豚鼠，分離豚鼠的抗血清，以五種rAg85b蛋白作為包被抗原，間接ELISA檢測五種抗血清之間的交叉反應情況。分離免疫豚鼠的脾淋巴細胞，分別用五種rAg85b蛋白進行刺激，測定淋巴細胞增殖情況及IFN-γ的分泌情況。 本研究獲得了具有較好免疫原性的五種rAg85b蛋白，間接ELISA結果表明，，五種分枝桿菌Ag85b蛋白之間均存在血清學交叉反應，并且每一種分枝桿菌的rAg85b蛋白均能夠刺激其他各實驗組的淋巴細胞增殖和誘導IFN-γ分泌水平的增加，顯示M.paratuberculosis、M.smegmatis、M.marinum、M.bovis、M.avium的Ag85b蛋白之間存在很嚴重的交叉反應，這將會給Ag85b作為牛結核病的候選疫苗的免疫監(jiān)測造成很大的障礙。因此，如果將Ag85b作為牛結核病新型疫苗的組分，應該加上可以鑒別疫苗免疫與自然感染的標記物，這對于疫苗的監(jiān)測及牛群的凈化將具有重要的意義。同時本研究還提示了Ag85b對結核分枝桿菌和非結核分枝桿菌還存在交叉保護作用，使得Ag85b可以作為一種良好的輔助物，在增強牛結核病疫苗的免疫應答的同時對NTM的感染提供免疫交叉保護效應。
[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- 緯 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- 緯 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.
【學位授予單位】：中國農業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392.1

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