本文選題：牛結(jié)核分枝桿菌 + 非結(jié)核分枝桿菌�。� 參考：《中國農(nóng)業(yè)科學(xué)院》2012年碩士論文

【摘要】：牛結(jié)核�。˙ovine Tuberculosis）是由牛結(jié)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(yán)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(jié)核分枝桿菌(Nontuberculous Mycobacteriua, NTM)，這些NTM引起的疾病也都呈現(xiàn)結(jié)核樣癥狀，對結(jié)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(jié)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(jié)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(yīng)，對牛結(jié)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(jié)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(gòu)建了五種分枝桿菌的原核和真核表達重組質(zhì)粒。誘導(dǎo)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(gòu)建的五種真核表達重組質(zhì)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(yīng)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(jié)果表明，，五種分枝桿菌Ag85b蛋白之間均存在血清學(xué)交叉反應(yīng)，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(dǎo)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(yán)重的交叉反應(yīng)，這將會給Ag85b作為牛結(jié)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(jié)核病新型疫苗的組分，應(yīng)該加上可以鑒別疫苗免疫與自然感染的標(biāo)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(jié)核分枝桿菌和非結(jié)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(jié)核病疫苗的免疫應(yīng)答的同時對NTM的感染提供免疫交叉保護效應(yīng)。
[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(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

【參考文獻】

相關(guān)期刊論文 前10條

1 薛瑩,李元,史皆然,師長宏,李別虎,范雄林,柏銀蘭,馬文煜;結(jié)核分枝桿菌分泌蛋白Ag85B的免疫學(xué)特性[J];第四軍醫(yī)大學(xué)學(xué)報;2002年13期

2 楊恩明;譚建明;楊順良;;Notch信號通路與免疫系統(tǒng)[J];華夏醫(yī)學(xué);2006年03期

3 王洪生,吳勤學(xué);非結(jié)核分枝桿菌感染與艾滋病[J];國外醫(yī)學(xué).皮膚性病學(xué)分冊;2005年03期

4 劉思國,王春來,張秀華,郭設(shè)平,郭洋,邵美麗;牛結(jié)核病病原生態(tài)學(xué)和流行病學(xué)研究[J];中國畜牧獸醫(yī);2005年03期

5 黃其文 ,黃重敏 ,覃后繼 ,龍顯科 ,何延專 ,覃亞勤 ,周耀南 ,陸春雷 ,徐湘;新型液體培養(yǎng)基用于結(jié)核分枝桿菌快速培養(yǎng)的研究[J];廣西醫(yī)學(xué);2005年06期

6 江山;;結(jié)核分枝桿菌潛伏性感染發(fā)生機制的研究進展[J];國外醫(yī)學(xué)(微生物學(xué)分冊);2003年01期

7 王巍;;非結(jié)核分枝桿菌病流行病學(xué)診斷治療近況[J];結(jié)核病健康教育;2008年02期

8 張萬江;鄧喜玲;吳芳;黃春;曹旭東;吳江東;杜燕;李蕾;;結(jié)核桿菌Ag85B基因疫苗的免疫保護效果研究[J];生命科學(xué)研究;2006年02期

9 徐廣賢;趙德明;;牛分支桿菌與肺結(jié)核分支桿菌基因組的比較[J];現(xiàn)代生物醫(yī)學(xué)進展;2006年07期

10 吳位珩;孫啟躍;徐景峨;楊莉;廖梅;史開志;楊茂生;;應(yīng)用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)技術(shù)檢測牛結(jié)核病[J];上海畜牧獸醫(yī)通訊;2012年01期

相關(guān)碩士學(xué)位論文 前2條

1 杜艷芬;牛結(jié)核病病原學(xué)調(diào)查及分子流行病學(xué)研究[D];中國農(nóng)業(yè)科學(xué)院;2009年

2 亓英芳;兩種基因型結(jié)核分枝桿菌的比較蛋白組學(xué)分析[D];中國農(nóng)業(yè)科學(xué)院;2010年

本文編號：1876592