本文選題：遼寧絨山羊 + 副結(jié)核分支桿菌��； 參考：《錦州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的副結(jié)核分支桿菌(Mycobacterium paratuberculosis)引起的羊副結(jié)核病給遼寧省遼寧絨山羊飼養(yǎng)業(yè)造成的危害日趨明顯,臨床病例不斷增多,一些優(yōu)質(zhì)母羊遭到淘汰,目前對(duì)副結(jié)核病的早期診斷容易誤診和漏診,研究出快速鑒別副結(jié)核分支桿菌的方法對(duì)羊副結(jié)核病的早期診斷以及治療具有重要意義。本研究采用分離培養(yǎng)和PCR檢測(cè)方法對(duì)疑似感染羊副結(jié)核分支桿菌病料樣本進(jìn)行鑒定得到副結(jié)核分支桿菌,并初步探究副結(jié)核分支桿菌MAP0862基因的反應(yīng)原性,為進(jìn)一步研究副結(jié)核分支桿菌的亞單位疫苗的開(kāi)發(fā)奠定了試驗(yàn)基礎(chǔ)。方法1、副結(jié)核分支桿菌的分離鑒定本研究對(duì)采自遼寧省某養(yǎng)殖場(chǎng)飼養(yǎng)遼寧絨山羊疑似感染羊副結(jié)核分支桿菌病料樣本,通過(guò)細(xì)菌的分離培養(yǎng)、PCR檢測(cè)等方法進(jìn)行鑒定。2、遼寧東南部地區(qū)羊副結(jié)核病血清學(xué)調(diào)查采用ELISA檢測(cè)方法對(duì)遼寧東南部地區(qū)的遼寧絨山羊開(kāi)展了副結(jié)核病血清學(xué)調(diào)查工作。3、副結(jié)核分支桿菌MAP0862基因的克隆與表達(dá)針對(duì)羊副結(jié)核分支桿菌主要抗原MAP0862基因設(shè)計(jì)利用DNAstar軟件設(shè)計(jì)一對(duì)特異性引物,引物兩端加入Bam HI和Hind III酶切位點(diǎn)。以分離的羊副結(jié)核分支桿菌DNA為模板進(jìn)行PCR擴(kuò)增,并對(duì)其產(chǎn)物進(jìn)行克隆,轉(zhuǎn)化,獲得的T-MAP0862質(zhì)粒,雙酶切后用T4連接酶與表達(dá)載體連接,構(gòu)建出原核表達(dá)載體PET-28a-MAP0862并轉(zhuǎn)化到表達(dá)菌BL21(DE3)感受態(tài)細(xì)胞中,經(jīng)IPTG誘導(dǎo)表達(dá)4小時(shí)后,經(jīng)SDS-PAGE電泳分析蛋白表達(dá)情況,用KCl法純化蛋白并用Westernblot檢測(cè)蛋白的免疫原活性。結(jié)果1、遼寧絨山羊副結(jié)核分支桿菌分離鑒定細(xì)菌分離培養(yǎng)所分離出的菌株為抗酸染色陽(yáng)性桿菌,參照MAPISMav2的因序列,利用Oligo6.0軟件對(duì)全基因組序列進(jìn)行比對(duì)分析,針對(duì)全基因組序列保守區(qū)域,設(shè)計(jì)一對(duì)特異性引物,通過(guò)PCR方法擴(kuò)增出來(lái)的目的片段大小為246bp,與預(yù)期的目的片段大小基本一致。鑒定結(jié)果表明從疑似羊副結(jié)核病羊所分離菌株為羊副結(jié)核桿菌。2、遼寧東南部地區(qū)羊副結(jié)核病血清學(xué)調(diào)查羊副結(jié)核病血清學(xué)調(diào)查結(jié)果表明,遼寧東南部地區(qū)絨山羊副結(jié)核病平均血清陽(yáng)性率為12.54%;其中3個(gè)規(guī)�；B(yǎng)殖場(chǎng)的平均血清陽(yáng)性率為16.06%,最高陽(yáng)性率為18.4%,最低為11.82%;5個(gè)散養(yǎng)戶的平均血清陽(yáng)性率為7.41%,最高陽(yáng)性率為10.71%,最低為4.44%。3、副結(jié)核分支桿菌MAP0862基因克隆表達(dá)(1)采用PCR方法擴(kuò)增MAP0862基因,所得條帶大小為1080bp,與預(yù)期大小基本一致,并成功構(gòu)建了PET-28a-MAP0862表達(dá)載體。(2)經(jīng)IPTG誘導(dǎo),SDS-PAGE電泳在38k Da的位置出現(xiàn)清晰的目的蛋白帶,結(jié)果表明重組基因獲得有效表達(dá),純化后的蛋白僅有唯一的目的蛋白帶。(3)用羊副結(jié)核陽(yáng)性血清進(jìn)行Westernblot檢測(cè),證明MAP0862重組蛋白具有良好的反應(yīng)原性。為進(jìn)一步研究MAP0862作為研制疫苗和檢測(cè)方法建立奠定了基礎(chǔ)。結(jié)論1、成功從患病羊群中分離培養(yǎng)出羊副結(jié)核分支桿菌,通過(guò)提取DNA鑒定該分離菌株為副結(jié)核分支桿菌。2、遼寧東南部地區(qū)羊副結(jié)核病血清學(xué)調(diào)查結(jié)果,3個(gè)規(guī)模化養(yǎng)殖場(chǎng)絨山羊的平均陽(yáng)性率16.06%,5個(gè)散養(yǎng)戶的平均陽(yáng)性率為8.64%,表明該地區(qū)規(guī)�；B(yǎng)殖場(chǎng)副結(jié)核病的感染率要明顯高于散養(yǎng)絨山羊。3、成功構(gòu)建了PET-28a-MAP0862重組表達(dá)載體,用表達(dá)菌BL21(DE3)表達(dá)獲得38k Da(含標(biāo)簽)目的蛋白,經(jīng)Western-blot檢測(cè)該蛋白具有較好反應(yīng)原性。
[Abstract]:Objective the harm of sheep by Mycobacterium tuberculosis (Mycobacterium paratuberculosis) to the cashmere goat breeding industry in Liaoning of Liaoning is becoming more and more obvious, the clinical cases are increasing, some high quality ewes are eliminated, and the early diagnosis of the secondary tuberculosis is easy to be misdiagnosed and missed. The method of bacilli is of great significance to the early diagnosis and treatment of sheep accessory tuberculosis. This study uses the method of isolation and culture and PCR detection to identify Mycobacterium subtubercular Mycobacterium tuberculosis samples from suspected infected sheep, and explore the preliminary study on the reactivity of MAP0862 gene of Mycobacterium subtuberculosis. The development of subunit vaccine of Mycobacterium subtubercular Bacillus laid the experimental basis. Method 1, the isolation and identification of Mycobacterium subtubercular Mycobacterium tuberculosis in a culture farm of Liaoning Province, Liaoning cashmere goat suspected to be infected with Mycobacterium tumefaciens samples, identified by isolation and culture of bacteria, PCR detection and other methods to identify.2, Southeast Liaoning The serological investigation of the local sheep accessory tuberculosis (ELISA) was used to carry out a serological investigation of the Liaoning cashmere goats in the southeast of Liaoning. The cloning and expression of the MAP0862 gene of the Mycobacterium accessory tuberculosis (MTB) was designed to design a pair of specificity against the MAP0862 gene design of the main antigen of the Mycobacterium tumefaciens using DNAstar software. Primers, both ends of the primers were added to the Bam HI and Hind III enzyme cutting sites. The isolated Mycobacterium tuberculosis DNA was used as a template for PCR amplification, and the product was cloned and transformed, and the T-MAP0862 plasmid was obtained. After double enzyme digestion, the T4 ligase was connected with the expression vector, and the prokaryotic expression vector was constructed and converted to BL21 (DE3) of the expression bacteria. After 4 hours of IPTG induced expression, the protein expression was analyzed by SDS-PAGE electrophoresis, the protein was purified by KCl method and the immunogenicity of the protein was detected by Westernblot. Results 1, the isolated strains isolated from Liaoning cashmere goat were isolated and identified as acid resistant positive bacilli, referring to MAPISMav2 The whole genome sequence was compared and analyzed by Oligo6.0 software. A pair of specific primers were designed for the conservative region of the whole genome sequence. The size of the target fragment amplified by the PCR method was 246bp, which was basically the same as the expected target fragment size. The identification results showed that the isolated strains isolated from the suspected sheep sub tuberculosis sheep were isolated. The serological survey of the serological investigation of sheep accessory tuberculosis in southeastern Liaoning showed that the positive rate of serological positive rate of cashmere goat accessory tuberculosis in southeastern Liaoning was 12.54%, of which the average positive rate of 3 scale farms was 16.06%, the highest positive rate was 18.4%, the lowest was 11.82%, and the lowest was 11.82%, 5. The average positive rate was 7.41%, the highest positive rate was 7.41%, the highest positive rate was 10.71%, the lowest was 4.44%.3. The clone expression of MAP0862 gene of Mycobacterium accessory tuberculosis (1) amplified MAP0862 gene by PCR method. The band size was 1080bp, and the PET-28a-MAP0862 expression vector was basically consistent with the expected size. (2) SDS-PAGE electricity was induced by IPTG and SDS-PAGE electricity. A clear target protein band in the position of 38K Da showed that the recombinant gene was effectively expressed and the purified protein was only the only target protein band. (3) the positive serum of the positive serum of sheep accessory tuberculosis was detected by Westernblot, which proved that the recombinant protein of MAP0862 has a good reactivity. It is a further study of MAP0862 as a vaccine and a vaccine. The foundation of the detection method was established. Conclusion 1, the Mycobacterium tuberculosis was successfully isolated and cultured from the sick sheep. Through the extraction of DNA, the isolated strain was identified as.2 of Mycobacterium accessory tuberculosis, the serological investigation of sheep accessory tuberculosis in southeastern Liaoning, the average positive rate of 3 large-scale farm Cashmere Goats was 16.06%, and 5 scattered households. The average positive rate was 8.64%, indicating that the infection rate of the sub tuberculosis in the large-scale breeding farm in this area was obviously higher than that of the scattered cashmere goat.3. The recombinant expression vector of PET-28a-MAP0862 was successfully constructed and the expression of 38K Da (labeled) target protein was obtained by the expression of BL21 (DE3), and the protein had good reactivity by Western-blot.
【學(xué)位授予單位】：錦州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.618

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王和;結(jié)核分支桿菌L型[J];中華結(jié)核和呼吸雜志;2002年10期

2 阮萍;結(jié)核分支桿菌研究進(jìn)展[J];紹興文理學(xué)院學(xué)報(bào)(自然科學(xué)版);2002年08期

3 吳守芝,宋俊峰;結(jié)核分支桿菌致病機(jī)制與免疫[J];中華結(jié)核和呼吸雜志;2003年02期

4 匡紅;孫薏;張聰;府偉靈;陳慶海;馮懷志;李碩;李玉紅;;結(jié)核分支桿菌擴(kuò)增體系的建立、優(yōu)化及評(píng)價(jià)[J];四川醫(yī)學(xué);2009年05期

5 胡忠義;我國(guó)結(jié)核分支桿菌L型實(shí)驗(yàn)研究現(xiàn)狀與亟待解決的問(wèn)題[J];中華結(jié)核和呼吸雜志;2002年10期

6 馬鴻達(dá),朱鈞,張乃鑫,趙天如,王欣,劉淑萍;結(jié)核分支桿菌DNA的單管巢式聚合酶鏈反應(yīng)檢測(cè)[J];中華結(jié)核和呼吸雜志;1998年07期

7 汪正清;結(jié)核分支桿菌抗拒巨噬細(xì)胞吞噬及在胞內(nèi)的生存機(jī)制[J];中華結(jié)核和呼吸雜志;2001年06期

8 孟祥紅;匡鐵吉;董梅;雷紅;梁艷;何菊芳;佟愛(ài)華;肖漓;;結(jié)核分支桿菌依賴鏈霉素的實(shí)驗(yàn)觀察[J];軍醫(yī)進(jìn)修學(xué)院學(xué)報(bào);2006年03期

9 吳雪瓊,張俊仙,莊玉輝,張曉剛,李國(guó)利,何秀云,張軍芝,賈樹(shù)林;結(jié)核分支桿菌利福平耐藥基因突變的研究[J];中華結(jié)核和呼吸雜志;1998年06期

10 代雪玲;務(wù)孔興;魯玲;吳宇明;;結(jié)核分支桿菌特異蛋白抗原研究進(jìn)展[J];兵團(tuán)醫(yī)學(xué);2010年01期

相關(guān)會(huì)議論文 前10條

1 糜祖煌;秦玲;;結(jié)核分支桿菌三種不同靶基因傳統(tǒng)PCR、巢式PCR檢測(cè)比較研究[A];第五屆全國(guó)優(yōu)生科學(xué)大會(huì)論文匯編[C];2000年

2 糜祖煌;秦玲;;結(jié)核分支桿菌三種不同靶基因傳統(tǒng)PCR巢式PCR檢測(cè)比較研究(摘要)[A];中華醫(yī)學(xué)會(huì)第六屆全國(guó)結(jié)核病學(xué)術(shù)大會(huì)論文匯編[C];2000年

3 李洪敏;徐明;王濤;安慧茹;劉真;趙玉梅;;建立長(zhǎng)期保存結(jié)核分支桿菌的方法[A];中國(guó)防癆雜志2003第25卷增刊——2003年中國(guó)防癆協(xié)會(huì)全國(guó)學(xué)術(shù)會(huì)議論文集[C];2003年

4 張永勝;李書琳;李昕;張小剛;何秀云;;結(jié)核分支桿菌耐異煙肼分子機(jī)制的研究[A];中國(guó)防癆協(xié)會(huì)全國(guó)學(xué)術(shù)會(huì)議大會(huì)學(xué)術(shù)報(bào)告[C];2001年

5 梁建琴;吳雪瓊;李洪敏;張俊仙;張靈霞;;結(jié)核分支桿菌耐乙胺丁醇分子機(jī)制的研究[A];中國(guó)防癆協(xié)會(huì)全國(guó)學(xué)術(shù)會(huì)議大會(huì)學(xué)術(shù)報(bào)告[C];2001年

6 李衛(wèi)民;劉忠全;裴秀英;錢明;王蘇民;趙冰;端木宏謹(jǐn);;北京、廣東、寧夏結(jié)核分支桿菌DNA指紋的應(yīng)用研究[A];新世紀(jì)預(yù)防醫(yī)學(xué)面臨的挑戰(zhàn)——中華預(yù)防醫(yī)學(xué)會(huì)首屆學(xué)術(shù)年會(huì)論文摘要集[C];2002年

7 矯慶輝;韓中波;李月龍;;對(duì)幾種結(jié)核分支桿菌檢測(cè)方法的初步探討[A];中國(guó)防癆雜志2003第25卷增刊——2003年中國(guó)防癆協(xié)會(huì)全國(guó)學(xué)術(shù)會(huì)議論文集[C];2003年

8 熊志紅;莊玉輝;李國(guó)利;;利用抑制消減雜交技術(shù)研究結(jié)核分支桿菌強(qiáng)毒株和弱毒株的基因差異[A];中國(guó)的遺傳學(xué)研究——中國(guó)遺傳學(xué)會(huì)第七次代表大會(huì)暨學(xué)術(shù)討論會(huì)論文摘要匯編[C];2003年

9 陳子芳;勞海黎;李永德;;痰多管接種培養(yǎng)結(jié)核分支桿菌陽(yáng)性率的實(shí)驗(yàn)觀察[A];結(jié)核與肺部疾病論文集[C];2006年

10 郭振興;;100例結(jié)核分支桿菌陽(yáng)性涂片再觀察[A];中國(guó)防癆協(xié)會(huì)全國(guó)學(xué)術(shù)會(huì)議大會(huì)學(xué)術(shù)報(bào)告[C];2001年

相關(guān)博士學(xué)位論文 前1條

1 黃海榮;結(jié)核分支桿菌利福平敏感性分析的研究[D];北京市結(jié)核病胸部腫瘤研究所;2001年

相關(guān)碩士學(xué)位論文 前10條

1 孫曉可;羊副結(jié)核分支桿菌分離鑒定及MAP0862基因克隆表達(dá)[D];錦州醫(yī)科大學(xué);2017年

2 李洋;聚合酶鏈反應(yīng)—單鏈構(gòu)象多態(tài)性技術(shù)鑒別結(jié)核分支桿菌異煙肼耐藥基因[D];吉林大學(xué);2005年

3 安慧茹;結(jié)核分支桿菌耐喹諾酮類藥物的分子機(jī)制及其耐藥基因檢測(cè)方法的研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2004年

4 程曉東;multi-PCR-SSCP檢測(cè)耐異煙肼結(jié)核分支桿菌方法建立和初步臨床應(yīng)用[D];中國(guó)人民解放軍第四軍醫(yī)大學(xué);2003年

5 溫書香;PMA-qPCR技術(shù)用于結(jié)核分支桿菌耐藥性快速檢測(cè)的初步研究[D];石河子大學(xué);2014年

6 吳靜希;結(jié)核分支桿菌PE/PPE特異性基因的克隆表達(dá)及初步應(yīng)用[D];西北農(nóng)林科技大學(xué);2011年

7 陳效友;TaqMan聚合酶鏈反應(yīng)技術(shù)檢測(cè)結(jié)核分支桿菌DNA及其臨床應(yīng)用[D];北京市結(jié)核病胸部腫瘤研究所;2000年

8 劉艷群;結(jié)核分支桿菌H37Rv與H37Ra的差異表達(dá)基因研究[D];重慶醫(yī)科大學(xué);2010年

9 馮雪鳴;結(jié)核分支桿菌H_（37）R_v多肽抗原的提純及臨床應(yīng)用價(jià)值的初步研究[D];浙江大學(xué);2005年

10 崔艷麗;煙臺(tái)地區(qū)結(jié)核分支桿菌rpoB、KatG和rpsL三種耐藥基因的檢測(cè)與耐藥相關(guān)性的研究[D];青島大學(xué);2006年

，

本文編號(hào)：2037838