本文選題：奶牛乳房炎 + 表皮葡萄球菌�。� 參考：《塔里木大學(xué)》2015年碩士論文

【摘要】：表皮葡萄球菌（Staphylococcus epidermidis）是一種引起奶牛隱性乳房炎的重要病原菌，其致病性主要是通過形成生物膜（Biofilm Formation，，BF）。目前發(fā)現(xiàn)在表皮葡萄球菌生物膜形成的過程中主要依賴胞外多糖黏附因子（polysaccharide intercellular adhesion PIA）、胞外DNA（extracellular DNA，eDNA）和聚集相關(guān)蛋白（accumulation-associated protein，Aap）等。其中Aap蛋白是表皮葡萄球菌細(xì)胞壁表面重要的蛋白黏附分子，約90%的表皮葡萄球菌攜帶aap基因，其表達(dá)與生物膜形成有關(guān)，因此aap基因可以作為研究表皮葡萄球菌生物膜形成的重要靶點(diǎn)，這將為奶牛乳房炎病的預(yù)防控制和進(jìn)一步研究有效的治療方案提供理論指導(dǎo)。 首先，以危害新疆奶牛養(yǎng)殖業(yè)健康發(fā)展的表皮葡萄球菌為研究對(duì)象，對(duì)其進(jìn)行aap特征性基因片段的PCR擴(kuò)增和生物膜形成能力檢測。aap特征性基因片段預(yù)期大小為486bp，經(jīng)PCR擴(kuò)增，共檢測得到15株表皮葡萄球菌含有aap基因，占所檢菌株的26.3%。采用微量板半定量法對(duì)分離獲得的表皮葡萄球菌的生物膜形成表型進(jìn)行檢測，共得到10株生物膜形成陽性菌株，占所檢菌株的18.2%，在這10株生物膜陽性菌株中50%為aap陽性（aap+）菌株，且生物膜形成能力普遍高于aap陰性（aap-）菌株。在上述實(shí)驗(yàn)的基礎(chǔ)上篩選出生物膜陽性、aap陽性（BF+，aap+）和生物膜陽性、aap陰性（BF+，aap-）菌株，同時(shí)針對(duì)與生物膜形成有關(guān)的上述三種主要成分對(duì)（BF+，aap+）和（BF+，aap-）菌株生物膜形成依賴型進(jìn)行檢測，結(jié)果表明本地區(qū)分離得到的表皮葡萄球菌其生物膜構(gòu)成包含蛋白質(zhì)、多糖及胞外DNA，且三者對(duì)表皮葡萄球菌生物膜形成的抑制率分別達(dá)99.6%、82.6%和99.7%。說明蛋白質(zhì)、胞外DNA和胞外多糖均是本地區(qū)奶牛乳房炎性表皮葡萄球菌生物膜構(gòu)成中的重要組成部分，因此本研究以本地區(qū)臨床表皮葡萄球菌生物膜中的三大成分為研究對(duì)象進(jìn)行了較為深入的研究和探索。 其次，為進(jìn)一步驗(yàn)證aap基因的功能，對(duì)aap基因進(jìn)行原核表達(dá)載體的構(gòu)建并對(duì)其進(jìn)行誘導(dǎo)表達(dá)：通過PCR擴(kuò)增表皮葡萄球菌臨床分離株aap基因，應(yīng)用基因重組技術(shù)構(gòu)建原核表達(dá)載體pET32a(+)-aap，轉(zhuǎn)化宿主菌DH5a后再轉(zhuǎn)化E. coli BL21，并進(jìn)行重組蛋白的誘導(dǎo)表達(dá)。結(jié)果表明，當(dāng)0.75mmol/L IPTG誘導(dǎo)4h時(shí)，便可得到最高表達(dá)量的重組蛋白。重組蛋白分子量約為180KDa，與預(yù)期大小一致。利用鎳離子親和層析法對(duì)Aap蛋白進(jìn)行純化。結(jié)果發(fā)現(xiàn)，當(dāng)洗雜緩沖液中咪唑濃度為40mM、洗脫緩沖液中咪唑濃度為200mM時(shí)可以獲得單一的目的蛋白。為進(jìn)一步驗(yàn)證所獲得蛋白的準(zhǔn)確性，對(duì)其進(jìn)行了Western-blot檢測，因?yàn)樵赼ap基因上下游分別引入了載體上特有的組氨酸標(biāo)簽，在180KDa左右，Aap蛋白能有效地與組氨酸標(biāo)簽抗體結(jié)合，證明了該蛋白的準(zhǔn)確性。 最后，使用微量板半定量法檢測了Aap蛋白對(duì)表皮葡萄球菌生物膜形成的影響，并根據(jù)生物膜的三大主要成分分別檢測了Aap蛋白對(duì)生物膜形成的影響機(jī)制。實(shí)驗(yàn)證實(shí)Aap蛋白對(duì)標(biāo)準(zhǔn)菌株和臨床株生物膜形成均具有很好的抑制作用，而且在初始黏附期就開始發(fā)揮其抑制作用，當(dāng)Aap蛋白濃度分別為1.0μg/mL及0.4μg/mL時(shí)即可以完全抑制住ATCC35984及臨床株生物膜的形成。為了進(jìn)一步摸清Aap蛋白抑制生物膜形成的機(jī)制，選擇ATCC35984作為研究對(duì)象分別進(jìn)行了以下實(shí)驗(yàn)：通過Aap蛋白處理ATCC35984基因組DNA實(shí)驗(yàn)得知Aap蛋白對(duì)ATCC35984生物膜的抑制不是通過降解eDNA而實(shí)現(xiàn)的；通過TLC分析其全細(xì)胞多糖和胞外多糖組分，得知Aap蛋白對(duì)其多糖分泌種類沒有影響；對(duì)Aap蛋白進(jìn)行熒光標(biāo)記，并在顯微鏡下觀察，發(fā)現(xiàn)標(biāo)記的Aap蛋白確實(shí)與細(xì)胞接觸，并將其包裹住，從而使其呈現(xiàn)熒光綠色，經(jīng)流式細(xì)胞儀統(tǒng)計(jì)發(fā)現(xiàn)隨著Aap蛋白濃度的增加，熒光強(qiáng)度增加，被FITC著染的陽性細(xì)胞數(shù)逐漸增多。當(dāng)Aap蛋白濃度為1.0μg/mL時(shí)ATCC35984細(xì)胞著色率比對(duì)照組增加了95.91%，由此可推測，Aap蛋白可以通過對(duì)ATCC35984的包裹而起到抑制其生物膜形成的作用。 綜上所述，Aap蛋白對(duì)生物膜具有很強(qiáng)的抑制作用，因此aap基因是一個(gè)潛在的制備抑制奶牛乳房炎性表皮葡萄球菌生物膜感染藥劑的候選基因，可為奶牛乳房炎病的生防治療和進(jìn)一步研究提供幫助，這也為進(jìn)一步研究aap基因的功能奠定了一定的理論基礎(chǔ)。
[Abstract]:Staphylococcus epidermidis (Staphylococcus epidermidis) is an important pathogen causing recessive mastitis in dairy cows. Its pathogenicity is mainly through the formation of the biofilm (Biofilm Formation, BF). It is found that in the process of the formation of Staphylococcus epidermidis, it is mainly dependent on the extracellular polysaccharide adhesion factor (polysaccharide intercellular adhesio). N PIA), extracellular DNA (extracellular DNA, eDNA) and aggregation related proteins (accumulation-associated protein, Aap), among which Aap protein is an important protein adhesion molecule on the surface of Staphylococcus epidermidis, and about 90% of Staphylococcus epidermidis carries the AAP gene, and its expression is related to the formation of biofilm, so the AAP gene can be used as a study of the epidermis. The important target of Staphylococcus biofilm formation will provide theoretical guidance for the prevention and control of dairy cow mastitis and further study of effective treatment options.
First, in order to study the Staphylococcus epidermidis which endangering the healthy development of Xinjiang dairy farming industry, the PCR amplification and the biofilm formation ability of the AAP characteristic gene fragments are expected to be 486bp. After PCR amplification, 15 strains of Staphylococcus epidermidis were detected to contain AAP gene, which accounted for 26.3 of the tested strains. The microplate semi quantitative method was used to detect the biofilm phenotype of Staphylococcus epidermidis isolated from the isolated Staphylococcus epidermidis, and 10 biofilm positive strains were obtained, which accounted for 18.2% of the detected strains, and 50% of the 10 biofilm positive strains were AAP positive (aap+) strains, and the biofilm formation ability was generally higher than that of AAP negative (aap-) strains. On the basis of the experiment, the biofilm positive, AAP positive (BF+, aap+) and biofilm positive, AAP negative (BF+, aap-) strain were screened, and the dependence of the three main components related to biofilm formation on the biofilm formation of (BF+, aap+) and (BF+, aap-) strains were detected. The results showed that the isolated Staphylococcus epidermidis produced in this area was born. The membrane consists of proteins, polysaccharides and extracellular DNA, and the inhibition rates of the formation of Staphylococcus epidermidis by three are 99.6%, 82.6% and 99.7%., respectively. The extracellular DNA and extracellular polysaccharide are important components in the biofilm formation of the dairy cow mastitis. The three major components of Staphylococcus epidermidis biofilm were studied and explored in depth.
Secondly, in order to further verify the function of the AAP gene, the prokaryotic expression vector of the AAP gene was constructed and induced to be induced: to amplify the AAP gene of the clinical isolates of Staphylococcus epidermidis by PCR, and to construct the prokaryotic expression vector pET32a (+) -aap by gene recombination technology, and then convert the host bacterium DH5a to E. coli BL21, and reorganize it. The results showed that the highest expression of recombinant protein was obtained when 4H was induced by 0.75mmol/L IPTG. The molecular weight of the recombinant protein was about 180KDa, which was in accordance with the expected size. The nickel ion affinity chromatography was used to purify the Aap protein. The results showed that the concentration of imidazole in the detergent buffer was 40mM, and imidazole in the elution buffer solution was found. A single target protein was obtained when the concentration was 200mM. In order to further verify the accuracy of the protein, it was detected by Western-blot, because the specific histidine label on the AAP gene was introduced into the AAP gene, and the protein could be effectively combined with the histidine labeled antibody at about 180KDa. It's true.
Finally, the effect of Aap protein on the biofilm formation of Staphylococcus epidermidis was detected by the semi quantitative microplate method, and the effect mechanism of Aap protein on biofilm formation was detected according to the three major components of the biofilm. The experiment proved that Aap protein had a good inhibitory effect on the formation of the standard strain and the clinical strain of the biofilm. The initial adhesion period began to play its inhibitory effect. When the concentration of Aap protein was 1 mu g/mL and 0.4 micron g/mL, the formation of ATCC35984 and the biofilm of clinical strain could be completely suppressed. In order to further understand the mechanism of Aap protein inhibition of biofilm formation, the following experiments were carried out by selecting ATCC35984 as the research object: through Aap eggs. The white treated ATCC35984 genome DNA experiment showed that the inhibition of the Aap protein to the ATCC35984 biofilm was not achieved by degradation of eDNA; by TLC analysis of the whole cell polysaccharide and the extracellular polysaccharide component, it was found that the Aap protein had no effect on its polysaccharide secretory type; the Aap protein was labeled with fluorescence and observed under the microscope, and found the marked A. The AP protein did contact with the cell and wrapped it into a fluorescent green. The flow cytometry found that with the increase of the concentration of Aap protein, the fluorescence intensity increased and the number of positive cells infected by FITC increased gradually. When the concentration of Aap protein was 1 u g/mL, the coloring rate of ATCC35984 cells increased by 95.91% than that of the control group. It is speculated that Aap protein can inhibit the formation of biofilm by encapsulated ATCC35984.
In conclusion, the Aap protein has a strong inhibitory effect on the biofilm. Therefore, the AAP gene is a potential candidate gene for the preparation of the drug to inhibit the infection of the biofilm of the dairy cow mastitis. It can provide help for the biocontrol and further research of dairy cow mastitis. It also lays the foundation for the further study of the function of the AAP gene. A certain theoretical basis.

【學(xué)位授予單位】：塔里木大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 黃忍;;奶牛乳房炎研究進(jìn)展[J];山東畜牧獸醫(yī);2009年06期

2 寨鴻瑞;田甜;朱偉;王開功;周碧君;文明;;奶牛乳房炎研究進(jìn)展[J];貴州農(nóng)業(yè)科學(xué);2009年08期

3 沈冬梅;郭宇;張秉升;;金黃色葡萄球菌引起的奶牛乳房炎的診治[J];獸醫(yī)導(dǎo)刊;2014年04期

4 劉翠平;葉蕾;吳正鈞;王蔭榆;郭本恒;;干酪乳桿菌LC2W胞外多糖提取工藝研究[J];華北農(nóng)學(xué)報(bào);2008年S2期

5 劉東明;陳建國;胡長敏;丁明星;;奶牛隱性乳房炎的檢測方法[J];今日畜牧獸醫(yī);2009年01期

6 周慶民;馮萬宇;徐馨;侯美如;黃健;;奶牛乳房炎防治現(xiàn)狀[J];今日畜牧獸醫(yī);2014年09期

7 郭抗抗;張為民;張彥明;王晶鈺;李勇;;奶牛隱性乳房炎主要病原菌的分離鑒定及耐藥性測定[J];西北農(nóng)業(yè)學(xué)報(bào);2010年07期

8 任曉鏷;陳偉;張利莉;;不同環(huán)境因素對(duì)表皮葡萄球菌生物膜形成的影響[J];西北農(nóng)業(yè)學(xué)報(bào);2011年12期

9 喻華英;張苗;陳偉;郭亞莉;;阿拉爾地區(qū)某奶牛場隱性乳房炎的病原菌分離鑒定[J];新疆農(nóng)業(yè)科學(xué);2009年04期

10 宋亞攀;楊利國;;中國奶牛乳房炎防治研究進(jìn)展[J];中國奶牛;2010年12期

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