新疆南疆地区奶牛乳房炎性表皮葡萄球菌aap基因的原核表达及功能研究

发布时间：2018-05-04 14:27

本文选题：奶牛乳房炎 + 表皮葡萄球菌　；参考：《塔里木大学》2015年硕士论文

【摘要】：表皮葡萄球菌（Staphylococcus epidermidis）是一种引起奶牛隐性乳房炎的重要病原菌，其致病性主要是通过形成生物膜（Biofilm Formation，，BF）。目前发现在表皮葡萄球菌生物膜形成的过程中主要依赖胞外多糖黏附因子（polysaccharide intercellular adhesion PIA）、胞外DNA（extracellular DNA，eDNA）和聚集相关蛋白（accumulation-associated protein，Aap）等。其中Aap蛋白是表皮葡萄球菌细胞壁表面重要的蛋白黏附分子，约90%的表皮葡萄球菌携带aap基因，其表达与生物膜形成有关，因此aap基因可以作为研究表皮葡萄球菌生物膜形成的重要靶点，这将为奶牛乳房炎病的预防控制和进一步研究有效的治疗方案提供理论指导。首先，以危害新疆奶牛养殖业健康发展的表皮葡萄球菌为研究对象，对其进行aap特征性基因片段的PCR扩增和生物膜形成能力检测。aap特征性基因片段预期大小为486bp，经PCR扩增，共检测得到15株表皮葡萄球菌含有aap基因，占所检菌株的26.3%。采用微量板半定量法对分离获得的表皮葡萄球菌的生物膜形成表型进行检测，共得到10株生物膜形成阳性菌株，占所检菌株的18.2%，在这10株生物膜阳性菌株中50%为aap阳性（aap+）菌株，且生物膜形成能力普遍高于aap阴性（aap-）菌株。在上述实验的基础上筛选出生物膜阳性、aap阳性（BF+，aap+）和生物膜阳性、aap阴性（BF+，aap-）菌株，同时针对与生物膜形成有关的上述三种主要成分对（BF+，aap+）和（BF+，aap-）菌株生物膜形成依赖型进行检测，结果表明本地区分离得到的表皮葡萄球菌其生物膜构成包含蛋白质、多糖及胞外DNA，且三者对表皮葡萄球菌生物膜形成的抑制率分别达99.6%、82.6%和99.7%。说明蛋白质、胞外DNA和胞外多糖均是本地区奶牛乳房炎性表皮葡萄球菌生物膜构成中的重要组成部分，因此本研究以本地区临床表皮葡萄球菌生物膜中的三大成分为研究对象进行了较为深入的研究和探索。其次，为进一步验证aap基因的功能，对aap基因进行原核表达载体的构建并对其进行诱导表达：通过PCR扩增表皮葡萄球菌临床分离株aap基因，应用基因重组技术构建原核表达载体pET32a(+)-aap，转化宿主菌DH5a后再转化E. coli BL21，并进行重组蛋白的诱导表达。结果表明，当0.75mmol/L IPTG诱导4h时，便可得到最高表达量的重组蛋白。重组蛋白分子量约为180KDa，与预期大小一致。利用镍离子亲和层析法对Aap蛋白进行纯化。结果发现，当洗杂缓冲液中咪唑浓度为40mM、洗脱缓冲液中咪唑浓度为200mM时可以获得单一的目的蛋白。为进一步验证所获得蛋白的准确性，对其进行了Western-blot检测，因为在aap基因上下游分别引入了载体上特有的组氨酸标签，在180KDa左右，Aap蛋白能有效地与组氨酸标签抗体结合，证明了该蛋白的准确性。最后，使用微量板半定量法检测了Aap蛋白对表皮葡萄球菌生物膜形成的影响，并根据生物膜的三大主要成分分别检测了Aap蛋白对生物膜形成的影响机制。实验证实Aap蛋白对标准菌株和临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开始发挥其抑制作用，当Aap蛋白浓度分别为1.0μg/mL及0.4μg/mL时即可以完全抑制住ATCC35984及临床株生物膜的形成。为了进一步摸清Aap蛋白抑制生物膜形成的机制，选择ATCC35984作为研究对象分别进行了以下实验：通过Aap蛋白处理ATCC35984基因组DNA实验得知Aap蛋白对ATCC35984生物膜的抑制不是通过降解eDNA而实现的；通过TLC分析其全细胞多糖和胞外多糖组分，得知Aap蛋白对其多糖分泌种类没有影响；对Aap蛋白进行荧光标记，并在显微镜下观察，发现标记的Aap蛋白确实与细胞接触，并将其包裹住，从而使其呈现荧光绿色，经流式细胞仪统计发现随着Aap蛋白浓度的增加，荧光强度增加，被FITC着染的阳性细胞数逐渐增多。当Aap蛋白浓度为1.0μg/mL时ATCC35984细胞着色率比对照组增加了95.91%，由此可推测，Aap蛋白可以通过对ATCC35984的包裹而起到抑制其生物膜形成的作用。综上所述，Aap蛋白对生物膜具有很强的抑制作用，因此aap基因是一个潜在的制备抑制奶牛乳房炎性表皮葡萄球菌生物膜感染药剂的候选基因，可为奶牛乳房炎病的生防治疗和进一步研究提供帮助，这也为进一步研究aap基因的功能奠定了一定的理论基础。
[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.

【学位授予单位】：塔里木大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【参考文献】