牛源金黄色葡萄球菌FnBPA-A分子不同遗传多态性重组质粒免疫生物学特性研究

发布时间：2019-06-21 17:21

【摘要】：奶牛乳房炎是危害奶牛产业健康发展的重要疾病之一,给奶牛业造成了巨大经济损失。金黄色葡萄球菌是导致奶牛乳房炎重要的致病菌,耐药菌株的出现严重阻碍了该病的治疗。黏附素可在金黄色葡萄球菌早期感染过程中发挥重要作用,FnBPA是该菌重要的黏附素之一。本研究以金黄色葡萄球菌FnBPA-A不同遗传多态性重组质粒为研究对象,旨在阐明FnBPA-A分子遗传多态性与其免疫生物学特性的关系。本研究在对牛乳源金黄色葡萄球菌黏附素FnBPA-A序列分析的基础上,利用构建的FnBPA-A不同遗传多态性重组质粒对小鼠进行免疫,比较分析其免疫生物学特性。检测结果表明,FnBPA-A不同遗传多态性重组质粒免疫小鼠后抗血清对不同金黄色葡萄球菌的识别能力不同。检测结果还显示,FnBPA-A不同遗传多态性重组质粒免疫后的抗血清可对金黄色葡萄球菌粘附纤维蛋白原(Fg)表现显著抑制作用(P0.05),但FnBPA-A不同遗传多态性重组质粒抗血清之间在抗粘附效果方面差异不显著(P0.05),不同重组质粒刺激淋巴细胞增殖能力和IFN-γ水平表现不同程度的升高。不同重组质粒免疫后小鼠免疫保护力不同,并且与细胞免疫水平相关。本研究选用奶牛乳腺组织块接种法与胰蛋白酶消化分离培养及纯化牛乳腺上皮细胞。通过对特异性的角蛋白18进行鉴定,证明纯化的细胞为牛乳腺上皮细胞。并进一步测定得出纯化后的牛乳腺上皮细胞的生长曲线。利用荧光染料CFSE和DiI分别对金黄色葡萄球菌与乳腺上皮细胞进行染色,在激光共聚焦显微镜下观察金黄色葡萄球菌对牛乳腺上皮细胞的粘附。比较4株不同金黄色葡萄球菌分离株对牛乳腺上皮细胞的粘附能力,结果表明不同金黄色葡萄球菌分离株对牛乳腺上皮细胞的粘附能力不同,其中GY278分离株对乳腺上皮细胞的粘附能力最强,并且与其它分离株差异显著(P0.05)。进一步利用培养的牛乳腺上皮细胞检测与分析FnBPA-A不同遗传多态性重组质粒免疫后抗血清对4株不同金黄色葡萄球菌分离株粘附乳腺上皮细胞的影响。结果显示,FnBPA-A不同遗传多态性重组质粒免疫后抗血清能够抑制金黄色葡萄球菌粘附乳腺上皮细胞,不同的FnBPA-A遗传多态性抗血清抑制能力存在差异,同一FnBPA-A遗传多态性抗血清抑制不同金黄色葡萄球菌粘附乳腺上皮细胞差异不显著(P0.05)。本研究为金黄色葡萄球菌免疫学研究提供实验依据。
[Abstract]:Dairy cow mastitis is one of the important diseases which is harmful to the healthy development of dairy cattle industry, and has caused great economic loss to the dairy cattle industry. Staphylococci are the most important pathogenic bacteria in the mastitis of dairy cows, and the emergence of drug-resistant strains has seriously hindered the treatment of the disease. The adhesive can play an important role in the early infection of Staphylococcus aureus, and the FnBPA is one of the important adhesin of the bacterium. The purpose of this study was to study the relationship between the molecular genetic polymorphism of FnBPA-A and its biological characteristics. On the basis of the analysis of the FnBPA-A sequence of the Staphylococcus aureus in the milk source, the mice were immunized with the constructed FnBPA-A different genetic polymorphism recombinant plasmid, and the biological characteristics of the mice were compared. The results showed that the identification ability of the antiserum to different S. aureus was different after the recombinant plasmid of FnBPA-A with different genetic polymorphism. The results also showed that the anti-serum of FnBPA-A with different genetic polymorphisms (FnBPA-A) could significantly inhibit the adhesion of fibrinogen (Fg) to Staphylococcus aureus (P0.05). However, there was no significant difference in the anti-adhesion effect between the recombinant plasmid of FnBPA-A and the recombinant plasmid (P0.05). The immune protective force of the mice after immunization with different recombinant plasmids was different and correlated with the cellular immune level. In this study, bovine mammary gland epithelial cells were isolated and cultured with trypsin digestion. The purified cells are proved to be bovine mammary epithelial cells by the identification of specific keratin 18. And further determining the growth curve of the purified bovine mammary epithelial cell. Staphylococcus aureus and mammary epithelial cells were stained with CFSE and DiI respectively, and the adhesion of Staphylococcus aureus to bovine mammary epithelial cells was observed under a laser confocal microscope. The adhesion ability of 4 strains of different S. aureus isolates to bovine mammary epithelial cells was compared. The results showed that the adhesion of different S. aureus isolates to bovine mammary epithelial cells was different, among which GY278 isolates had the strongest adhesion to the epithelial cells of the breast. And the difference with other isolates was significant (P0.05). The effect of the antiserum on the adhesion of 4 strains of different S. aureus isolates to the mammary epithelial cells was further investigated and analyzed using the cultured bovine mammary epithelial cells. The results showed that the anti-serum of FnBPA-A can inhibit the adhesion of the Staphylococcus aureus to the mammary epithelial cells, and the inhibitory ability of the different FnBPA-A genetic polymorphism antisera is different. The antisera of the same FnBPA-A polymorphism did not significantly inhibit the adhesion of different S. aureus to the epithelial cells (P0.05). This study provides an experimental basis for the immunological study of Staphylococcus aureus.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.611

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