摘要
目的分析牛支原体(Mycoplasma bovis)核糖磷酸焦磷酸激酶(ribose-phosphate pyrophosphokinase, prsA)对宿主细胞的黏附功能。方法参照牛支原体HB0801株prsA基因序列设计特异性引物,PCR扩增获得牛支原体武威分离株prsA基因,构建原核表达载体pET-prsA,转化入大肠埃希菌Rosetta(DE3)感受态细胞后加入IPTG诱导表达His-prsA。表达产物纯化后免疫新西兰兔,制备抗血清,应用Western blot和间接ELISA分析prsA蛋白在牛支原体细胞中的分布;通过抗体依赖补体杀菌试验分析多抗血清介导的补体杀支原体活性;利用黏附及黏附抑制试验分析prsA蛋白的宿主细胞黏附功能。结果 SDS-PAGE显示,pET-prsA转染DE3细胞表达的重组蛋白His-prsA分子质量单位约36 ku,且主要以可溶性形式表达;间接ELISA检测重组His-prsA免疫兔血清抗体效价为1∶24 000;Western blot和ELISA结果表明,prsA蛋白存在于牛支原体细胞膜和细胞浆,且细胞浆中较多;抗体依赖的补体杀菌试验结果表明,prsA抗血清具有杀牛支原体活性;黏附及黏附抑制试验证实prsA蛋白与牛支原体黏附宿主细胞有关。结论牛支原体prsA蛋白具有良好的免疫原性和宿主细胞黏附功能,为深入研究牛支原体的致病机制奠定了基础。
Objective To analyze the adhesion of Mycoplasma bovis ribose-phosphate pyrophosphokinase(prsA) to host cells. Methods In accordance with the prsA gene sequence of the HB0801 strain of M. bovis in GenBank(AFM51575.1), a pair of specific primers was designed and the prsA gene of the Wuwei strain of M. bovis was amplified using PCR. The prokaryotic expression vector pET-prsA was constructed and transformed into E.coli Rosetta(DE3) competent cells. Then, expression of the recombinant protein His-prsA was induced with isopropyl β-D-1-thiogalactopyranoside(IPTG), and the expressed product was purified. The purified protein was used to immunize New Zealand rabbits to prepare anti-His-prsA serum. The distribution of prsA protein in M. bovis cells was then analyzed using Western blotting and indirect ELISA. Complement-mediated mycoplasmicidal activity of anti-prsA serum was analyzed using a bactericidal assay, adhesion of the prsA protein was verified using an adhesion inhibition assay. Results SDS-PAGE indicated that the recombinant protein was successfully expressed in a soluble form, and its relative molecular mass was about 36 ku. Indirect ELISA indicated that the titer of the polyclonal antibody with respect to His-prsA was 1:24 000. Western blotting and ELISA indicated that the prsA protein was located in both the cytomembrane and cytoplasm of M. bovis, but it was more prevalent in the cytoplasm. The bactericidal assay indicated that anti-His-prsA serum had potent mycoplasmicidal activity, with mycoplasmicidal action of 42.7%. An adhesion inhibition assay indicated that adhesion of M. bovis to host cells was significantly inhibited by anti-His-prsA rabbit serum, and the rate of inhibition was as high as 54.2%. This confirmed that the prsA protein was related to the adhesion of M. bovis to host cells. Conclusion The prsA protein of M. bovis has excellent immunogenicity and adhesion. The current results have laid the foundation for further research on the pathogenesis of M. bovis.
作者
张阳阳
邢小勇
武小椿
贺健
张生英
刘佳
温峰琴
潘阳阳
包世俊
余四九
ZHANG Yang-yang;XING Xiao-yong;WU Xiao-chun;HE Jian;ZHANG Sheng-ying;LIU Jia;WEN Feng-qin;PAN Yang-yang;BAO Shi-jun;YU Si-jiu(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2021年第7期747-752,共6页
Journal of Pathogen Biology
基金
甘肃省教育厅产业支撑引导项目(No.2019C-03)。
关键词
牛支原体
磷酸核糖焦磷酸激酶
黏附
Mycoplasma bovis
ribose-phosphate pyrophosphokinase
adhesion
