一种新的牛支原体膜蛋白p59的表达与鉴定  

Identification and expression of Mycoplasma bovis P59 as a new membrane protein

作　　者：蒋仁新 王基隆 朱曼玲[1,2] 程凯慧[2] 解晓莉 杨宏军[2] 丁家波[3] 刘磊[1] 张亮[2] JIANG Ren-xin;WANG Ji-long;ZHU Man-ling;CHEN Kai-hui;XIE Xiao-li;DING Jia-bo;YANG Hong-jun;LIU Lei;ZHANG Liang(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou,China 730070;Bovine Research Center,Shandong Academy of Agricultural Sciences;China Institute of Veterinary Drug Control)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]山东省农业科学院奶牛研究中心 [3]中国兽医药品监察所

出　　处：《中国病原生物学杂志》2019年第11期1261-1267,1272,共8页Journal of Pathogen Biology

基　　金：十三五国家重点研发计划项目(No.2017YFD0500904,2016YFD0500902,2016YFD0500904);现代农业(奶牛)产业技术体系科学家岗位项目(No.CARS-36);山东省农业科学院农业科技创新工程项目(No.CXGC2016A10)

摘　　要：目的寻找新的牛支原体(Mycoplasma bovis)抗原蛋白并进行原核表达,为牛支原体疫苗的制备奠定基础。方法采用生物信息学方法分析牛支原体PG45株基因组,发现一种新的膜蛋白p59,并对其理化性质、蛋白定位、是否存在信号肽及其互作蛋白进行分析预测。通过PCR扩增p59蛋白编码基因,经酶切连接表达载体pET-30a(+)后转化入大肠埃希菌BL21(DE3)感受态细胞,利用IPTG进行诱导表达并进行优化,利用不同浓度咪唑洗脱液来洗脱蛋白。通过Western blot进行鉴定。制备p59重组蛋白兔多抗,通过粘附试验和支原体生长抑制试验验证该重组蛋白的功能。结果生物信息学分析该蛋白可能为ABC转运系统的组成蛋白,其互作蛋白为糖、微量元素摄取的ABC转运系统相关蛋白,可能参与牛支原体摄入营养物质的过程。PCR扩增p59基因,双酶切及测序鉴定pET-30a(+)-p59原核表达载体构建正确,SDS-PAGE分析重组载体转化BL21表达p59重组蛋白,相对分子质量为66×10^3。Western blot鉴定该蛋白为膜蛋白,免疫新西兰兔可刺激产生特异性抗体,即具有抗原性。p59蛋白粘附MDBK细胞试验显示,用p59兔多抗作为一抗时的MDBK(Madin-Darby bovinekidney,牛肾细胞)细胞表面能够粘附更多的P59蛋白,表明p59重组蛋白具有一定的粘附能力。生长抑制试验表明,p59兔多抗能抑制牛支原体在固体培养基中的生长,生长抑制率为45.53%。结论新鉴定的牛支原体膜蛋白p59可能是一种粘附相关蛋白,且具有抗原性,该蛋白多抗血清能抑制支原体的生长,为研究支原体膜蛋白的功能奠定了基础,为牛支原体亚单位疫苗的研制提供了新的靶蛋白。Objective To find a new Mycoplasma bovis antigenic protein and to express it in a prokaryotic expression system in order to the foundation for preparation of an M.bovis vaccine.Methods The genome of the PG45 strain of M.bovis was analyzed bioinformatically,and a new membrane protein,p59,was discovered.Its physicochemical properties,protein localization,whether it had signal peptides,and interacting proteins were analyzed and predicted.The p59 protein-encoding gene was amplified with PCR,and the expression vector pET-30 a(+)was inserted into Escherichia coli BL21(DE3)competent cells.Expression of the protein was induced with different concentrations of IPTG and optimized.An imidazole eluate was used to elute the protein,and the purified recombinant protein was subjected to SDS-PAGE electrophoresis and identified with Western blotting.New Zealand white rabbits were immunized with purified recombinant protein to prepare polyclonal antibodies.The cell membrane,p59 protein,and nucleus were labeled with red fluorescent dye,green fluorescent dye,and blue fluorescent dye,respectively,and adhesion of p59 recombinant protein to MDBK cells was observed using laser confocal microscopy.The p59 rabbit polyclonal antibody was mixed with different titers of mycoplasma,incubated in a 37℃incubator,and then smeared on mycoplasma solid medium.The growth of mycoplasma on the solid medium was observed under a microscope to determine whether p59 rabbit polyclonal antibody inhibited mycoplasma growth.Results The CDD database predicted that the p59 protein is an M.bovis ABC transporter protein.The STRING database predicted that five proteins interact with p59,which is associated with the ABC transport system that takes up sugars and trace elements and that may be involved in the process of nutrient intake by M.bovis.Analysis of Gram-negative bacteria in the TMHMM 2.0 database predicted that the p59 protein is localized extracellularly,and the protein was found to have no signal peptides according to Signal P 4.1 database analysis.The p

关 键 词：牛支原体 p59重组蛋白 生物信息学分析 

分 类 号：R375[医药卫生—病原生物学]