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

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 p59 gene was amplified with PCR,and the prokaryotic expression vector pET-30 a(+)-p59 was correctly constructed according to double enzyme digestion and sequencing.The recombinant vector was transformed into BL21 expressing p59 recombinant protein according to SDS-PAGE.The recombinant’s relative molecular mass was 66×10^3.Expression of the recombinant plasmid was higher when the final concentration of IPTG was 0.5 mmol/L.Expression was induced at 37℃for 4 h,and the concentration of imidazole was 100 mmol/L.Western blot analysis was performed using purified p59 recombinant protein,which was recognized by positive bovine sera,and the reaction band was located at 66×10~3.Western blot analysis indicated that the protein is a membrane protein.Immunization of New Zealand rabbits stimulated the production of specific antibodies.An experiment involving p59 protein adhesion to Madin-Darby bovine kidney(MDBK)cells indicated that more P59 protein was able to adhere to the surface of MDBK with p59 rabbit polyclonal antibody,indicating that p59 recombinant protein has some ability to adhere to cells.A M.bovis growth inhibition test indicated that p59 rabbit polyclonal antibody inhibited the growth of mycoplasma in solid medium.In this experiment,the average value of three inhibition tests was 252 for rabbit negative serum,28 for rabbit polyclonal antiserum,and 492 for the mycoplasma blank control group;growth was inhibited at a rate of 45.53%in rabbit antiserum.Conclusion The newly identified M.bovis membrane protein p59 may be an adhesion-related protein,and it is antigenic.Rabbit antiserum against this protein inhibited the growth of mycoplasma.This finding lays the foundation for study of the function of mycoplasma membrane proteins and ir provides a new target protein for development of a vaccine against M.bovis.