牛支原体LRR5蛋白原核表达及其在牛支原体入侵宿主细胞过程中的作用分析

Prokaryotic expression of Mycoplasma bovis LRR5 protein involved in the invasion of Mycoplasma bovis into host cells

致病性支原体具有入侵宿主细胞的能力,这是其发挥致病作用的关键。介导支原体入侵宿主细胞的自身功能蛋白可能是一种潜在的药物或疫苗靶标。【目的】克隆表达牛支原体(Mycoplasma bovis)MBOVPG45_0564基因编码蛋白(命名为LRR5蛋白),并探究其在M.bovis入侵宿主细胞过程中的作用。【方法】利用NCBI数据库对MBOVPG45_0564基因进行同源性分析,用Discovery Studio Client系统对LRR5蛋白进行蛋白结构预测;原核表达LRR5蛋白并制备其小鼠多克隆抗体,利用免疫电镜对LRR5蛋白进行亚细胞定位;通过平板计数、激光共聚焦显微镜观察LRR5抗体封闭后M.bovis对胎牛肺(embryonic bovine lung,EBL)细胞入侵率的变化;将LRR5蛋白偶联至荧光微球表面后,以激光共聚焦显微镜及高内涵活细胞成像系统观察微球进入EBL细胞情况。【结果】MBOVPG45_0564基因在牛支原体属中为保守基因,其编码蛋白LRR5为膜相关蛋白,空间构象呈典型的月牙状,多个重复的亮氨酸基序以超螺旋方式组装并形成螺线管蛋白质结构单元。LRR5抗体封闭后,M.bovis对EBL细胞的入侵率显著降低(P<0.05),荧光微球偶联LRR5蛋白后,荧光微球可成功进入EBL细胞。【结论】MBOVPG45_0564基因编码的LRR5蛋白定位在M.bovis膜上,在M.bovis入侵宿主细胞过程中发挥着重要作用。

Pathogenic mycoplasma has the ability to invade host cells,which is a key mechanism to their pathogenicity.Functional proteins that mediate the entry of mycoplasma may serve as potential drug or vaccine targets.[Objective]To clone and express the LRR5 protein encoded by MBOVPG45_0564 in Mycoplasma bovis and to explore its role in the invasion of host cells by M.bovis.[Methods]The NCBI database was used for the homology analysis of MBOVPG45_0564,and the structure of LRR5 protein was predicted by the Discovery Studio Client system.After prokaryotic expression of LRR5 protein,the mouse polyclonal antibody was prepared,and the subcellular localization of LRR5 protein was observed by immunoelectron microscopy.The invasion of M.bovis into embryonic bovine lung(EBL)cells after LRR5 antibody blocking was observed by plate counting and laser confocal microscopy.After conjugation of LRR5 protein to the surface of fluorescent microspheres,the entry of the microspheres into EBL cells was observed by laser confocal microscopy and a high-content live-cell imaging system.[Results]MBOVPG45_0564 was a conserved sequence in M.bovis and encoded LRR5,a membrane-associated protein with a typical crescent-like spatial conformation.Multiple repeating leucine motifs were assembled in a supercoiled manner to form a solenoid protein structural unit.After LRR5 antibody blocking,the invasion rate of M.bovis to EBL cells reduced(P<0.05),and the fluorescent microspheres conjugated with LRR5 protein could successfully enter EBL cells.[Conclusion]The LRR5 protein encoded by MBOVPG45_0564 is localized on the M.bovis membrane and plays a role in the invasion of M.bovis into host cells.