Leachii支原体LppA蛋白的表达及其粘附特性鉴定  

Expression and adhesion property of LppA protein of Mycoplasma leachii

作　　者：王冠博[1] 常继涛[2] 刘云[1] 于力[2] 

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/牛羊传染病研究创新团队,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第8期641-645,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金项目(C201348);黑龙江省科技计划重大项目(GA12B301);国家科技计划课题(2012BAD12B03-3)

摘　　要：Leachii支原体可以引起犊牛多发性关节炎,与犊牛肺炎有关。为研究M.leachii LppA蛋白粘附特性,本研究采用大肠杆菌表达系统表达M.leachii LppA蛋白,并制备抗LppA多克隆抗体。胰酶敏感试验结果显示,全菌体蛋白经胰酶处理后,LppA蛋白条带消失,表明LppA蛋白是位于M.leachii细胞表面的膜脂蛋白。通过粘附抑制试验,利用激光共聚焦显微镜可以观察到抗LppA血清能够抑制M.leachii对胎牛关节滑膜细胞的粘附作用;同时,采用Columbas分析软件对共聚焦图片进行图像分析,结果显示1:25倍稀释的LppA多克隆抗体对M.leachii的粘附抑制率为58.3%,而同比稀释的M.leachii全菌体多抗的粘附抑制率为79.26%。此外,ELISA试验结果表明,重组LppA蛋白可以与关节滑膜细胞膜蛋白发生特异性粘附作用,并且这种粘附作用可以被抗LppA多抗特异性抑制。这些结果表明,LppA蛋白是M.leachii的一个粘附相关的外膜脂蛋白。Mycoplasma leachii causes polyarthritis and pneumonia in calves. To identify the adhesion property of lipoprotein LppA in M.leachii, the LppA protein of M.leachii was expressed in E.coli and the anti-LppA antibodies were prepared in mice. Trypsin sensitivity experiment showed that the LppA was undetectable in M.leachii total proteins after digestion with trypsin, which indicated that the LppA was an outer membrane lipoprotein. In addition, the results of cells adherence inhibition assay showed that the adhesion property of M.leachii was specifically inhibited by the anti-LppA antibodies. Meanwhile, the image analysis by Columbas software showed that the adherence inhibition rates of 1：25 diluted anti-LppA antibodies and anti-M.leachii antibodies were 58.3% and 79.26%, respectively. Furthermore, the ELISA detection showed that the LppA protein possessed the ability of adhesion to joint synovial cell membrane proteins, and this adhesion was specifically inhibited by the anti-LppA antibodies, in a dose-depend manner. These data demonstrated that LppA is an adhesion-related outer membrane lipoprotein of M. leachii.

关 键 词：Leachii支原体 膜脂蛋白 LppA蛋白 粘附特性 

分 类 号：S852.62[农业科学—基础兽医学]