牛支原体免疫相关性蛋白硫锌酰胺脱氢酶的筛选与鉴定  

Screening and identification of immuno-related protein of Mycoplasma bovis

作　　者：孙文静[1,2] 李媛[2] 宋志强[2] 邹晓辉[2] 刘洋[2] 陈维[1] 周玉梅[2] 张秀英[1] 辛九庆[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/动物细菌病研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2012年第10期761-765,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：兽医生物技术国家重点实验室基本科研业务费(NKLVBP200809)

摘　　要：为鉴定牛支原体(M.bovis)菌体免疫相关蛋白及其免疫原性,本实验以M.bovis湖北株为样品,通过建立M.bovis全菌蛋白的双向电泳体系,获得了分辨率及重复性良好的双向电泳(2-DE)图谱。利用western blot技术进行筛选,鉴定得到多个M.bovis蛋白,其中一个分子量为68 ku,经过质谱分析和氨基酸一级结构比对确定该蛋白为硫锌酰胺脱氢酶(LipDH)。经原核重组表达,LipDH重组蛋白与M.bovis阳性血清的western blot反应为阴性,而Dot blot及以重组表达蛋白作为包被抗原的ELISA鉴定结果均为阳性。本研究为进一步研究LipDH的功能以及采用其重组蛋白用于该蛋白缺失的M.bovis疫苗株的鉴别检测方法的建立奠定了基础。To identify the immuno-associated proteins of Mycoplama bovis, the two-dimensional gel electrophoresis （2-DE） for M. bovis proteome separation was established and followed by western blot identification for immuno-related proteins of M. bovis Hubei-1 strain. Many proteins were recognized by M. bovis positive serum, one of which was identified as lipoamide dehydrogenase （LipDH） by mass spectrometry analysis. Furthermore, the P68 （LipDH） was expressed in E. coll. The dot-bolt and ELISA assays showed that the recombinant LipDH was reacted with M. bovis positive serum, while it was failed to be detected by western blot assay, indicating that this protein is probably a conformation dependent immunoprotein in M. bovis. This study has established the foundation of the function research of LipDH and the detection method to M. bovis.

关 键 词：牛支原体 双向电泳 免疫 质谱 

分 类 号：S852.6[农业科学—基础兽医学]