猪肺炎支原体P97 C末端蛋白单克隆抗体的制备及其抗原表位分析  

Preparation for monoclonal antibodies of P97 C-terminal protein of Mycoplasma hyopneumoniae and analysis of epitope of the McAb

作　　者：张美晶[1] 王亮[1] 李媛[1] 董慧[1] 姜海芳[1] 陈超[1] 曹培丽[1] 郭丹[1] 辛九庆[1] 

机构地区：[1]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国家牛传染性胸膜肺炎指定检测实验室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2010年第2期150-157,共8页Chinese Veterinary Science

基　　金：兽医生物技术国家基本科研项目(NKLVBP200809)

摘　　要：以大肠杆菌表达的pET-30a-P97C重组蛋白为免疫原免疫BALB/c小鼠,制备针对猪肺炎支原体P97C末端蛋白的单克隆抗体,并将其与截短表达的P97C末端蛋白进行免疫印迹分析,初步鉴定了P97C末端蛋白的抗原表位。进一步采用肽探针扫描技术确定最小抗原决定区域,并通过生长抑制试验及抗黏附特性试验对抗原表位进行了分析。结果显示,获得2株能稳定分泌特异性抗体的杂交瘤细胞株A3C9和B4D5,并成功鉴定出P97C末端蛋白的2个独立线性表位F905PMAFSY911和G991TPNQGKKAE1000。2株单抗对猪肺炎支原体的生长均有一定的抑制作用,但对其黏附特性没有显著抑制作用。BALB/c mice were immunized with the purified P97 C-terminal recombinant protein of Mycoplasma hyopneumoniae expressed in Escherichia coli in order to prepare monoclonal antibodies and identify their biological properties.Immunoblotting assay was made with P97 C-terminal proteins expressed from different overlapping fragments and the monoclonal antibodies.To map initially epitopes of the C-terminal protein,Pepscan was used to exactly identify the minimum epitope of the C-terminal protein.Growth inhibition and adherence inhibition assays were established to analyze the epitopes of the C-terminal protein.The results showed that two strains of monoclonal hybridoma cells stably secreting the specific antibodies were developed and two independently linear epitopes of C-terminal protein of P97 adhesin,905-911aa（F^905PMAFSY^911）and 991-1000aa（G^991TPNQGKKAE^1000）,were identified successfully.The two antibodies were able to inhibit the growth of M.hyopneumoniae cells in vitro,but were not able to significantly inhibit the binding of M.hyopneumoniae cells.

关 键 词：猪肺炎支原体 单克隆抗体 抗原表位 肽扫描 生长抑制试验 

分 类 号：S852.62[农业科学—基础兽医学]