猪肺炎霉形体热休克蛋白Hsp70单克隆抗体的制备及鉴定  

Preparation and identification of monoclonal antibodies against HSP70 of Mycoplasma hyopneumoniae

作　　者：乔祖健[1] 王亮[2] 李媛[2] 陈超[2] 辛九庆[2] 李继昌[1] 

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国家牛传染性胸膜肺炎参考实验室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2008年第8期701-706,共6页Chinese Veterinary Science

基　　金：中国农业科学院哈尔滨兽医研究所所长基金项目(2007)

摘　　要：以肺炎霉体体(Mhp)Yin-1株为模板,利用所合成的引物扩增了伴侣蛋白DnaK(热休克蛋白Hsp70)C末端基因,将得到的目的片段分别克隆至表达载体pET-28a和pGEX-6P-1中,原核表达获得了重组蛋白6P-1-DnakC和28a-DnakC,经Western-blot分析,均能被Mhp抗血清识别。以28a-DnakC作为包被抗原,建立了间接ELISA检测方法,以6P-1-DnakC为免疫抗原,免疫BALB/c小鼠。筛选和鉴定了2株分泌抗DnaK的杂交瘤细胞株,命名为1AD10、2AF11。Western-blotting结果表明,这2株单抗与Mhp发生特异性反应而不与其他相关霉形体发生反应。抗体亚类鉴定结果显示,1AD10、2AF11分别为IgG2b和IgG2a亚类,且轻链均为κ链。相加ELISA试验表明,2株单抗能识别不同的抗原表位。The chaperones protein DnaK（HSP70） gene in C-terminal was amplified from Mycollasma hyolneurnoniae （Mhp）strain Yin-1 by PCR using the synthesized primers and cloned into prokaryotic ex pression vectors pET-28a and pGEX-6P-1, respectively. The expressed recombinant proteins 6P-1-DnakC and 28a-DnakC could be recognized by Mhp antiserum in Western-blotting. An indirect ELISA was estab lished using the 28a-DnakC as coating antigen. BALB/c mice were immunized with the 6P-1-DnakC. Spleno- cytes of the immunized mice were fused with SP2/0 cells with PEG3250 after the 3rd immunization. Two hybridoma cell lines secreting monoclonal antibodies（McAbs） against the DnaK protein,designated lAD10 and 2AFll, were obtained and identified. Western blot analysis showed that the two McAbs specifically recognized Mhp but did not recognize other mycoplasmas. McAbs lAD10 and 2AFll represented subtypes IgG2b and IgG2a respectively, and their light chains were k chain. Additive ELISA showed that the two McAbs could recognize different epitopes.

关 键 词：猪肺炎霉形体 伴侣蛋白DnaK 热休克蛋白70 单克隆抗体 

分 类 号：S852.62[农业科学—基础兽医学] Q813.2[农业科学—兽医学]