牛霉形体免疫相关性蛋白P51的筛选与鉴定

Screening and identification of immuno-related protein P51 of Mycoplasma bovis

为了筛选免疫原性蛋白,建立牛霉形体的快速诊断方法,利用二维凝胶(2-D)电泳、Western-blot分析及蛋白质谱分析,筛选出一个免疫相关性蛋白P51。以牛霉形体中国分离株Hubei-1为模板,扩增了p51基因,并克隆到原核表达载体pET32a上。结果显示,重组蛋白pET32a-p51与牛霉形体阳性血清的Dot-blotting试验结果为阳性,而Western-blotting试验结果为阴性。以pET32a-p51重组蛋白为包被抗原的ELISA试验表明其能与自然感染和人工感染牛霉形体的阳性血清发生特异性反应,而与灭活疫苗免疫的阳性血清不发生特异性反应,与牛传染性胸膜肺炎的阳性血清没有交叉反应。表明P51可能是牛霉形体特有的一种构象依赖性免疫相关蛋白,是一种有前景的诊断用抗原。

To select the immuno related proteins of Mycoplasma boris and establish the quickly diagnostic method of M. boris,2 DE,Western-blot and mass spectrometric were used to filter out an immuno- protein P51. A pS1 gene was amplified by PCR from a M. boris strain isolated from Hubei-1 ,and was cloned into pET32a vector to express. The highly purified protein was obtained by Ni-NTA system. Dot-blot showed that recombinant protein pET32a-pS1 could be probed with M. boris positive bovine antiserum and could be considered to have the immunocompetence although Western blot could not be get the result. The purified protein was coated to microtiter ELISA plates. The ELISA result indicated that pET32a-p51 could react with the M. boris positive bovine antiserum made by natural infection and experimental infection,but could not react with the M. boris positive bovine antiserum immunized by inactivated vaccine and Mycoplasma mycoides subsp, mycoides small-colony type positive bovine antiserum. In conclusion,P51 maybe an immuno-related protein of conformation dependent and a prospect antigen for diagnosis of M. boris.