基于绵羊肺炎支原体Hsp70 C末端蛋白间接ELISA方法的建立

Establishment of indirect ELISA method based on Mo HSP70 C terminal protein

为探讨建立基于Mo Hsp70C末端蛋白检测绵羊肺炎支原体（Mo）感染或疫苗免疫抗体有效方法的可能性。本研究对Mo Hsp70C末端蛋白基因进行原核表达,采用亲和层析技术对表达产物进行蛋白纯化,Western blot和琼脂扩散试验检测纯化效果和抗原反应性。基于纯化目的蛋白构建检测Mo血清抗体的间接ELISA方法。结果显示：成功表达并纯化出Mo Hsp70C末端蛋白,大小约27 000,琼脂扩散试验表明纯化的目的蛋白具有良好的抗原反应性。基于纯化的Mo Hsp70C末端蛋白,成功构建间接ELISA方法,其最佳体系蛋白质量浓度为10mg/L、血清稀释度1∶5;抗原包被条件37℃1h、室温12h、封闭液5%脱脂奶粉、封闭时间1.5h;临界值为0.588。间接ELISA方法性能评价显示本研究所建立的ELISA方法具有较好的特异性、敏感性和可重复性,可用于临床样本检测,为Mo疫苗免疫和疫病诊断提供可行手段。

To establish an effective method for the detection of antibodies of Mo intecuon or vac- cines,this study explores the possibility of Mo Hsp70 based on C-terminal proteins. The Mo Hsp70 C-terminal protein was expressed prokaryotic. The product was purified by affinity chroma- tography. Western blotting and agar diffusion test were used to detect the purification effect and antigenicity. An indirect ELISA method for the detection of Mo serum antibody was established based on purified protein. The results showed that the Mo Hsp70 C-terminal protein was success- fully expressed and purified at a size of about 27 000. The agar diffusion test showed that the puri- fied protein had good antigenic reactivity. Based on the purified Mo Hsp70 C-terminal protein, an indirect ELISA method was successfully established. The optimal conditions were as follows：pro- tein concentration 1.0 fig / 100 μL,serum dilution 1 ： 5,antigen coating condition 37℃ lh,room temperature 12 h,5% skimmed milk powder,closed time 1.5 h,the critical value of 0. 588. The re- suits of indirect showed that the ELISA method established in this paper has good specificity, sen- sitivity and repeatability, and can be used for clinical sample detection, which provides feasible means for immunization Mo vaccine of and disease diagnosis.