基于0507蛋白的山羊支原体山羊肺炎亚种抗体间接ELISA检测方法的建立

Establishment of Indirect ELISA for Detection of Mccp Antibody Based on 0507 Protein

为建立山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp.capripneumoniae,Mccp)抗体检测方法,对Mccp的0507基因进行克隆与载体构建,通过原核表达技术制备重组0507蛋白,建立Mccp抗体间接ELISA检测方法,验证该方法的特异性、灵敏性与重复性,并对临床收集的血清样品进行检测。结果显示,重组0507蛋白大小为39 ku,Western blot证明重组0507蛋白具有良好的反应原性;建立的ELISA检测方法抗原包被量为3μg/mL,血清稀释倍数为1∶200,酶标二抗稀释倍数为1∶2500,阴阳临界值为0.221。用该方法对山羊流产衣原体、立克次氏体与牛支原体的阳性血清进行检测,结果均为阴性;Mccp阳性血清最大稀释倍数为1024倍;批内、批间变异系数均小于10%。对陕西省部分羊场收集到的282份血清进行检测,Mccp阳性率为13.1%。该研究建立了一种检测Mccp抗体间接ELISA检测方法,为Mccp的实验室诊断和流行病学调查提供了技术支持。

In order to establish a detection method for antibody against Mycoplasma capricolum subsp.capripneumoniae,(Mccp),0507 gene was cloned and vector was constructed.The recombinant 0507 protein was prepared by prokaryotic expression technology.The specificity,sensitivity and repeatability of Mccp antibody was verified by indirect ELISA,and serum samples collected in clinic were tested.The results showed that the recombinant 0507 protein was 39 ku in size,and Western blot showed that the recombinant 0507 protein had good reactivity.The results indicated that diluting the antigen to 3μg/mL and the serum to 1∶200 and the marked IgG to 1∶2500 were the best reaction condition.,and the cut-off value was 0.221.The positive sera of Chlamydia abortica,Rickettsia rickettsia and Mycoplasma bovis were detected by this method,and the results were negative.The maximum dilution of Mccp positive serum was 1024 times.The coefficient of variation within and between batches was less than 10%.The positive rate of Mccp was 13.1%in 282 serum samples collected from sheep farms in Shaanxi province.This study established an indirect ELISA for Mccp antibody detection,which provided technical support for laboratory diagnosis and epidemiological investigation of Mccp.