基于丝状支原体丝状亚种P35反应原性的验证及其间接ELISA方法的建立

Validation of reactogenicity of Mycoplasma mycoides subsp Mycoides P35 and establishment of indirect ELISA method

为建立检测丝状支原体丝状亚种(Mmm)的间接ELISA方法,本研究利用生物信息学软件对Mmm的全基因组序列进行了分析,选定大小为540bp的0584基因,该基因是编码分子量为35ku的脂蛋白,并对其免疫反应原性进行研究。利用原核表达系统获得了0584基因的重组蛋白rP35,以牛传染性胸膜肺炎(CBPP)阳性血清为一抗进行western blot分析,试验结果为阴性,但Dot-blot试验结果为阳性,证明该蛋白可能是Mmm特有的一种构象依赖性免疫相关蛋白且具有反应原性。以rP35蛋白为包被抗原,通过反应条件优化初步建立了检测CBPP血清抗体的间接ELISA方法,特异性为88.0%,敏感性为89.8%,批内和批间变异系数均小于10%,与商品化试剂盒符合率为84.8%。本研究为研制CBPP血清检测试剂盒提供了一种候选蛋白。

In order to establish an indirect ELISA method for detecting Mycoplasma mycoides subsp Mycoides(Mmm),the whole genome sequence of Mmm was analyzed by bioinformatics software,and a 540bp gene 0584 was selected,which encodes a lipoprotein with a molecular weight of 35ku,and its reactogenicity was studied.The recombinant protein rP35 of 0584 gene was obtained by prokaryotic expression system.Using a positive serum of Contagious bovine pleuropneumonia(CBPP)as the primary antibody the results were negative in western blot analysis,but it was positive in dot-blot test,which proved that the protein might be a conformation-dependent immuno-related protein specific to Mmm and possess reactogenicity.An indirect ELISA method for detecting serum antibodies to CBPP was established using the rP35 protein as coated antigen.After optimizing the reaction conditions,the specificity and sensitivity of the method were 88.0%and 89.8%respectively.The intra-batch and inter-batch variation coefficients were less than 10%and the coincidence rate with the commercial kit was 84.8%.This study provided a candidate protein for the development of bovine infectious pleuropneumonia serum detection kit.