基于脂蛋白P80的滑液支原体抗体ELISA检测方法的建立及应用

【目的】研究滑液支原体(Mycoplasma synoviae, MS)脂蛋白P80的免疫反应性及其在MS血清抗体ELISA检测中的应用。【方法】对MSP80的氨基酸序列进行生物信息学分析、原核表达和纯化,并用免疫印迹法分析其与6种不同MS分离株阳性血清的免疫反应性以及与其他禽病原血清的交叉反应性;运用纯化的MS P80表达蛋白作为包被抗原建立了MS血清抗体的间接ELISA检测方法,对其敏感性和重复性进行检测;比较检测了与美国爱德士检测试剂盒对50份临床血清样品的阳性符合率。【结果】生物信息学分析预测MS P80蛋白为脂蛋白且含有信号肽,其在MS种内同源性高达98%-100%,与其他种属P80蛋白同源性在25%-34%之间,成功表达和纯化了MS P80重组蛋白(rMS P80);Western blotting分析表明纯化的rMS P80具有良好的免疫反应性和特异性;运用rMS P80建立的MS血清ELISA抗体检测方法可对不同株MS阳性血清进行抗体效价检测,而对其他禽病原阳性血清均无交叉反应性;该检测方法的批内变异系数小于5%,批间变异系数小于10%,重复性良好;与美国IDEXX检测试剂盒比较,本文建立的ELISA抗体检测方法敏感性更高,阳性符合率为75%,阴性符合率为89.47%,总样本符合率为86%。【结论】MS P80具有较好的免疫反应性、种内保守性和种间特异,并且可用作MS抗体检测的靶标抗原。

Establishment and application of ELISA method for detection of Mycoplasma synoviae antibody based on lipoprotein P80

Objective] We studied the immunoreactivity of Mycoplasma synoviae lipoprotein P80 (MS P80) and applied it to detect ELISA antibody.Methods] Firstly, we performed bioinformatics analysis, prokaryotic expression and purification on MS P80, and analyzed its immunoreactivity with 6 positive sera against different MS isolates and other avian pathogens by Western blotting. Then we used the purified rMS P80 as coating antigen to establish an indirect ELISA assay for detection of MS antibodies, and tested its specificity, sensitivity, reproducibility and coincidence rates compared with US IDEXX ELISA kit.Results] The MS P80 protein was predicted as a lipoprotein that shared 98%-100% homology between different MS strains and 25% to 34% homology with other species. Western blotting analysis proved that rMS P80 protein had good immunoreactivity and specificity. An indirect ELISA assay based on rMS P80 was established and showed no cross-reaction with positive sera against other pathogens. The intra- and inter-assay variation coefficients were less than 5% and 10%, respectively. When compared with the commercial kit, the established ELISA assay was more sensitive, and the total coincidence rate was 86%.Conclusion] MS P80 has good immunoreactivity, intraspecies conservation and interspecies specificity. Therefore, it can be used as a target antigen for MS antibody detection.