基于Mhp336蛋白的猪肺炎支原体ELISA抗体检测方法的建立

Development of an ELISA method for detection of antibodies against Mycoplasma hyopneumoniae based on Mhp336 protein

本研究拟建立一种高特异性的猪肺炎支原体酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)抗体检测方法。首先,构建重组菌大肠杆菌BL21(DE3)-p ET-32a(+)-mhp336,诱导重组蛋白Mhp336表达并纯化,获得的纯化蛋白作为包被抗原;然后,确定ELISA方法的抗原包被浓度、封闭液和封闭时间、血清和二抗稀释比例和孵育时间、显色时间和判定标准;之后进行重复性试验、与猪的其他主要病原抗血清的交叉反应性和确定血清最大稀释倍数;最后,用建立的方法检测226份猪血清样品,并与商品化猪肺炎支原体ELISA抗体检测试剂盒和猪肺炎支原体ELISA恢复期血清抗体检测方法进行比较,确定检测方法的灵敏性和特异性。最终确定Mhp336纯化蛋白的最佳包被浓度为0.05μg/m L,封闭液为含2.5%脱脂奶粉的PBS,封闭时间为1 h,血清稀释比例为1:500,孵育时间为0.5 h,二抗稀释比例为1:10000,孵育时间为1 h,显色时间为5 min。检测方法的cut off值为0.332。试剂盒的批内和批间变异系数均低于7%。猪血清样品的检测结果表明,本研究建立的检测方法的灵敏性和特异性均优于商品化猪肺炎支原体ELISA抗体检测试剂盒和猪肺炎支原体ELISA恢复期血清抗体检测方法。本研究成功建立了基于Mhp336蛋白的猪肺炎支原体ELISA抗体检测方法,灵敏性和特异性均较好,为猪场猪支原体肺炎的净化提供了检测方法。

This study aims to establish an ELISA method with high specificity for the detection of antibodies against Mycoplasma hyopneumoniae.Firstly,we constructed a recombinant strain Escherichia coli BL21(DE3)-pET-32a(+)-mhp336 to express the recombinant protein Mhp336 and used the purified Mhp336 as the coating antigen.Then,we optimized the ELISA parameters,including antigen concentration,blocking buffer,blocking time,dilution of serum,incubation time with serum,secondary antibody dilution,secondary antibody incubation time,colorimetric reaction time,and cut-off value.Afterwards,reproducibility experiments were conducted,and the cross reactivity of Mhp366 with other antisera of porcine major pathogens and the maximum dilution ratios of the sera were determined.Finally,226 porcine serum samples were detected using the method established in this study,a commercial ELISA kit for M.hyopneumoniae antibody detection,and a convalescent serum ELISA kit for M.hyopneumoniae antibody detection.The detection results of the three methods were compared to evaluate the sensitivity and specificity of the ELISA method established in this study.For this method,the optimal antigen concentration,blocking buffer,blocking time,dilution of serum,incubation time with serum,secondary antibody dilution,secondary antibody incubation time,and colorimetric reaction time were 0.05μg/mL,PBS containing 2.5%skim milk,1 h,1:500,0.5 h,1:10000,1 h,and 5 min,respectively.Validation of the ELISA method based on Mhp336 showed a cut-off value of 0.332.The coefficients of variation of both intra-batch and inter-batch kits were below 7%.The detection results of porcine serum samples indicated that the method established in this study outperformed the commercial ELISA kit and the convalescent serum ELISA kit for M.hyopneumoniae antibody detection in terms of sensitivity and specificity.We successfully established an ELISA method for detecting the antibodies against M.hyopneumoniae based on Mhp336 protein.This method demonstrated high sensitivity and specificity,serving as a tool for the prevention of mycoplasmal pneumonia of swine in pig farms.