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本文主要研究了重组蛋白Dna K(rDnaK)的免疫反应原性及其作为ELISA方法中诊断抗原的应用价值。将已构建的含有鸡毒支原体(MG)DnaK基因的重组质粒pET-30a-DnaK进行原核表达纯化,以多份MG的标准阳性血清和阴性血清作为一抗,进行Western blot分析,鉴定rDnaK的免疫反应原性,结果显示rDnaK与MG的阳性血清发生特异性反应,与MG的阴性血清无反应条带,表明rDnaK具有较好的免疫反应原性。将其作为包被抗原包被酶标板,用HRP标记的兔抗鸡Ig G作为酶标二抗,分别对抗原包被浓度、血清稀释度、酶标二抗稀释度及工作时间等反应条件进行了优化,建立了一种稳定检测鸡毒支原体(MG)抗体的间接ELISA方法,并且与商品化的试剂盒比较结果显示,该方法的特异性性为95.4%,敏感性为92.6%。交叉反应性试验表明包被抗原rDna K不与NDV、IBV、ILTV等几种常见的禽呼吸道疾病的阳性血清发生交叉反应。抗体持续期试验结果显示该方法最早能在鸡群免疫MG疫苗后一周检测到MG抗体,说明该诊断方法适用于MG感染的早期检测。采用该方法检测了来自全国五个省份的568份临床样品,其结果表明MG的流行率在23%～51%之间,对我国MG的流行状况做了初步的调查。
In this paper, the immunogenicity of recombinant protein Dna K (rDnaK) and its application as a diagnostic antigen in ELISA were studied. The constructed recombinant plasmid pET-30a-DnaK containing Mycoplasma gallisepticum (MG) DnaK gene was purified by prokaryotic expression. Western blot analysis was performed using standard positive and negative serums of MG as the primary antibody to identify the immunogenicity of rDnaK The results showed that rDnaK reacted specifically with the positive serum of MG and negative with serum MG, indicating that rDnaK has good immunogenicity. Antigen-coated ELISA plates were coated with HRP-labeled rabbit anti-chicken Ig G as enzyme-labeled secondary antibody. The reaction conditions were as follows: antigen concentration, serum dilution, dilution of ELISA secondary antibody and working time An indirect ELISA method was established for the stable detection of Mycoplasma gallisepticum (MG) antibody. Compared with the commercially available kit, the specificity and specificity of this method were 95.4% and 92.6%, respectively. Cross-reactivity test showed that the coated antigen rDna K does not cross-react with the positive sera of several common avian respiratory diseases such as NDV, IBV and ILTV. Antibody duration test results showed that this method can detect MG antibody as early as one week after the chicken immunized MG vaccine, indicating that the diagnostic method is suitable for early detection of MG infection. Using this method, 568 clinical samples from five provinces in China were detected. The results showed that the prevalence of MG was between 23% and 51%. The prevalence of MG in our country was preliminary investigated.