经济动物学报

为建立一种具有良好特异性和敏感性的鹦鹉热衣原体iELISA检测方法。根据Cps ompA基因序列构建重组质粒PfastBac1-dx，利用bac to bac杆状病毒表达系统表达目的蛋白并用His镍柱进行纯化，经Western blot鉴定后，将纯化的MOMP蛋白作为检测抗原进行包被，成功建立了一种iELISA方法。通过对包被条件、封闭液种类、封闭时间及血清孵育时间等关键反应条件进行系统优化，进一步对该方法的临界值、特异性及灵敏性进行了全面验证。Western blot分析结果表明，MOMP蛋白能够与Cps阳性血清发生特异性结合反应。优化后的ELISA反应条件为：10μg/mL抗原4℃包被12 h，1%明胶4℃封闭12 h，1∶50稀释的血清37℃孵育2 h，1∶0.1μg/mL的G蛋白二抗37℃孵育2 h。该方法灵敏性较高，Cps阳性血清稀释度为1∶800时仍有明显反应。与IHA试剂盒相比，其总符合率为85.07%。本研究建立了一种以Cps MOMP蛋白为检测抗原的可同时检测牛和羊的CpsiELISA方法，该方法具有良好的敏感性和特异性，能运用于Cps的血清学检测，为鹦鹉热衣原体疫苗的质量控制提供技术支撑。

To establish an indirect ELISA method with good specificity and sensitivity for the detection of Chlamydia psittaci. The recombinant plasmid PfastBac1-dx was constructed according to the sequence of Cps ompA gene， and the target protein was expressed by bac to bac baculovirus expression system and purified by His nickel column. After identification by Western blot， the purified MOMP protein was used as detection antigen for coating. An indirect ELISA method was successfully established. The critical value， specificity and sensitivity of the method were further verified by systematically optimizing the key reaction conditions such as coating condition， blocking solution type， blocking time and serum incubation time. Western blot analysis showed that MOMP protein could specifically bind to Cps positive serum. The optimized ELISA reaction conditions were as follows: 10μg/mL antigen was coated at 4℃ for 12 h， 1% gelatin was blocked at 4℃ for 12 h， diluted serum was incubated at 37℃ for 2 h， and 1∶0.1ug/ml G-protein secondary antibody was incubated at 37℃ for 2 h. The sensitivity of this method is high， and the reaction is still obvious when the dilution of Cps positive serum is 1∶800. Compared with IHA kit， the total coincidence rate was 85.07%. This study established an indirect ELISA method with Cps MOMP protein as the detection antigen， which can simultaneously detect cattle and sheep Cps. The method has good sensitivity and specificity， and can be used for the serological detection of Cps， which provides technical support for the quality control of Chlamydia psittaci vaccine.