基于鸽鹦鹉热衣原体外膜蛋白的间接ELISA抗体检测方法的建立及应用

doi:10.16431/j.cnki.1671-7236.2025.10.037

摘要/Abstract

摘要： 【目的】建立一种适用于鸽鹦鹉热衣原体(Chlamydia psittaci，Cps)抗体的间接酶联免疫吸附试验(enzyme-linked immunosorbent assay，ELISA)检测方法。【方法】试验对Cps的主要外膜蛋白(major outer membrane protein,MOMP)编码基因进行密码子优化，在N-端加入触发因子(trigger factor，TF)后连接至pET-30a(+)载体构建重组质粒pET-30a-M，经IPTG诱导表达和纯化后，利用SDS-PAGE和Western blotting对重组蛋白进行可溶性分析及鉴定。通过棋盘滴定法选择最佳抗原包被浓度和血清稀释度，优化鸽Cps抗体的间接ELISA法。比较本研究建立的间接ELISA法与间接血凝试验(indirect hemagglutination assay,IHA)的敏感性。通过检测鸽其他常见疾病阳性血清评估该方法的特异性，检测Cps阳性和阴性血清各3份以评估其重复性，并利用253份鸽源血清(174份临床血清样品、79份Cps灭活疫苗免疫后血清样品)评估该间接ELISA法与IHA法的符合率。【结果】SDS-PAGE和Western blotting结果显示，在诱导超声破碎后上清中成功表达大小约100 ku的MOMP蛋白,表明该蛋白具有可溶性。间接ELISA法优化后条件为：2 μg/mL抗原4 ℃包被过夜，5%脱脂奶粉37 ℃封闭2 h，血清稀释度为1∶200、血清孵育时间为1 h，辣根过氧化物酶(HRP)标记兔抗鸽IgY孵育30 min，底物反应时间为10 min。鸽Cps阳性血清稀释度为1∶16时，间接ELISA法检测结果仍为阳性，敏感性高于IHA法。间接ELISA法与其他常见鸽病阳性血清均无交叉反应。批内和批间重复试验结果显示，间接ELISA法变异系数均＜10%，表明该方法具有良好的重复性。本研究建立的间接ELISA法和IHA法检测临床样品和免疫后血清样品的符合率分别为80.72%和85.71%，Kappa值分别为0.60和0.69。【结论】本研究建立的间接ELISA法敏感性高、特异性强、重复性好，适用于Cps血清抗体的检测。

关键词: 鸽; 鹦鹉热衣原体; 间接ELISA; 间接血凝试验(IHA); 抗体

Abstract: 【Objective】 This experiment aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) method applicable to detecting antibodies against Chlamydia psittaci (Cps) in pigeons.【Method】 The codons of the gene encoding the major outer membrane protein (MOMP) of Cps were optimized.After adding trigger factor (TF) at the N-terminus,it was ligated to the pET-30a(+) vector to construct the plasmid pET-30a-M.After induction with IPTG,expression and purification were carried out.Solubility analysis and identification were performed using SDS-PAGE and Western blotting.A checkerboard titration was used to determine the optimal antigen coating concentration and serum dilution,thereby optimizing the indirect ELISA method for detecting pigeon Cps antibodies.The sensitivity of the indirect ELISA method established in this study were compared with that of the indirect hemagglutination assay (IHA).The specificity of this method was evaluated by detecting the positive sera of other common diseases in pigeons.Three positive and three negative sera for Cps were detected respectively to assess its repeatability.The coincidence rate of this indirect ELISA method and the IHA method was evaluated by using 253 pigeon-derived sera (174 clinical serum samples and 79 serum samples after immunization with Cps inactivated vaccine).【Result】 SDS-PAGE and Western blotting results showed that the MOMP protein with a size of approximately 100 ku was successfully expressed in the supernatant after induction and sonication,indicating that this protein was soluble.The optimized conditions of the indirect ELISA method were as follows:Coating with 2 μg/mL antigen at 4 ℃ overnight,blocking with 5% skimmed milk at 37 ℃ for 2 h,serum dilution of 1∶200,serum incubation time of 1 h,HRP-labeled rabbit anti-pigeon IgY incubation for 30 min,and substrate reaction time of 10 min.When the dilution of the positive serum of pigeon Cps was 1∶16,the detection result of the indirect ELISA method was still positive,and the sensitivity was higher than that of the IHA method.There was no cross-reaction between the indirect ELISA method and the positive sera of other common pigeon diseases.Both intra- and inter-assay test results showed that the coefficients of variation of the indirect ELISA method were all less than 10%,indicating that this method had good repeatability.The coincidence rates of the indirect ELISA method established in this study and the IHA method for detecting clinical samples and serum samples after immunization were 80.72% and 85.71% respectively,and the Kappa values were 0.60 and 0.69 respectively.【Conclusion】 The indirect ELISA method established in this experiment exhibited high sensitivity,strong specificity,and excellent repeatability,and was suitable for the detection of serum antibodies against Cps in pigeons.

Key words: pigeons; Chlamydia psittaci; indirect ELISA; indirect hemagglutination assay (IHA); antibodies

中图分类号:

S852.69

张紫萱, 杨志远, 刘月焕, 杨宝琳, 高洁, 冯倩倩, 罗伟珏, 林健, 赵际成, 黄程, 胡格. 基于鸽鹦鹉热衣原体外膜蛋白的间接ELISA抗体检测方法的建立及应用[J]. 中国畜牧兽医, 2025, 52(10): 4920-4929.

ZHANG Zixuan, YANG Zhiyuan, LIU Yuehuan, YANG Baolin, GAO Jie, FENG Qianqian, LUO Weijue, LIN Jian, ZHAO Jicheng, HUANG Cheng, HU Ge. Establishment and Application of an Indirect ELISA Antibody Detection Method Based on the Outer Membrane Protein of Chlamydia psittaci in Pigeons[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4920-4929.

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