一种基于鸽源鹦鹉热衣原体MOMP蛋白的抗体间接ELISA检测方法的建立  

Establishment of an indirect ELISA detection method for antibodies based on MOMP protein of Chlamydia psittaci from pigeon

作　　者：路文彬 庞洪泽 刘琳敏 田甜[1] 张武超 袁万哲[1,2] LU Wen-bin;PANG Hong-ze;LIU Lin-min;TIAN Tian;ZHANG Wu-chao;YUAN Wan-zhe(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001

出　　处：《中国兽医科学》2023年第11期1363-1370,共8页Chinese Veterinary Science

基　　金：河北省“三三三人才工程”项目(A20221003);校企合作项目(3109120001)。

摘　　要：为建立一种检测鸽源鹦鹉热衣原体(Chlamydia psittaci,Cps)抗体的间接ELISA方法,将Cps的主要外膜蛋白(Major outer membrane protein,MOMP)基因分别克隆至2个原核表达载体,获得了2种带有不同标签的重组蛋白MOMP-GST和MOMP-His,利用MOMP-His重组蛋白免疫制备多克隆抗体,MOMPGST重组蛋白经抗MOMP的家兔源多抗验证后作为包被抗原,经条件优化,建立基于鸽源Cps的MOMP蛋白的间接ELISA方法。结果显示,成功表达了大小约为51 ku的MOMP-His和65 ku的MOMP-GST重组蛋白,制备的抗MOMP家兔源多克隆抗体效价在1∶409 600以上。该ELISA方法的最适条件为:抗原包被质量浓度为0.5 mg/L,抗原包被液为CBS,抗原包被条件为37℃1 h;封闭液为50 mL/L FBS,封闭条件为37℃2 h;血清稀释度为1∶200,血清孵育时间为30 min;酶标二抗稀释度为1∶8 000,酶标二抗孵育时间为1 h;底物反应时间为5 min。特异性试验结果显示,该方法仅能检测鸽源Cps阳性血清,与鸽Ⅰ型副黏病毒等其他常见鸽病毒阳性血清均无交叉反应。敏感性试验结果显示,当鸽阳性血清稀释到1∶1 600时检测结果仍为阳性,且批内批间的重复变异系数均在6%以内。结果表明,本研究建立的检测Cps的MOMP抗体的间接ELISA方法特异性强、敏感性高和重复性好,且可用于鸽Cps疫苗免疫抗体水平检测与流行病学调查。In order to establish an indirect ELISA method for the determination of antibodies against Chlamydia psittaci(Cps)of pigeon origin,the major outer membrane protein(MOMP)gene of Cps was cloned into two prokaryotic expression vectors,respectively,and two recombinant proteins MOMP-GST and MOMP-His with different tags were obtained.Polyclonal antibodies were prepared using recombinant MOMP-His protein immunization(New Zealand Large White rabbit or domestic rabbit),and recombinant MOMP-GST protein was verified by rabbit-sourced polyantibody against MOMP as a coating antigen,and conditions were optimized to establish an indirect ELISA method based on the MOMP of pigeon-derived Cps.The results showed that recombinant proteins MOMP-His about 51 ku in size and MOMP-GST 65 ku in size were successfully expressed,and the potency of the prepared anti-MOMP rabbit-derived polyclonal antibody was above 1∶409600.The optimal conditions of this ELISA method were as follows:the antigen encapsulation mass concentration was 0.5 mg/L,the antigen encapsulation solution was CBS,the antigen encapsulation condition was 37℃for 1 h,and the blocking solution was 50 mL/L FBS,the blocking condition was 37℃for 2 h,serum dilution was 1∶200,the incubation time of serum was 30 min,and the enzyme-labeled secondary antibody was diluted at 1∶8000,and the enzyme-labeled secondary antibody incubation time was 1 h.Substrate reaction time was 5 min.The results of specificity test showed that the method could only detect the positive serum of pigeon Cps,and there was no cross-reactivity with the positive sera of other common pigeon viruses,such as pigeon paramyxovirus typeⅠ.The results of the sensitivity test showed that the test result was still positive when the pigeon positive serum was diluted to 1∶1600,and the coefficients of variation for the replicates intra-and inter-batches were all within 6%.The results showed that the indirect ELISA method for the detection of MOMP antibodies to Cps was highly specific,sensitive and reproducible,an

关 键 词：鹦鹉热衣原体 MOMP蛋白 间接ELISA 

分 类 号：S852.67[农业科学—基础兽医学]