鸡传染性喉气管炎病毒gG蛋白间接竞争ELISA方法的建立及初步应用  

Establishment and preliminary application of an indirect competitive ELISA for detecting the gG protein of infectious laryngotracheitis virus

作　　者：郭鹏宇 赵妍[1] 刘胜旺[1] GUO Peng-yu;ZHAO Yan;LIU Sheng-wang(State Key Laboratory for Animal Disease Control and Prevention/Avian Respiratory Diseases Division,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所,动物疫病防控全国重点实验室,禽呼吸道传染病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2023年第7期704-709,728,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金(团队项目)(TD2021C001);现代农业产业技术体系岗位科学家专(CARS-40-K18)。

摘　　要：鸡传染性喉气管炎是由鸡传染性喉气管炎病毒(ILTV)引起鸡的一种急性、高度接触性呼吸道传染病。为建立检测该病毒g G蛋白的间接竞争ELISA方法,本研究以原核系统表达ILTV g G蛋白,并采用切胶纯化该重组g G蛋白(rg G)后经western blot检测纯化的rg G (P-rg G)与本研究室制备的g G蛋白单克隆抗体(MAb) 3C8的反应原性。将MAb 3C8采用Protein G亲和层析柱纯化后与HRP偶联(HRP-3C8),利用间接ELISA法测定纯化HRP-3C8的效价。结果显示,P-rg G与MAb 3C8发生了特异性反应,在30 ku处出现特异性条带。纯化的HRP-3C8效价达1∶51 200。以P-rg G作为包被抗原,以纯化的HRP-3C8作为检测抗体,通过对各反应条件优化后初步建立了ILTV g G蛋白间接竞争ELISA (ic-ELISA)检测方法。条件优化结果显示,P-rg G包被量为12.5 ng/孔,HRP-3C8抗体稀释度为1∶10 000,将待检样品与HRP-3C8抗体在37℃孵育40 min后加入ELISA板再反应20 min,TMB底物反应10 min。将待检样品的抑制率(PI)≥31.08%判为阳性;采用该方法分别检测ILTV、新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、鸡痘病毒(FPV),通过计算PI,评估该方法的特异性;按照Reed-Muench法测定ILTV K317株的鸡胚半数感染量(EID_(50)),再将该病毒2倍倍比稀释(1∶10~1∶2 560)后,以该ic-ELISA方法检测,评估该方法的敏感性;利用同批次和不同批次P-rg G分别包被ELISA板,采用该ic-ELISA方法检测ILTV阳性及阴性样品,评估该方法的重复性。结果显示,该方法除了能检测ILTV外,其余相关病毒均为阴性结果;将ILTV稀释至1∶320时检测结果仍为阳性,相当于75 EID_(50)/0.1 m L;批内、批间重复性试验的变异系数均小于10%。利用该方法及荧光定量PCR (q PCR)分别检测50份临床ILTV、IBV和FPV感染鸡的口咽拭子和喉头组织、10份ILTV活疫苗,该方法检测结果显示,阳性检测率为73.3%(44/60),阴性率为26.7%(16/60)。q PCR的检测结果显示,阳性检测率为81.7%(Infectious laryngotracheitis is an acute and highly contagious respiratory disease caused by infectious laryngotracheitis virus (ILTV).In order to establish an indirect competitive ELISA (ic-ELISA) method for the detection of g G protein of the virus,ILTV g G protein was expressed in prokaryotic system,and the recombinant g G protein (rg G) was purified(P-rg G) by gel slices,and the reactivity of P-rg G with monoclonal antibody (MAb) 3C8 of g G protein prepared in our laboratory was detected by western blot.MAb 3C8 was purified by Protein G affinity chromatography and coupled with HRP (HRP-3C8).The titer of the purified HRP-3C8 was determined by indirect ELISA.The results showed that P-rg G reacted specifically with MAb3C8,and specific bands appeared at 30ku.The titer of purified HRP-3C8 was 1∶51200.The P-rg G and purified HRP-3C8 were used as the coated antigen and the detection antibody,respectively.An ic-ELISA method for ILTV g G protein was established by optimizing the reaction conditions.The results showed that the coated amount of P-rg G was 12.5ng/well and the dilution of HRP-3C8 antibody was 1∶10000.The samples to be tested were incubated with HRP-3C8 antibody at 37℃for 40min and then added to ELISA plate for 20min,and the reaction time of TMB substrate was 10min.The inhibition rate (PI)≥31.08%of tested samples was considered as positive.The method was used to detect ILTV,Newcastle disease virus,infectious bronchitis virus(IBV) and fowlpox virus (FPV),and the specificity of the method was evaluated by calculating the inhibition rate PI.The 50%egg infectious dose (EID_(50)) of ILTV K317 strain was measured by Reed-Muench method,and then the virus was diluted by a 2×ratio(1∶10-1∶2560),and then detected by ic-ELISA method to evaluate the sensitivity of the method.The P-rg G from the same batch and different batches were coated with ELISA plates respectively,and the positive and negative ILTV samples were detected by the ic-ELISA method to evaluate the repeatability of the method.The results sho

关 键 词：鸡传染性喉气管炎病毒 gG蛋白 单克隆抗体 间接竞争ELISA 

分 类 号：S852.65[农业科学—基础兽医学]