摘要
为了建立一种猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)抗体检测方法,将PDCoV CH/XJYN/2016毒株N基因序列插入原核表达质粒载体pColdⅡ中,表达重组蛋白PDCoV-N1和PDCoV-N2,经SDS-PAGE分析和Ni+-NTA纯化后,检测重组原核蛋白PDCoV-N1和PDCoV-N2与猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)阳性血清的反应原性和特异性。同时对重组真核蛋白PDCoV-N1和PDCoV-N2进行Western blotting和间接免疫荧光实验验证分析。最后使用重组原核蛋白PDCoV-N2建立间接ELISA方法,优化反应条件,检测敏感性、特异性及重复性,并对102份临床血清进行了检测。重组原核蛋白PDCoV-N2与PEDV抗血清交叉反应较低。以PDCoV-N2蛋白制备的间接ELISA方法的最优条件为:抗原包被浓度1.25μg/mL,37℃包被1 h,BSA封闭液4℃过夜,血清最佳稀释浓度1:50,37℃孵育1 h,酶标二抗最佳稀释比例为1:80000,37℃孵育1 h,加入四甲基联苯胺(tetramethylbenzidine,TMB)显色液37℃反应10 min。待检样品的S/P值(样本值–阴性对照值)/(阳性对照值–阴性对照值)≥0.45为阳性,S/P值≤0.38为阴性,S/P值介于0.45和0.38之间,则为可疑。检测猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)、猪圆环病毒2型(porcine circovirus 2,PCV2)、猪传染性胃肠炎病毒(transmissiblegastroenteritis of swine,TGEV)、口蹄疫病毒(foot-and-mouth disease virus,FMDV)、非洲猪瘟病毒(african swine fever virus,ASFV)阳性血清,结果显示,其他冠状病毒的S/P值均小于0.38,表明其具有良好的特异性;PDCoV抗血清800倍稀释后仍为阳性;批间和批内重复性实验结果显示,本方法变异系数均小于10%;临床血清样本检测结果显示,该方法与中和实验的符合率为94.12%。本研究基于截短的PDCoV N蛋白,建立了能够检测抗PDCoV特异性IgG抗体的ELISA方法,且该方法敏感、特异、稳定、重复性好,为PDCoV的临床诊断提供了新的方法。
This study aims to establish an antibody detection method for porcine deltacoronavirus(PDCoV).The recombinant proteins PDCoV-N1 and PDCoV-N2 were expressed via the prokaryotic plasmid pColdII harboring the N gene sequence of the PDCoV strain CH/XJYN/2016.The reactivity and specificity of PDCoV-N1 and PDCoV-N2 with anti-PEDV sera were analyzed after the recombinant proteins were analyzed by SDS-PAGE and purified by the Ni-NTA Superflow Cartridge.Meanwhile,Western blotting and indirect immunofluorescence assay were carried out separately to validate the recombinant proteins PDCoV-N1 and PDCoV-N2.Finally,we established an indirect ELISA method based on the recombinant protein PDCoV-N2 after optimizing the conditions and tested the sensitivity,specificity,and reproducibility of the method.Then,the established method was employed to examine 102 clinical serum samples.The recombinant protein PDCoV-N2 showed low cross-reactivity with anti-PEDV sera.The optimal conditions of the indirect ELISA method based on PDCoV-N2 were as follows:the antigen coating concentration of 1.25μg/mL and coating at 37°C for 1 h;blocking by BSA overnight at 4°C;serum sample dilution at 1:50 and incubation at 37°C for 1 h;secondary antibody dilution at 1:80000 and incubation at 37°C for 1 h;color development with TMB chromogenic solution at 37°C for 10 min.The S/P value≥0.45,≤0.38,and between 0.45 and 0.38 indicated that the test sample was positive,negative,and suspicious,respectively.The testing results of the antisera against porcine epidemic diarrhea virus(PEDV),porcine circovirus 2(PCV2),transmissible gastroenteritis virus(TGEV),foot-and-mouth disease virus(FMDV),and African swine fever virus(ASFV)showed that the S/P values were all less than 0.38.The testing results of the 800-fold diluted anti-PDCoV sera were still positive.The results of the inter-and intra-batch tests showed that the coefficients of variation of this method were less than 10%.Clinical serum sample test results showed the coincidence rate between this method and neutralization test was 94.12%.In this study,an ELISA method for the detection of anti-PDCoV antibodies was successfully established based on the truncated N protein of PDCoV.This method is sensitive,specific,stable,and reproducible,serving as a new method for the clinical diagnosis of PDCoV.
作者
王东升
于瑞明
张莉萍
白英杰
刘霞
王永录
杜晓华
刘新生
WANG Dongsheng;YU Ruiming;ZHANG Liping;BAI Yingjie;LIU Xia;WANG Yonglu;DU Xiaohua;LIU Xinsheng(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,Gansu,China;State Key Laboratory for Animal Disease Control and Prevention,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,Gansu,China)
出处
《生物工程学报》
北大核心
2025年第7期2760-2773,共14页
Chinese Journal of Biotechnology
基金
甘肃省科技重大专项(23ZDNA007)。
