猪δ冠状病毒抗体间接ELISA检测方法的建立  

Establishment of indirect ELISA for detection of porcine deltacoronavirus antibody

作　　者：尚再卓 于瑞明 张莉萍 王永录[2] 潘丽[2] 杜晓华[3] 刘霞[1] 刘新生[2] SHANG Zaizhuo;YU Ruiming?;ZHANG Liping;WANG Yonglu;PAN Li;DU Xiaohua;LIUXia;LIU Xinsheng(College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,China;Lanzhou Institute of Veterinary Medicine,Chinese Academy of Agricultural Sciences,Lanzhou 730030,China;College of Animal Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学生命科学技术学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,甘肃兰州730030 [3]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《中国兽医学报》2022年第12期2339-2346,共8页Chinese Journal of Veterinary Science

基　　金：“十三五”国家重点研发计划资助项目(2016YFD0501505)。

摘　　要：以大肠杆菌表达系统表达的猪δ冠状病毒(porcine deltacoronavirus,PDCoV)截短抗原S1-CTD蛋白作为包被抗原,建立能够检测血清特异性IgA抗体的间接ELISA方法。以本实验室分离保存的PDCoV流行株CH/XJYN/2016(MN064712)的S基因为模板设计合成特异性引物,通过RT-PCR扩增出S基因抗原表位区S1-CTD(877~1299 bp),构建原核表达载体pET24a-S1-CTD,经IPTG诱导表达重组蛋白S1-CTD,纯化后作为包被抗原建立了检测PDCoV特异性IgA抗体的间接ELISA方法。结果显示,抗原最佳包被质量浓度为8 mg/L,血清和酶标二抗最佳稀释比例为1∶10和1∶5000;S/P≥0.489判定为阳性,S/P<0.489判定为阴性。该方法与猪流行性腹泻病毒、猪嵴病病毒、口蹄疫病毒及猪瘟病毒的标准阳性血清均无交叉反应,特异性良好;批内和批间变异系数均小于10%,具有较好的重复性。间接ELISA检查方法的建立为PDCoV的血清学调查及免疫效果评价提供了有效的检测手段,为进一步开发临床检测试剂盒奠定基础。Using porcine deltacoronavirus(PDCoV)truncated antigen S1-CTD protein expressed in E.coli expression system as coating antigen,an indirect ELISA method of detecting serum specific IgA antibody was established.The S gene of the PDCoV endemic strain CH/XJYN/2016(MN064712)isolated and preserved in our laboratory was used as a template to design and synthesize specific primers,and the S gene antigenic epitope region S1-CTD(877-1299 bp)was amplified by RT-PCR to construct the prokaryotic expression vector pET24 a-S1-CTD,which was induced by IPTG to express the recombinant protein.Finally,the protein S1-CTD was purified and used as the encapsulated antigen to establish an indirect ELISA method for the detection of PDCoV-specific IgA antibodies.It was shown that the optimal concentration of antigen encapsulation was 8 mg/L,and the optimal dilution ratios of serum and enzyme secondary antibody were 1∶10 and 1∶5000.S/P≥0.489 was judged as positive and S/P<0.489 was judged as negative.Moreover,the method was specific and without any cross react with standard positive serum of porcine epidemic diarrhea virus,porcine crest disease virus,foot-and-mouth disease virus and classical swine fever virus.The variable coefficient of intra-and inter-batch were less than 10%,which indicated that repeatability was good.In summary,we provide an effective method for the serological investigation and immune effect evaluation of PDCo V,and lay the foundation for further development of clinical test kits.

关 键 词：猪δ冠状病毒 S1-CTD蛋白 间接ELISA IGA抗体 

分 类 号：S852.65[农业科学—基础兽医学]