猪δ冠状病毒S1-CTD的截短表达及间接ELISA抗体方法的建立  

Truncated Expression of the S1-CTD Fragment of Porcine Deltacoronavirus and Establishment of an Indirect ELISA for Detecting Its Antibody

作　　者：瞿欢 李成 陈汭 廖艺杰 曹三杰 文翼平[1] 颜其贵 黄小波 QU Huan;LI Cheng;CHEN Rui;LIAO Yi-jie;CAO San-jie;WEN Yi-ping;YAN Qi-gui;HUANG Xiao-bo(Research Center of Swine Disease,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130;Sichuan Science-observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technology,Ministry of Agriculture and Rural Affairs,Chengdu 611130;National Teaching and Experiment Center of Animal,Sichuan Agricultural University,Chengdu 611130)

机构地区：[1]四川农业大学动物医学院猪病研究中心,成都611130 [2]农业农村部兽用药物与兽医诊断技术四川科学观测实验站,成都611130 [3]四川农业大学国家级动物类实验教学示范中心,成都611130

出　　处：《生物技术通报》2021年第5期273-280,共8页Biotechnology Bulletin

基　　金：四川省科技计划(2020YFS0011)。

摘　　要：猪δ冠状病毒(Porcine deltacoronavirus,PDCoV)是一种能引起仔猪呕吐、腹泻和脱水的新的猪肠道冠状病毒,表达了PDCoV的S基因抗原表位区(S1-CTD)并建立了检测抗体的间接ELISA方法。根据S基因序列设计引物,扩增含中和抗原表位的S1-CTD区(位于S基因的832-1 848 bp),构建原核表达载体pET28a-S1-CTD,表达的重组S1-CTD蛋白经SDS-PAGE鉴定大小约41 kD,Western Blot证明其有良好的反应原性。以重组S1-CTD 蛋白为抗原建立间接ELISA,抗原最佳包被浓度为1μg/孔,待检血清的最佳稀释度为1∶50,阴阳性临界值为OD_(450nm)≥0.377。该ELISA检测其他8种常见猪病阳性血清无交叉反应,特异性好;批内和批间变异系数均小于10%,重复性好。ELISA与病毒中和试验的总符合率达83.3%。用建立的ELISA检测了2012-2017年四川部分猪场490份临床血清,结果显示血清中PDCoV抗体阳性率为49.18%。Porcine deltacoronavirus(PDCoV)is a novel porcine intestinal coronavirus that causes vomiting,diarrhea and dehydration of piglets.The epitope region(S1-CTD)expressing S gene of PDCoV was studied,and an indirect ELISA for the detection of PDCoV antibody was established.The primer was designed according to the S gene sequence,the S gene fragment containing epitope region(S1-CTD)(located at 832-1848 bp in S gene)was amplified and inserted into pET28a,and the recombinant plasmid pET28a-S1-CTD was constructed.The expressed recombinant S1-CTD protein was about 41 kD by SDS-PAGE and good reactogenicity was proved by Western Blot.The indirect ELISA was developed by using the recombinant S1-CTD protein as coating antigen,and the optimal conditions were as follows:the coating concentration of S1-CTD protein was 1μg/well,the dilution concentration of serum was 1∶50,the detection positive threshold was OD_(450nm)≥0.377.The ELISA was very specific,because there was no cross reaction with positive sera of other eight common swine diseases.The method was of high duplicability with the<10% variation of intra-and inter-batch coefficients.The coincidence rate between ELISA and virus neutralization test(VNT) was 83.3%.A total of 490 clinical swine sera samples collected from Sichuan province from 2012 to 2017 were detected by the ELISA,and the PDCoV-antibody positive rate was 49.18%.

关 键 词：猪丁型冠状病毒 S1-CTD蛋白 原核表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]