Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (5): 273-280.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1181

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

QU Huan1(), LI Cheng1, CHEN Rui1, LIAO Yi-jie1, CAO San-jie1,2,3, WEN Yi-ping1, YAN Qi-gui1, HUANG Xiao-bo1,2,3()   

  1. 1. Research Center of Swine Disease,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130
    2. Sichuan Science-observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technology,Ministry of Agriculture and Rural Affairs,Chengdu 611130
    3. National Teaching and Experiment Center of Animal,Sichuan Agricultural University,Chengdu 611130
  • Received:2020-09-18 Online:2021-05-26 Published:2021-06-11
  • Contact: HUANG Xiao-bo E-mail:1157759897@qq.com;rsghb110@126.com

Abstract:

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 832-1 848 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 1 μg/well,the dilution concentration of serum was 1∶50,the detection positive threshold was OD450nm≥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%.

Key words: porcine deltacoronavirus, S1-CTD protein, prokaryotic expression, indirect ELISA