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| Truncated Expression of Porcine deltacoronavirus M Protein and Establishment of Indirect ELISA |
| SONG Dai-Li1, YANG Fu-Sheng1, CHEN Yu-Qiao1, SUN Fan-Yuan1, CHEN Rui1, CAO San-Jie1,2,3, HUANG Xiao-Bo1,2,3,* |
1 Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China;
2 Sichuan Science-observation Experimental station of Veterinary Drugs and Veterinary Diagnostic Technology, Ministry of Agriculture and Rural affairs, Wenjiang 611130, China;
3 National Teaching and Experiment Center of Animal, Sichuan Agricultural University, Wenjiang 611130, China |
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Abstract Porcine deltacoronavirus (PDCoV) is a new swine (Sus scrofa) enteropathogenic coronavirus, which can cause severe dehydration, vomiting and watery diarrhea in piglets, with worldwide distribution. In order to develop an indirect ELISA method for rapid detection of PDCoV antibody, based on the bioinformatics analysis of PDCoV M gene sequence, the M gene located at 76~651 bp was amplified and inserted into the expression vector pET-32a to construct the recombinant plasmid pET-32a-M. The pET-32a-M was transformed into Escherichia coil Transetta (DE3) and the recombinant M protein was expressed, and a target truncated protein with a size of 35 kD was obtained. Western blot test showed that M protein had good reactivity with porcine PDCoV positive serum. An indirect ELISA method was constructed based on purified recombinant M truncated protein, and the critical value was determined to be 0.271, and had no cross reaction with Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV-2), Pseudorabies virus (PRV), Foot-and-mouth disease virus (FMDV), Porcine epidemic diarrhea virus (PEDV), Swine transmissible gastroenteritis virus (TGEV). The coefficient of variation of intra- and inter-batch repeatability tests were less than 10%, and the total coincidence rate with the indirect ELISA based on PDCoV recombinant S1-CTD protein as coating antigen was 96%. The above results showed that the established indirect ELISA method had good specificity, sensitivity, and repeatability. The positive rate of PDCoV antibody was 49.11% in 507 pig serum samples collected in some areas of Sichuan province from 2012 to 2018, which preliminarily proved that the disease had been seriously infected in Sichuan. This study established an indirect ELISA method based on recombinant M truncated protein, which could provide technical support for PDCoV epidemic diagnosis and serum epidemiological investigation, and is of certain significance for PDCoV prevention and control.
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Received: 11 May 2021
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Corresponding Authors:
*rsghb110@126.com
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