Development and evaluation of an indirect ELISA based on recombinant nucleocapsid protein for detecting antibodies against porcine deltacoronavirus

Abstract:

[Objective] Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. PDCoV outbreak was first reported in the United States swine in 2014, and has subsequently been reported in many Asian countries. Nowadays, PDCoV has been becoming a great threat to swine industry worldwide. Using the recombinant nucleocapsid (N) protein expressed in E. coli Rosetta(DE3) as the coated antigen to establish an indirect ELISA for the detection of PDCoV antibody, providing a useful tool for antibody detection and epidemiology surveillance of PDCoV. [Methods] The full-length cDNA of PDCoV N gene was amplified by RT-PCR based on the PDCoV strain CHN-HN-2014. The RT-PCR production was inserted into prokaryotic expressing vector pET-30a to generate a recombinant plasmid pET30a-N. Then pET30a-N was transformed into E. coli Rosetta(DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG). The expressed N protein was purified and used as coated antigen to develop an indirect ELISA for detecting antibodies against PDCoV. After the specificity, sensibility, and repeatability of the developed ELISA method were evaluated, this method was used to test clinical serum samples. [Results] SDS-PAGE and Western blotting analysis showed that the N protein was efficiently expressed in supernatant and was specific reactions with antiserum against PDCoV. The established N-ELISA was highly specific, sensitive and repeatable. A total of 148 serum samples collected from pigs with a known immunization history and 102 serum samples collected from pigs with unknown PDCoV status were detected using both N-ELISA and neutralization test. The results showed that the positive agreement rate was 88.99%, the negative agreement rate was 92.90%, and overall coincidence rate was 91.20%. The positive rate of 267 clinical serum samples examined by N-ELISA was 66.67%. [Conclusion] The established ELISA method can be used as a tool for antibody detection and epidemiology surveillance of PDCoV.

Key words:porcine deltacoronavirus, nucleocapsid protein, indirect ELISA, neutralizing test