Abstract:

To establish a rapid detection method for swine acute diarrhea syndrome coronavirus (SADS-CoV) serum antibodies, N gene was amplified by PCR, and then the purified fragment was cloned into pET-32a (+) prokaryotic expression vector to construct the recombinant plasmid pET-32a-N. The expression of recombinant N protein was determined by SDS-PAGE and western blot. The purified recombinant N protein was applied as the coating antigen. Rabbit anti-pig HRP-IgG was used as the secondary antibody. We used the square-array titration to optimize the reaction conditions of the indirect ELISA method targeting SADS-CoV N protein antibodies. In addition, we identified the sensitivity, specificity and repeatability of this method, and evaluated its clinical applicability. SDS-PAGE and western blot results showed that the recombinant N protein was about 70 ku and had good reactivity. The antibody titer of SADS-CoV positive serum detected by this method was 1: 3 200, and there was no cross reaction with PEDV, TGEV, PDCoV, PRRSV, PoRV or PCV positive pig serum, which exhibited good sensitivity and specificity. The coefficient of variation of intra-batch and inter-batch repeatability tests were both less than 10%, demonstrating favorable reproducibility of this method. Fifty randomly selected porcine clinical serum samples were tested by ELISA and immunofluorescence assay, and the coincidence rate of results was 98%. In conclusion, we established an indirect ELISA based on N protein to test SADS-CoV clinical serum antibodies, which is useful for epidemiological surveillance, and is of great significance for prevention and control of SADS-CoV.

Key words: swine acute diarrhea syndrome coronavirus (SADS-CoV), N protein, prokaryotic expression, indirect ELISA