[Prokaryotic expression of the nucleocapsid protein of caprine parain-fluenza virus type 3 and establishment of a double-antigen sandwich ELISA method].

This study aims to establish a double-antigen sandwich ELISA method for detecting serum antibodies against caprine parainfluenza virus type 3(CPIV3), thereby supporting the clinical diagnosis of CPIV3 infection. According to the reference nucleocapsid (N) gene sequence (NCBI accession number: MT756864.1) published in NCBI, the N gene was synthesized and inserted into pET-30a to construct the recombinant plasmid pET-30a-N. The N protein was expressed in Escherichia coli and purified by Ni2+-affinity chromatography via its His tag. Reactivity of the purified N protein was verified by indirect ELISA and Western blotting. The His tag was removed with thrombin (1:1 000, W/W). The optimal coating concentration of the capture antigen, dilution of the HRP-conjugated detection antigen, and serum dilution were determined by checkerboard titration. Ultimately, a double-antigen sandwich ELISA method was established for clinical CPIV3 samples. Indirect ELISA and Western blotting confirmed that the N protein exhibited excellent antigenicity. The His tag was completely removed without altering the reactivity of the N protein. Checkerboard assays established the following optimal parameters: coating antigen concentration of 0.5 µg/mL, HRP-conjugated antigen dilution of 1:10 000, and serum dilution of 1:20. The cut-off value for the assay was set at 0.237 (OD450). No cross-reactivity was observed with positive sera against foot-and-mouth disease virus type O (FMDV-O), sheep/goat pox virus (GTPV), orf virus (ORFV), or Brucella. The sensitivity of the developed ELISA reached 1:512, being significantly higher than that of a commercial kit. Both intra- and inter-assay coefficients of variation were below 10%. Parallel testing of 150 field sheep sera with the Shengbo indirect ELISA kit yielded an overall concordance rate of 87%. The established double-antigen sandwich ELISA method demonstrates high specificity, sensitivity, and reproducibility. It provides an effective technical tool for large-scale surveillance, sero-epidemiological investigations, and disease control of CPIV3 infection.