Prokaryotic expression and polyclonal antibody preparation of Nesfatin-1 protein in Micropterus salmoides

Nesfatin-1 protein can affect fish adipogenesis and metabolism by regulating gene expression and signaling pathways. To investigate its physiological functions in largemouth bass (Micropterus salmoides), we constructed and purified a prokaryotic expression plasmid, and prepared a murine polyclonal antibody to this protein. The pET32a-Nesfatin-1 recombinant plasmid was obtained by amplifying the gene sequence of Nesfatin-1 protein and constructing a prokaryotic expression vector. The protein expression was induced by isopropyl-β-D-thiogalactosid (IPTG), and the fusion protein was purified by nickel ion affinity chromatography and immunized in Balb/c mice to obtain a polyclonal antibody against Nesfatin-1. The results show that the Nesfatin-1 protein had a gene sequence length of 246 bp; the concentration of the Nesfatin-1 fusion protein in the supernatant of the bacterial broth was 1.05 mg·mL⁻¹, and its relative molecular mass was 30 kD. Western blot analysis shows that the polyclonal antibody could specifically bind to the recombinant protein; the potency of the antibody reached over 1: 204 800 by indirect ELISA. The results of immunofluorescence detection indicates that the antibody specifically recognized the protein in the hepatopancreas of largemouth bass and was diffusely distributed in the hepatopancreas, showing strong positive reaction around the blood vessels. In this study, the Nesfatin-1 fusion protein of largemouth bass was successfully expressed and purified, and a specific murine polyclonal antibody was prepared, which can provide the functional protein and specific antibody for further study of the biological role.