Perparation of zebrafish zgc113227 polyclonal antibody and Trachinotus ovatus EVM0008813 peptide antibody

【Objective】To prepare the zebrafish zgc113227 polyclonal antibody and Trachinotus ovatus EVM0008813 polypeptide polyclonal antibody, so as to provide an important tool for further study on the functional characteristics of zebrafish zgc113227 gene and T. ovatus EVM0008813 gene.【Method】The zebrafish homologous gene zgc113227 (NP_001014341) was obtained by BLASTp search using the EVM0008813 gene of T. ovatus as the query sequence. The prokaryotic expression vectors were constructed by homologous recombination and transformed into competent cells of Escherichia coli B21 for induction and expression. The fusion proteins obtained by induction expression were used as antigens to immunize Japanese white rabbits to prepare polyclonal antibodies. The titers of the polyclonal antibody were determined by ELISA, the polyclonal antibody was identified by Western blotting and the specificity of polyclonal antibody against zgc113227 protein was detected by SDS-PAGE. The polyclonal antibody against EVM0008813 polypeptide was purified and identified by high performance liquid chromatography mass spectrometry(HPLC-MS).【Result】Both zebrafish zgc113227 and T. ovatus EVM0008813 were hydrophilic proteins, and their amino acid sequence similarity was 58.12%. At the same time, they showed many potential antigenic sites, strong flexibility, high surface accessibility, and no transmembrane structure and signal peptide were found. The conserved domains of zgc113227 and EVM000881 contained 5 conserved motifs(Motif 1-Motif 5), and one conserved domain(DDE_Tnp_4) was found. Therefore, it was speculated that they belonged to the DDE_Tnp_4 family. The titer of zgc113227 polyclonal antibody prepared by mixing zgc113227 protein with equal volume of Freund's complete adjuvant was 1:256000, which had good specificity and could be purified by Protein G affinity chromatography column. The titer of EVM0008813 polypeptide antibody was 1:256000, which was prepared by coupling EVM0008813 polypeptide with bovine serum albumin(BSA) as antigen. The purity of the antibody was 95.35% by HPLC-MS purification with good specificity.【Conclusion】The polyclonal antibodies of zebrafish zgc113227 protein and T. ovatus EVM0008813 polypeptide are prepared with high purity, high antibody titer and good specificity, which can provide a useful tool for the functional study of zebrafish zgc113227 gene and T. ovatus EVM0008813 gene.