Prokaryotic expression and polyclonal antibody preparation of Danio rerio angiogenesis related factor PTPRB

【Objective】Prokaryotic expression of zebrafish(Danio rerio) protein tyrosine phosphatase receptor B(PTPRB) and preparation of polyclonal antibodies were conducted to lay the foundation for studying the function of zebrafish PTPRB gene and signal transduction pathways related to vascular development.【Method】The zebrafish PTPRB gene was inserted into the prokaryotic expression vector pET-B2m to construct the recombinant expression plasmid by seamless cloning technology. The recombinant plasmid was transformed into Escherichia coli B21 competent cells, and induced to express by IPTG. Then the polyclonal antibody was prepared by immunizing rabbits with the induced fusion protein. The specificity and immune titer of the polyclonal antibody were detected by Western blotting and ELISA.【Result】The average hydrophilicity of zebrafish PTPRB protein was-0.490, which belonged to hydrophilic protein. It had abundant potential epitopes, distributed evenly and had no typical transmembrane region. The zebrafish PTPRB gene was inserted into the prokaryotic expression vector pET-B2m, and the recombinant expression plasmid pET-B2-PTPRB was successfully constructed. The recombinant expression plasmid was transformed into B21 competent cells and induced by IPTG to express a 35.0 kD fusion protein. The fusion protein PTPRB mainly existed in the form of inclusion bodies;the purified fusion protein PTPRB was used to immunize big-eared rabbits, and the serum antibody titer was 1:2048000, indicating that the fusion protein PTPRB could effectively stimulate the big-eared rabbits to produce a stronger immune response and obtain PTPRB polyclonal antibody with high titer. Western blotting results showed that PTPRB polyclonal antibody has good antigen specificity. High purity polyclonal antibody against PTPRB was obtained by using protein A/G affinity chromatography column. The concentration of the purified PTPRB polyclonal antibody was above 10 mg/mL.【Conclusion】The constructed zebrafish PTPRB gene prokaryotic expression vector can efficiently express the fusion protein PTPRB with good immunogenicity. High titer and high specificity polyclonal antibody against PTPRB can be obtained by immunizing big-eared rabbits with the fusion protein PTPRB, which provides a tool for studying the function of zebrafish PTPRB protein and a way to reveal the mechanism of PTPRB in fish vascular development.