Prokaryotic Expression and Purification of JAK3 Protein in Golden Pompano (Trachinotus ovatus)

【Objective】The purpose of this study was to obtain purified recombinant protein of Trachinotus ovatus JAK3 (TroJAK3)to lay a foundation for understanding the function, interaction and antibody preparation of TroJAK3 protein. 【Method】In this study, the recombinant plasmid pET-32a-TroJAK3 was constructed by molecular biology,the BL21 competent cells of E. coli were transformed, and. SDS-PAGE and Western blot were used to detect the expression of TroJAK3 induced by IPTG.【Result】The PCR amplification fragment was 3 333 bp in length, and the recombinant plasmid pET-32a-TroJAK3 was successfully constructed, which was confirmed by double enzyme digestion, nucleotide sequencing and open reading frame. After being induced with a final concentration of 1 mmol/L IPTG at 37℃ for 4 h, SDS-PAGE assay showed that TroJAK3 recombinant protein was highly expressed in a form of inclusion body, with a molecular weight of about 140 ku.The purified recombinant protein was obtained by Ni-IDA resin column. Western blot detection showed that here was a band of 140 ku, indicating that TroJAK3 could be recognized by anti-his antibodies.【Conclusion】The recombinant plasmid pET-32a-TroJAK3 was successfully constructed and purified to obtain TroJAK3 fusion protein with high purity.