PROKARYOTIC EXPRESSION AND POLYCLONAL ANTIBODY PREPARATION OF CU HOMEOSTASIS-RELATED GENES CCS AND COX17 IN YELLOW CATFISH TACHYSURUS FULVIDRACO

CCS (Copper chaperone for superoxide dismutase) and COX17 (Cytochrome c oxidase copper chaperone 17) proteins are two common copper (Cu) chaperone proteins that play an important role in the regulation of Cu homeostasis. In order to further investigate the regulatory mechanism of Cu homeostasis in yellow catfish Tachysurus fulvidraco, 810 and 192 bp coding sequences of T. fulvidraco ccs (Pfccs) and T. fulvidraco cox17 (Pfcox17) genes were cloned from the cDNA of yellow catfish, and the recombinant expression vectors pET32a(+)-Pfccs and pET32a(+)-Pfcox17 were successfully constructed. Recombinant plasmids were transformed into E. coli Rosetta (DE3) competent cells, IPTG (Isopropyl-β-D-thiogalactopyranoside) was used as an inducer to induce expression, and the expression of recombinant proteins was identified by SDS-PAGE. We found that PfCCS protein was mainly expressed in the sediment, and PfCOX17 protein was mainly expressed in the supernatant. The purified recombinant PfCCS and PfCOX17 proteins were obtained by inclusion and affinity chromatography, respectively. Polyclonal antibodies were prepared by immunizing Balb/C mice three times with purified PfCCS and PfCOX17 proteins. Western Blot analysis showed that PfCCS and PfCOX17 antisera could specifically recognize recombinant PfCCS and PfCOX17 proteins, and could also specifically recognize endogenous CCS and COX17 proteins of yellow catfish, respectively. The distribution of CCS and COX17 proteins in gill, heart and liver was detected by Western Blot, and the expression levels of CCS and COX17 proteins were higher in liver, the expression levels of CCS protein in gill were the lowest, and the expression levels of COX17 protein in gill and heart were very low. In conclusion, this study successfully prepared polyclonal antibodies that can specifically recognize recombinant PfCCS and PfCOX17 proteins, and murine PfCCS and PfCOX17 antibodies can also be well used in the Western Blot detection of CCS and COX17 protein of yellow catfish, which laid a foundation for further study on the roles of CCS and COX17 protein in Cu transport and regulatory mechanism of Cu homeostasis in yellow catfish.