PROKARYOTIC EXPRESSION AND POLYCLONAL ANTIBODY PREPARATION OF AUTOPHAGY-RELATED GENES LC3B AND BECLIN1 OF YELLOW CATFISH PELTEOBAGRUS FULVIDRACO

In order to investigate the mechanism of autophagy in yellow catfish Pelteobagrus fulvidraco, we cloned P. fulvidraco LC3B (PfLC3B) and Beclin1 (PfBeclin1) genes that were 372 bp and 1344 bp, respectively, and constructed the plasmid pET32a(+)-PfLC3B and pET32a(+)-PfBeclin1 encoding recombinant PfLC3B and PfBeclin1 protein, respectively. The recombinant PfLC3B was highly purified by affinity chromatography, while the recombinant PfBeclin1 protein was highly purified under inclusion body conditions. The polyclonal anti-PfLC3B and anti-PfBeclin1 antibodies were generated by immunizing Balb/C mouse with recombinant PfLC3B and PfBeclin1 protein, respectively. The results of western blot showed that the polyclonal anti-PfLC3B and anti-PfBeclin1 antibodies could recognize recombinant and endogenous PfLC3B and PfBeclin1 protein, respectively. PfLC3 and PfBeclin1 were predominantly expressed in the liver, but not in the kidney and spleen. In conclusion, we generated polyclonal anti-PfLC3B and anti-PfBeclin1 antibodies, which provided powerful tools to investigate the mechanism of autophagy in yellow catfish.