Molecular Cloning, Prokaryotic Expression And Enzymatic Activity Analysis Of CAT From Cristaria Plicatea

Cristaria plicala is one of the most important "Freshwater pearl mussel" in our country. But recently Cristaria plicata is vulnerable to disease frequently, which makes a great influence to the ability of the pearl. The catalase cDNA of C. plicata (cpCAT) was cloned and the full length was expressed with Prokaryotic expression in the paper.The cpCAT cDNA full sequence has been cloned using homologous cloning and RACE-PCR. The gene is 4863 bp long and has a total of two introns and three exons. In addition the full-length cDNA of cpCAT contained 2618 bp, The cDNA contained a 5'untranslated region (UTR) of 136 nucleotides, the 3'UTR of 979 bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1503 bp, encoding 501 amino acid residues with 56.86 kDa predicted molecular weight. The theoretical isoelectric point was 6.77. BLAST analysis showed that the deduced amino acid sequence of cpCAT had significant homology to catalases from animals, plants and bacteria. The deduced amino acid sequence of cpCAT had characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77).heme-ligand signature motif (351RLYSYSDTH359), two glycosylation sites (N145, N436), NADPH binding site and the three catalytic amino acid residues (His72, Asnl45 and Tyr355). It had no signal peptide. The phylogenetic tree indicated that cpCAT gene was very close to the gene of scallops, Chlamysfarreri.Expression of cpCAT after injected by Aeromonas hydrophila was determined by RT-PCR. The results showed that after injected by A. hydrophila. The expression of cpCAT in different tissues of Cristaria plicata were determined by RT-PCR and the results showed the expression of cpCAT were detected in mantel, conductor muscle, hepatopancreas, sexual gland and hemocyte.and the best expression in hepatopancreas. The results showed that after injected by A. hydrophila, the expression of cpCAT were increased in all the tissues, the expression of cpCAT were increased at first 12h, and decreased thereafter, however recovery the original level at 48h, which proved the cpCAT was important immune system in C. plicata. According to the ORF, designed site (KpnI and EcoRI) primers, the constructed recombinant expression plasmids were induced by the chemical inducer IPTG. The expression products were analyzed by SDS-PAGE. The results indicated that recombination cpCAT was successfully expressed in Escherichia Coli. The expression of protein was 56.86 KDa. The enzymatic activity of purified recombinant cpCAT was 11194.4±40.4 U/mg, it might resist against H2O2.The recombinant enzyme holded higher thermal stability, the optimum temperature was 25℃, The stability of the recombinant enzyme were higher between pH 5 and 10, and the optimal pH value was 7.0. when cpCAT was treated with 2-4moL/L urea and 1%～3% SDS, the activity was also stable, it kept more than 80% activity. With the increasing of the concentration of denaturation agent, the activity of cpCAT reduced gradually.