Molecular Cloning, Characterization And Expression Analysis Of Cathepsins L, B From Cristaria Plicata

Cathepsin is one of the crucial enzyme superfamilies and involved in multiplicate process of physiological and pathological. The cpCathL and cpCathB cDNA full sequence and the Cathepsin L genome sequence has been cloned using degenerate primers by the rapid amplification of cDNA ends (RACE)PCR. mRNA expression of cathepsin L and cathepsin B in different tissue and after Aeromonas hydrophila stimulation in hemocytes, hepatopancreas and gills were determined using Real time quantitative RT-PCR analysis.1. The full-length cDNA of cpCathL contained1144bp, the cDNA contained a5’ untranslated region (UTR) of34nucleotides, the3’UTR of108bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of1002bp, encoding333amino acid residues with37.65kDa predicted molecular weight. The theoretical isoelectric point was8.61. The cpCathL gene is9353bp long and has a total of six introns and seven exons. The full-length cDNA of cpCathB contained1825bp, the cDNA contained a5’untranslated region (UTR) of36nucleotides, the3’UTR of745bp with a polyA tail, and an open reading frame (ORF) of1044bp, encoding347amino acid residues with38.55kDa predicted molecular weight. The theoretical isoelectric point was5.77. The prepro-cathepsin L and prepro-cathepsin B consist of a typical signal peptide, a pro-region peptideand a mature peptide, the three active centers formed by Cys、His and Asn, and the potential N-glycosylation site.2.CathL and CathB mRNA were expressed in every organizations, and the higher expressed level in hepatopancreas. Compared with PBS challenge, the expression of the cpCathL mRNA in hemocytes and hepatopancreas was inceased after Aeromonas hydrophila, the expression of the cpCathL mRNA in gill was significantly lower after Aeromonas hydrophila; The expression of the cpCathB mRNA in hemocytes and hepatopancreas was inceased after Aeromonas hydrophila, the expression of the cpCathB mRNA in gill was not significantly different after Aeromonas hydrophila.3.cDNA of cpCathL and cpCathB directional inserted into the pET32,and the recombinant expression plasmids CathepsinL-pET-32a and CathepsinB-pET-32a were constructed and plentiful expression. The polyclonal antibodies were maked by immunizing rabbits of the recombinant protein. Detection of antibody titer of cpCathB and cpCathL genes, maximum titers were as follows:1:1280000and1:640000.