Prokaryotic expression, antibody preparation, and identification of a cyclin B protein in the Chinese mitten crab Eriocheir sinensis and Malaysian giant prawn Macrobrachium rosenbergii

Abstract

We amplified the open reading frames(ORFs) of the cyclin B gene from the ovarian cDNA of the Chi-nese mitten crab Eriocheir sinensis and the Malaysian giant prawn Macrobrachium rosenbergii.The ORFs from these two species were then ligated into the expression plasmids pGEX-2T and pET32a,respectively.Following transfer of the recombinant plasmids into a host bacteria,Escherichia coli BL21(DE3),the recombinant proteins were expressed as inclusion bodies by induction with IPTG.The optimum concentration of IPTG was 0.1 mmol/L for 4 h at 37?C for GST-EsCB and 0.1 mmol/L PTG for 6 h at 30?C for Trx-MrCB.The recombinant proteins were subsequently purified by affinity chromatography.The cyclin B antibody was further refined by immunizing New Zealand white rabbits with the purified protein.The highest resulting antiserum titer was 1∶80 000(ELISA).We verified the specificity of the antibodies against the recombinant protein and ovarian cyclin B protein by western blot.We isolated two isoforms of the cyclin B protein from the immature crab ovaries,but only one form from the prawn ovaries.Thus,our data suggest that the role of cyclin B in the regulation of oocyte maturation may differ between crabs and prawns.The cyclin B antibody will be useful for further study of the role of cyclin B on MPF activation and oocyte maturation.