Abstract:Crustacyanin plays an importantant role in the formation and regulation of the color of crustacean aquatic products. To study gene structural feature and prokaryotic expression of Procambarus clarkii crustacyanin A2 (PcCRA2), the coding sequence of PcCRA2 gene (cra2) was obtained by gene cloning. The expression patterns of cra2 in 9 tissues of P. clarkii were detected by real-time fluorescence quantitative PCR (RT-qPCR). Escherichia coli BL21(DE3) was used as host, recombinant expression of PcCRA2 was performed. The results showed that the full length of cra2 cDNA was 573 bp, which encoded 190 amino acids. Bioinformatics analysis showed that the relative molecular weight of PcCRA2 was 21158.9Da, the theoretical isoelectric point was 5.59. A multiple protein sequence alignment revealed that the amino acid sequence of PcCRA2 has the highest similarity with C. quadricarinatus (91.58%). RT-qPCR showed that cra2 was expressed in all the tested tissues, with the highest expression in epidermis (P<0.05). The prokaryotic expression vector pET28a-cra2 was constructed and induced in E. coli BL21. The best expression conditions for recombinant PcCRA2 is that 0.5 mmol/L IPTG was added 4 h after inoculation and induced at 30 ℃ for 6 h. UV-vis results showed that PcCRA2 is bond to astaxanthin specifically, and the maximum absorption peak was 505 nm. These results provide a foundation for further study of the biological function of crustacyanin.
