【摘要】 虾青蛋白对甲壳类水产品色泽的形成和调控具有重要作用。为探究克氏原螯虾虾青蛋白A2(PcCRA2)的基因结构及原核表达，作者通过基因克隆获得PcCRA2基因编码序列（cra2），采用实时荧光定量PCR(RT-qPCR)检测cra2基因在克氏原螯虾9种组织中的表达模式，并以大肠杆菌BL21(DE3)为宿主，进行PcCRA2异源重组表达。结果表明，克氏原螯虾cra2基因cDNA全长573 bp，编码190个氨基酸。生物信息学分析显示，虾青蛋白A2相对分子质量为21 158.9，理论等电点为5.59。氨基酸序列比对发现，克氏原螯虾cra2编码蛋白质序列与红螯螯虾相似度最高，为91.58%。RT-qPCR结果显示，cra2在所检组织中均有表达，在表皮的表达量最高（P<0.05）。构建了pET28a-cra2原核表达载体，并在大肠杆菌BL21(DE3)诱导表达。其最佳诱导表达条件为：接种4 h后加入0.5 mmol/L IPTG于30℃诱导6 h;UV-vis结果表明重组蛋白质能与虾青素特异性结合，最大吸收峰为505 nm。该研究结果为进一步研究虾青蛋白质的生物功能奠定基础。更多还原

【Abstract】 Crustacyanin plays an important role in the formation and regulation of the color in crustacean aquatic products. To study the gene structural feature and prokaryotic expression of Procambarus clarkii(P. clarkii) crustacyanin A2(PcCRA2), the coding sequence of PcCRA2 gene(cra2) was obtained by gene cloning. The expression patterns of cra2 in 9 different tissues of P.clarkii were detected by real-time fluorescence quantitative PCR(RT-qPCR). Escherichia coli BL21(DE3) was used as the host to conduct the heterologous recombinant expression of PcCRA2. The results showed that the full length of cra2 cDNA was 573 bp, encoding 190 amino acids.Bioinformatics analysis indicated that the relative molecular weight of PcCRA2 was 21 158.9, with a theoretical isoelectric point of 5.59. Amino acid alignment revealed that the protein coding sequence of cra2 had the highest similarity to that of C. quadricarinatus(91.58%). RT-qPCR demonstrated that cra2 was expressed in all tested tissues, with the highest expression in epidermis(P<0.05). The prokaryotic expression vector pET28a-cra2 was constructed and induced in DE3. The optimal expression condition was adding 0.5 mmol/L IPTG at 30 ℃ for 6 h after 4 h of inoculation. Results indicated that the recombinant protein could specifically bind to astaxanthin, with a maximum absorption peak at 505 nm. These results provide a foundation for further studies on the biological function of crustacyanin.更多还原