草鱼Stefin 克隆表达、纯化及活性特征鉴定
Clone, expression, purification and activity characterization of grass carp (Ctenopharyngodon idellus) Stefin
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摘要: 文章克隆了草鱼(Ctenopharyngodon idellus) Stefin cDNA 全长序列, 全长294 bp, 编码97个氨基酸, 无二硫键, N端存在高度保守的Gly(3、4)残基及QXVXG(45~49)序列, 比对结果显示其氨基酸序列与Burtons mouthbrooder (Haplochromis burtoni) Stefin A1一致性最高, 为47.5% 。进化树分析表明草鱼Stefin A与Burtons mouthbrooder (Haplochromis burtoni) 、southern platyfish (Xiphophorus maculatus) 、Colisa chuna (Trichogaster chuna) 、lamprologini (Neolamprologus brichard) 、elephant shark (Callorhinchus milii) 及bicolor damselfish (Stegastes partitus) Stefin A 聚为一类。将构建的原核表达载体Stefin-Pet30a转入E.coli BL21, 以1 molL-1 IPTG 诱导表达重组Stefin 蛋白, 而后经梯度尿素洗涤和镍亲和层析纯化, 并分别利用SDS-PAGE 和TSK-GEL G2000SWxl 高效液相色谱检测诱导及纯化效果, SDS-PAGE 结果显示重组Stefin 蛋白得到高度纯化, 最终呈现相对分子量11.4 kD的单一条带; 其在高效液相上保留时间25.98 min处亦呈单一活性峰, 纯度为96.28% 。以荧光合成肽底物(Z-Phe-Arg-MCA)测活法鉴定重组草鱼Stefin对鲤鱼组织蛋白酶B、L的抑制活性, 发现该重组蛋白对二者均体现了明显的抑制活性。Abstract: We cloned the Stefin gene of grass carp which was 294 bp, encoding a mature polypeptide of 97 amino acids lacking of disulfide bond and containing the typical conserved domain of Stefin (family Ⅰ) , such as Gly (3 and 4) and QXVXG(45~49) . Homology analysis indicates that grass carp Stefin A shared the highest amino acid sequence identity of 47.5% with Burtons mouthbrooder (Haplochromis burtoni) Stefin A1. Phylogenetic tree analysis indicats that grass carp Stefin A held together with Burtons mouthbrooder, Colisa chuna (Trichogaster chuna) , lamprologini (Neolamprologus brichard) , elephant shark (Callorhinchus milii) and bicolor damselfish (Stegastes partitus) . Recombinant Stefin was expressed by 1 molL-1 IPTG and the target protein was gradiently washed by urea and purified by Ni2+-NTA agarose affinity chromatography. SDS-PAGE and HPLC of TSK-GEL G2000SWxl were conducted to examine the results of expression and purification. The purified protein appeared as a single band on the SDS-PAGE, corresponding to a molecular weight of approximately 11.4 kD. And it also appeared as a single active peak on TSK-GEL G2000SWxl with purity of 96.28%. The activity assay (Z-Phe-Arg-MCA as a substrate) was finally performed to characterize the inhibitory effect of the recombinant Stefin to Cathepsin B and Cathepsin L fromcarp. The results reveal that it can inhibit the activities of these two proteinases effectively.