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

文章克隆了草鱼（Ctenopharyngodon idellus）Stefin c DNA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Burton＇s 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Burton＇s 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 mol·L^-1IPTG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 k D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。

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 Burton＇s mouthbrooder（ Haplochromis burtoni） Stefin A1. Phylogenetic tree analysis indicats that grass carp Stefin A held together with Burton＇ s mouthbrooder,Colisa chuna（ Trichogaster chuna）,lamprologini（ Neolamprologus brichard）,elephant shark（ Callorhinchus milii） and bicolor damselfish（ Stegastes partitus）. Recombinant Stefin was expressed by 1 mol·L^- 1IPTG and the target protein was gradiently washed by urea and purified by Ni2＋-NTA agarose affinity chromatography. SDS-PAGE and HPLC of TSK-GEL G2000 SWxl were con-ducted 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 k D. And it also appeared as a single active peak on TSK-GEL G2000 SWxl 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 from carp. The results reveal that it can inhibit the activities of these two proteinases effectively.