海刺参组织蛋白酶L基因的克隆、重组表达及活性鉴定

通过RT-PCR和RACE PCR技术,从海刺参(Stichopus japonicus)肠中克隆得到编码组织蛋白酶L(SjCL)基因,其中全长cDNA 1 287 bp,5′非编码区(UTR)202 bp,3′UTR 87 bp,开放阅读框(ORF)999 bp,编码332个氨基酸(aa),包括组织蛋白酶L成熟肽316 aa和信号肽16 aa,分子质量预测为35.3 ku。用SjCL序列与相似物种海胆、海葵等进行对比,可知SjCL具有组织蛋白酶L所特有的氨基酸保守区域ERFNIN和GNFD,以及由Cys 139,His 278和Asn 299残基组成的保守活性位点。将编码SjCL基因克隆进原核表达载体pET32a(+)中,构建重组质粒pET32a(+)-SjCL,转化至大肠杆菌BL21(DE3)pLysS感受态细胞,再进行诱导表达、亲和纯化和透析复性,得到了纯化且具有活性的重组SjCL。酶活力检测结果表明,重组SjCL对组织蛋白酶L特异性底物Z-Phe-Arg-Nmec具有较高活性,而对组织蛋白酶B特异性底物Z-Arg-Arg-Nmec无活性。本实验对SjCL的cDNA序列进行了生物信息学分析并通过基因工程的手段表达出具有活性的重组SjCL,为进一步研究组织蛋白酶L在海刺参自溶过程中的作用提供理论基础。

Cloning, expression and assay of cathepsin L from the sea cucumber Stichopus japonicus

A cDNA coding cathepsin L was cloned from the intestine of the sea cucumber Stichopus japonicus by RT-PCR and RACE PCR techniques.The length of cDNA is 1 287 bp with 202 bp of 5′ UTR,87 bp of 3′ UTR and 999 bp of ORF.The ORF encoded protein of 332 aa including a signal peptide of 16 aa at the N-terminus and a mature peptide of 316 aa.The cathepsin L from S.japonicus showed significantly sequence homology with the sea urchin,sea anemone and others.Two highly conserved sequences ERFNIN and GNFD and three catalytic residues Cys 139,His 278 and Asn 299 were detected in the mutiple sequence alignment.To gain insight into the in vitro activities of SjCL,the gene encoding cathepsin L was subcloned into the expression vector pET32a(+) to construct the recombinant plasmid pET32a(+)-SjCL,and then transformed into E.coli BL21(DE3) pLysS.After induced by isopropylthio-β-D-galactoside(IPTG),the recombinant protein was expressed as inclusion bodies.The activity of recombinant SjCL protein was detected using the synthetic substrate Z-Phe-Arg-Nmec(substrate specific for cathepsin L),but not detected using synthetic substrate Z-Arg-Arg-Nmec(substrate specific for cathepsin B).