中国明对虾组织蛋白酶L 的原核重组表达及其组织分布

组织蛋白酶L属于半胱氨酸蛋白酶中的木瓜蛋白酶超家族,在哺乳动物中它与抗原提呈、肿瘤入侵和转移、骨质吸收、细胞凋亡等许多生命活动有关。但对其在无脊椎动物中的功能了解的不多。从中国明对虾(Fenneropenaeus chinensis)中克隆得到了组织蛋白酶L基因,为了进一步研究组织蛋白酶L的组织分布及其功能,对该基因进行了重组表达。将中国明对虾组织蛋白酶L基因的酶原片段亚克隆进原核表达载体pET30a中,转化大肠杆菌BL21(DE3),再进行诱导表达,表达产物经SDS-PAGE电泳检测,其分子量在40kD左右,与期望的中国明对虾组织蛋白酶L的分子量一致。表达产物形成包涵体,经过对包涵体变性和复性,并进行His-tag柱亲和纯化,得到了纯化的蛋白。利用重组蛋白制备了的兔抗血清。Western实验表明,组织蛋白酶L在中国明对虾的肝、胃、肠中有表达,而在卵巢中没有表达。

Recombinant expression and tissue distribution of cathepsin L from Chinese shrimp Fenneropenaeus chinensis

Cathepsin L,a member of the papain superfamily of cysteine proteases,has been linked to tumor invasion and metastasis,antigen presentation,osteoclastic bone resorption and apoptosis in mammals,but the knowledge of cathepsin L from invertebrates remains underdeveloped.A cDNA of cathepsin L has been cloned from Fenneropenaeus chinensis (FCL).In order to analyze the function of FCL,it was recombinantly expressed in Escherichia coli.The fragment of FCL cDNA was subcloned into pET30a expression vector, and the recombinant plasmid was transformed into E.coli BL21 (DE3) and then induced by isopropylthio-b-D-galactoside(IPTG).SDS-PAGE analysis conf irmed the expression of FCL and the molecular mass was about 40 kDa,which was in accord with the expected.However,the expressed product formed complete inclusion bodies in the cells.After denature with 8 mol/L urea and refolding by dialysis in Tris-HCl buffer, the soluble target protein was obtained,which could be purifi ed by His Bind resin chromatography.The purif ied FCL was used as an antigen to immunize rabbits.After several injections,the polyclonal antiserum against FCL was obtained and used in immunoblotting experiments.Western blot analysis revealed that FCL was distributed in hepatopancreas, stomach and gut, but not in ovary.