日本囊对虾组织蛋白酶B基因的原核表达及纯化

Prokaryotic expression and purification of cathepsin B in shrimp,Marsupenaeus japonicus

采用原核表达的方法得到日本囊对虾(Marsupenaeus japonicus)组织蛋白酶B基因的重组蛋白。以日本囊对虾卵巢组织为试验材料,采用双标签(GST和His)的方法,用带有EcoRⅠ和NotⅠ酶切位点以及6×His-tag的特异引物扩增Cathepsin B的开放阅读框,并连接至表达质粒pGEX-4T-2中。将重组表达质粒导入大肠杆菌BL21中,在30℃条件下,用终浓度为1mmol/L的IPTG(isopropylβ-D-1-thiogalactopyranoside)诱导5h得到最佳诱导量,His亲和柱进行纯化,纯化蛋白依次于8、6、4、2、1、0 mol/L尿素缓冲液中梯度透析,得到复性后可溶于水的重组蛋白,经SDS-PAGE检测,得到单一条带,其分子质量约为63ku。取冻干后的蛋白制备多克隆抗体,抗体效价达50 000,经Western blot检测,同样可在63ku处得到该条带,表明制备的组织蛋白酶B(CB)多克隆抗体具有特异性,该抗体可特异识别CB蛋白。

The recombinant protein of cathepsin B in Marsupenaeus japonicus was obtained by proKaryotic expression.Opening reading frame of the cathepsin B gene was amplified with specific primer which contain EcoR 1 and Not Ⅰrestriction sites and 6 × His-tag from the ovary, connected with expression vector pGEX-4T-2 and introduced into E. coli BL21.The optimal induction was acquired at 5 h with IPTG concentration of 1 mmol/L at 30 ℃.The recombinant protein was purified by His affinity column. A series of urea buffers （8 mol/L, 6 tool/L, 4 mol/L, 2 mol/L, 1 mol/L and 0 mol/L） were used for gradient dialysis.The soluble recombinant proteins were preserved in -80 ℃ after freeze-drying.The molecular weight of the protein was 63 ku measured by SDS-PAGE.The polyclonal antibody was prepared by using the freeze-dried proteins,and the antiserum titer of antibody was 50 000 by ELISA. The specificity of it can be detected by Western blot.The preparation of antibody laid the foundation for protein expression and gene function study of cathepsin B.