中华绒螯蟹卵巢EJO1基因在大肠杆菌BL21(DE3)中的表达

扩增中华绒螯蟹(Eriocheir sinensis)卵巢EJO1基因(GenBank检索号:AY185917)的cDNA全序列得到编码区序列,并将其插入原核表达载体pET28b中,构建成表达质粒pET28b-EJO1。该表达质粒在大肠杆菌(Esche-richia coli)BL21(DE3)中经IPTG诱导表达,得到EJO1-H IS6融合蛋白。SDS-PAGE电泳分析表明,该融合蛋白的相对分子质量约为32 000。经镍离子柱亲和层析进一步纯化得到纯度较高的EJO1-H IS6融合蛋白。

Expression of Eriocheir sinensis Ovarian EJO1 Gene in E. Coli BL21(DE3)

EJO1(GenBank accessing number:AY185917)is an ovarian gene of Chinese mitten crab(Eriocheir sinensis)presumably involved in the ovary development.The complete mature peptide-coding cDNA fragment of EJO1 was obtained by PCR.The product was inserted into the prokaryotic expression vector,pET28b,to produce the expression vector pET28b-EJO1.The recombinant plasmid was transformed into E.coli BL21(DE3).EJO1-HIS_6 fusion protein was obtained after the addition of IPTG into the growth media.SDS-PAGE analysis revealed that the molecular weight of EJO1-HIS_6 fusion protein was about 32 kDa.Purified fusion protein was obtained after purification with NI-NTA His-Bind resin column.