中国明对虾蜕皮抑制激素在大肠杆菌中的表达与分离纯化

采用大肠杆菌表达系统对中国明对虾蜕皮抑制激素(MIH)进行了体外重组表达研究.重组蛋白以包涵体的形式存在于菌体中.尿素-SDS-PAGE分析表明,经1mmol/L的IPTG诱导4h后,重组蛋白获得了大量表达,在分子量为13 kD处有一条与预测大小一致的特异性蛋白条带;经金属螯合柱纯化后,得到了电泳纯的融合蛋白.Western blotting检测结果表明:重组表达的融合蛋白与兔抗刀额新对虾MIH的多克隆抗体特异结合,证实该融合蛋白为中国明对虾MIH.通过胶内酶切与LC-ESI-MS分析进一步证实融合蛋白的部分肽段与日本对虾MIH一致.MIH融合蛋白的成功表达为进一步深入研究其在中国明对虾蜕皮过程分子作用机制奠定了基础.

Expression and purification of recombinant Fenneropenaeus chinensis molt-inhibiting hormone in E. coli

Molt-inhibiting hormone (MIH) of Fenneropenaeus chinensis was expressed in E.coli as inclusion body. Urea-SDS-PAGE analysis revealed that the fusion protein with a band of 13 kD was highly expressed after the induction with 1mmol/L IPTG for 4h. The interested protein was purified using the immobilized-metal affinity chromatography (IMAC) and a single band was seen by Urea-SDS-PAGE. The result of Western blotting indicated that the recombinant protein expressed by E.coli could specifically react with the antibody of MIH from Metapenaeus ensis. Furthermore, digestion in gel and identification using LC-ESI-MS confirmed that the recombinant protein was identical to partial fragment of MIH from Kuruma japonicus.The fusion protein will be proved useful for studies on the structure and function of MIH in F. chinensis.