人β神经生长因子在大肠杆菌中的高表达

THE HIGH EXPRESSION OF HUMAN β-NERVE GROWTH FACTOR IN E. COLI

将编码人β神经生长因子(Huβ-HGF)的基因克隆到由T7噬菌体启动子控制的pET11c大肠杆菌表达载体中,重组质粒经鉴定含有Huβ-NGF基因,未解聚的表达产物经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE),结果显示出二聚体27kD的蛋白带。而完全解聚的表达产物SDS-PAGE显示出一条13β5kD单体带。经凝胶电泳扫描,表达带占菌体总蛋白的14.5％。用兔抗鼠β—NGF的多克隆抗体进行的Western-Blot的结果表明,二聚体同单体都有免疫原性。在生物活性的鉴定中,菌体表达产物可以使小鸡鸡胚的背根神经节产生神经元突起,由此可以证明该表达产物有较高的生物活性。

The Hup-NGF gene encoding theβ subunit of mature human nerve growth factor was cloned into the pETllc vector under the control of T7 bacteriophage promoter. The SDS-PAGE electrophoresis results of nonreduced recombinant products showed a clear band corresponding to homodimeric (27kD) form of the molecule. But the SDS-PAGE results of reduced expression protein showed a single band corresponding to monomenc form of Hup-NGF (13.5kD). It represented appromximately 14.5% of the total cellular protein. Both of them were immunopositive on Western-Blots with rabbit anti-m β -NGF polyclonic antibodies. And the recombinant product was biologically active on cultured chicken dorsal root ganglion neurons. So it demonstrated the feasibility of synthesizing the biologically active forms of Hup-NGF in E. coli.