小鼠NGF基因治疗型DNA质粒的构建及中试工艺的优化

Construction of Therapeutic Mouse NGF DNA Plasmid and Optimization of Procedure for Pilot Production

目的构建小鼠NGF基因治疗型DNA质粒,并进行中试工艺的优化。方法用SignalP3.0 Server软件选择并设计与NGF融合的信号肽;根据哺乳动物密码子偏爱性,对小鼠NGF的编码序列进行优化;通过流加补料等方式优化中试生产工艺;体外瞬时转染测定DNA质粒的表达和体外生物学活性。结果所构建的含有IgG/ngf的重组质粒,通过中试规模的发酵和纯化,获得了大量的高纯度重组质粒,该重组质粒可以在体外表达出具有生物学活性的NGF。结论构建了具有与小鼠NGF相似的生物学活性的重组质粒,并建立了中试生产工艺。

Objective To construct a therapeutic mouse NGF DNA plasmid and optimize the procedure for pilot production of the plasmid.Methods The signal peptide fusing with mouse NGF was selected and designed by SignalP3.0 Server software. The ORF of fusion protein containing mouse NGF and signal peptide was optimized according to mammalian genetic coden bias. The pilotproduction procedure was optimized by fed-batch.The expression and biological activity in vitro of NGF DNA plasmid were analyzed by in vitro transient transfection-chick embryo dorsal root nerve ganglion culture. Results The recombinant plasmid containing IgG/ngf was successfully constructed and prepared in a large quantity by pilot fermentation and purification. The NGF with biological activity was expressed in vitro by using the constructed recombinant plasmid. Conclusion The constructed recombinant plasmid showed similar biological activity with that of mouse NGF,and a pilot production procedure was developed.