新型神经营养因子TAT—BDNF生物合成及生物活性研究

Biosynthesis and biological evaluation of a neotype neurotrophin TAT-BDNF

目的:合成新型神经营养因子TAT-BDNF并验证其生物活性,为进一步应用功能蛋白质治疗中枢神经损伤提供研究方法。方法:采用分子克隆方法构建带有TAT蛋白转导区序列及无信号肽序列人BDNF基因的原核表达载体pTAT-HA-BDNF。经原核表达系统表达、纯化、复性获得纯净TAT-BDNF融合蛋白。利用体外培养的新生大鼠小脑颗粒细胞,通过双重免疫荧光细胞染色检测其蛋白转导活性;Hoechst33342染色观察TAT-BDNF对颗粒细胞谷氨酸兴奋性毒性损伤神经保护作用。结果:重组质粒能有效的通过原核表达系统表达并获得高纯度TAT-BDNF。免疫荧光细胞染色显示TAT-BDNF能快速转导入小脑颗粒细胞中,并能显著减少由谷氨酸诱导的细胞凋亡坏死的比例,改善神经元的存活状态。结论:合成的新型神经营养因子TAT-BDNF具有蛋白转导活性及神经保护作用。

Objective： To synthesis a neotype neurotrophin TAT-BDNF and identify its biological activity, for further investigation of an alternative for treatment of central nervous system injury with functional protein. Methods：With molecular cloning technique, The recombinant vector termed pTAT-HA-BDNF was constructed, which could encode both TAT protein transduction domain and human brain-derived neurotrophic factor without signal peptide. Purified TAT-BDNF fusion protein were obtained by prokaryotic expression, purification and renaturation. Then it was added into cerebellar granule cells cultured in vitro, while Immunofluorescence analysis of the protein transduction effect of TAT-BDNF and observation of neuroprotective effect fi＇om glutamate-mediated excitotoxicity insulted with Hoechst33342 staining were performed. Results： The purified fusion proteins by prokaryotic expression were confirmed by SDS-PAGE. Double Immtmofluorescence assay showed that TAT-BDNF can rapidly be deliveried into cerebellum granular cell cultured in vitro. During the study ofexcitotoxic insult induced by glutamate, TAT-BDNF can decrease the apoptosis ratio and enhance neurons survival. Conclusions： TAT-BDNF fusion proteins produced by prokaryotic expression system possess biological effect of protein transduction and neuroprotection