细胞穿透肽TAT融合小鼠nNOS1-133重组蛋白的表达纯化及药效学研究

目的:表达、纯化有活性的TAT-nNOS1-133重组蛋白,并初步验证其神经保护作用;为开发新神经保护药物提供理论基础。方法:通过PCR、酶切和酶连的方法构建pET28a-TATnNOS1-133重组质粒,并转化入大肠杆菌中表达。表达产物经洗涤、复性、阴离子交换柱(DEAE-52)、超滤纯化并除盐。通过FITC标记目的蛋白评价其能否进入细胞,并通过检测细胞凋亡评价TAT-nNOS1-133对谷氨酸诱导的兴奋性毒性的保护作用。结果:获得了较高纯度的TAT-nNOS1-133重组蛋白,经SDS-PAGE电泳显示相对分子质量在17 kD附近有单一条带,且与理论分子质量一致。TAT-nNOS1-133可以进入神经细胞,并减少了谷氨酸诱导的PI阳性细胞数增多。结论:成功构建了TAT-nNOS1-133的原核表达系统并建立了其分离纯化方案。TAT-nNOS1-133可以进入细胞并且减轻了脑卒中病理模型谷氨酸诱导的兴奋性毒性。

Prokaryotic Expression, Purification and Pharmacodynamics Research of the Recombinant Protein Containing Cell-penetrating TAT Peptide and nNOS1-133

Objective： The expression and purification of the recombinant protein TAT-nNOS1-133 and itseffects were investigated. Methods： The prokaryotic expression vector pET28a-TAT-nNOS1-133 was construct-ed and transformed into the expression strain of E.coli BL21 （DE3）. Subsequently, the transformed bacteriawere induced by lactose for expression of the recombinant protein. The recombinant protein accumulatedmainly in inclusion body was purified by the sequential application of washing, renaturation, ion exchangeand ultrafiltration. The transduction of the fusion protein labeled with FITC （FITC-TAT-nNOS1 -133 andFITC-BSA） in cortical neurons in vitro and its neuroprotective effects were also investigated. Results：SDS-PAGE revealed that tthe recombinant protein TAT-nNOS1-133 was of high purity. TAT-nNOS1-133 canget into neurons and protect neurons against neurotoxicity. Conclusion： TAT-nNOS1-133 can be successfullyexpressed in prokaryotic expression system and it protects neurons in vitro.