目的 构建原核表达质粒,表达并纯化pTAT-HA-ARC和pTAT-HA-eGFP融合蛋白,初步探讨其转导神经元细胞的活性.方法 构建pTAT-HA-ARC和pTAT-HA-eGFP原核表达载体,将载体转化大肠杆菌,通过IPTG诱导表达目的蛋白,优化IPTG诱导浓度,通过镍亲和层析柱和脱盐柱纯化蛋白,SDS-PAGE和Western blot印迹鉴定目的蛋白;并通过荧光显微镜观察pTAT-HA-eGFP转导小鼠海马神经元细胞HT22的活性.结果 成功表达、纯化pTAT-HA-ARC和pTAT-HA-eGFP融合蛋白,本试验中优化的IPTG诱导浓度为0.1mM;pTAT-HA-eGFP融合蛋白能充分转导入HT22细胞中.结论 表达和纯化了能直接转导入HT22细胞的带有TAT-HA标签的融合蛋白,为进一步在体外和体内研究pTAT-HA-ARC蛋白对神经元的保护作用奠定了基础.%Objective To contruct the prokaryotic expression vectors,pTAT-HA-ARC and pTAT-HA-eGFP, to express and purify the fusion proteins and to investigate their transduction activities in neuronal cells. MethodsProkaryotic expression vectors,pTAT-HA-ARC and pTAT-HA-eGFP were constructed and then the constructed recombinant plasmids were transformed into E.coli and the target proteins were induced with IPTG. The inductive concentration of IPTG was optimized. The target proteins were purified by Ni+-NTA affinity chromatography and desalting columns,and identified by SDS-PAGE and Western blot assay. The transduction activities of pTAT-HA-eGFP on neuronal cells were observed directly under fluorescent microscope.ResultsThe fusion proteins of pTAT-HA-ARC and pTAT-HA-eGFP were successfully expressed and purified and the optimized inductive concentration of IPTG used in this experiment was 0.1mM. The fusion protein of pTAT-HA-eGFP could efficiently transduce into HT22 cells.ConclusionThe TAT-protein transduction technique was successfully established,and the fusion protein with TAT-HA tag which could directly transduce into HT22 cells have been successfully expressed and purified. It laid a foundation for the further investigation on the protective effects of fusion protein pTAT-HA-ARC in neuronal cells both in vivo and in vitro.
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