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构建p38Loop12(L12)的TAT融合表达载体,纯化原核表达的p38L12融合蛋白并鉴定其在真核细胞内的功能.利用PCR方法分别扩增出p38L12及其“TXY”双磷酸化位点的AF突变体p38L12(AF)片段,克隆入His标记的TATEGFP融合蛋白原核表达载体pHTE(pET14bHisTATEGFP),经酶切、测序鉴定正确后,将重组质粒转化原核表达菌,诱导表达纯化融合蛋白;将融合蛋白加入ECV304细胞后于荧光显微镜下观察并行Western印迹分析,检测融合蛋白的细胞内转导活性;通过检测内源性ATF2磷酸化水平,鉴定高渗刺激下p38L12对内源性p38活性的影响.成功构建了p38L12和p38L12(AF)片段与TAT的融合表达载体,并获得相应的融合蛋白.在ECV304细胞中可见导入的HTEp38L12和HTEp38L12(AF)融合蛋白具有较高的细胞内转导活性和转导效率,并可竞争性抑制高渗刺激对内源性p38的活化.基于HIV1TAT细胞转导系统证实p38L12可竞争性抑制高渗刺激诱导的内源性p38对ATF2的活化,从而发挥对p38激活特异性抑制的功能.
The TAT fusion expression vector p38Loop12 (L12) was constructed, and the prokaryotic expression of p38L12 fusion protein was purified and its function in eukaryotic cells was identified.The AF mutation of p38L12 and its “TXY” double phosphorylation sites were amplified by PCR The p38L12 (AF) fragment was cloned into the prokaryotic expression vector pHTE (pET14bHisTATEGFP) of His tagged TATEGFP fusion protein. After identification by restriction analysis and sequencing, the recombinant plasmid was transformed into prokaryotic expression vector to express and purify the fusion protein. ECV304 cells were observed under a fluorescence microscope and parallel Western blot analysis to detect the intracellular transduction activity of the fusion protein; By detecting the level of endogenous ATF2 phosphorylation, the effect of p38L12 on endogenous p38 activity was identified under hypertonic stimulation. Fusion expression vectors of p38L12 and p38L12 (AF) fragments with TAT were obtained, and the corresponding fusion proteins were obtained.In the ECV304 cells, the introduced HTEp38L12 and HTEp38L12 (AF) fusion proteins showed high intracellular transduction activity and transduction efficiency , And competitive inhibition of hypertonic stimulation of endogenous p38 activation based on HIV1TAT cell transduction system confirmed p38L12 competitive inhibition of hypertonic stimulation-induced ATF2 activation of endogenous p38, and thus play activation of p38 specific inhibition function.