p38L12-TAT融合肽对山梨糖醇引起的ECV304细胞ATF2磷酸化的抑制作用

Inhibition of ATF2 Phosphorylation by p38L12-TAT Fusion Peptide in ECV304 Cell Stimulated with Sorbitol

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.

A prokaryotic expression vector p38 Loop-12 fused with HIV-1 TAT sequence was constructed for the expression and purification of the fusion proteins for cellular transduction and functional identification in eukaryotic cell. Fragments of p38L12 and its mutant of dual phosphorylation sites （p38L12 （AF）） were amplified with PCR method, and then were inserted into a His-tagged TAT-EGFP fusion protein expression vector pET14b-His-TAT-EGFP （pHTE）. After identification with restriction digestion and DNA sequencing, two recombinant vectors were transformed into BL21 （DE3）. After the indution with IPTG, the recombinant protein of HTE-p38L12 and HTE-p38L12（AF） were isolated and resolved by SDS-PAGE. The fusion proteins were added to ECV304 ceils for the observation with fluorescence microscopy and evaluation of the transduction efficiency by Western blot assay. Furthermore, the effect of HET-p38L12 on endogenous p38 activity was determinted by the assay of ATF2 phosphorylation level after the stimulation with sorbitol on ECV304 cells. The expression vector of TAT fused with p38L12 or p38L12（AF） fragment were successfully constructed. Fusion proteins of HTE-p38L12 and HTE-p38L12（AF） were purified successfully and showed high efficiency of cellular transduction activity, resulting in a significant inhibition on the intracellular activity of ATF2 in ECV304 cells on the stimulation with sorbitol. Using the TAT-based intracellular transduction system, we confirmed that p38L12 and its dual phorsphorylation site mutant could inhibit the activity of endogenous p38 on the phosphorylation of ATF2 in ECV304 ceils with high osmotic stress.