摘要
目的探讨胰腺相关转录因子Pax4是否具有蛋白质转导功能,及其进入细胞后的生物学功能。方法Ni柱亲和层析纯化原核表达的含His标签的Pax4全长蛋白质及一系列Pax4突变体与EGFP的融合蛋白。融合蛋白孵育的细胞,分别用于进胞检测和Pax4转导后的功能检测。结果外源纯化的Pax4蛋白能够转导进入HELF细胞。Paired-EGFP融合蛋白可以进入包括大鼠胰岛细胞在内的多种细胞。转导进入细胞的Pax4蛋白能够减少TNFα引发的胰岛细胞凋亡,增加胰岛细胞体外培养的存活率。结论研究结果表明转录因子Pax4具有蛋白质转导功能,完整的Paired结构域是Pax4的蛋白质转导结构域（PTD）,外源纯化的Pax4蛋白在转导入胰岛细胞有抑制细胞凋亡的作用。
Objective To examine whether the purified Pax4 protein can be transduced into cells,and to explore whether transduced Pax4 protein maintains its functional effect as the endogenous Pax4.Methods We constructed His-tagged intact Pax4 and several EGFP-tagged mutant Pax4 proteins.Each fusion protein was purified using a ninitrilotriacetic acid column from a bacterial expression system.The internalization of the protein was investigated by immunostaining and Western blot analysis.MTT and TUNEL assay were performed to characterize the effect of Pax4 treatment on protecting islet cell against apoptosis in culture.Results Pax4 protein was detected in HELF cells by Western blot analysis using anti-6-histidine antibody.Paired-EGFP but not the other fusion proteins can permeate into various cell types including pancreatic islets.Transduced Pax4 protein can protect TNFα-induced apoptosis and prolonged islet cell survival in culture.Conclusion These data suggest that Pax4 protein can permeate into HELF cells,the Paired Domain ofPax4 serves as a novel protein transduction domain（PTD）,and Pax4 protein transduction could be a safe and valuable strategy for protecting islet cell in culture from apoptosis.
