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构建了鼠源Reg3α蛋白基因工程表达菌E coli(T7 Expression),经IPTG诱导以包涵体形式表达。通过菌体裂解、洗涤和镍柱亲和层析等纯化步骤,SDS-PAGE和HPLC鉴定制得的重组Reg3α蛋白纯度为97.6%。细胞MTT和BrdU标记检测显示重组Reg3α蛋白能有效促进MIN6细胞和原代胰岛的增殖,Western blot方法证明其能够显著提高Erk1/2和Akt信号分子的磷酸化水平从而刺激细胞增殖。在重组Reg3α蛋白抵抗链脲霉素(Streptozotocin,Stz)诱导MIN6细胞凋亡实验中,未能检出剪切型Caspase-3水平的降低,但MTT结果显示其能维持活细胞数量。在毒胡萝卜内酯(Thapsigargin,Tg)诱导MIN6细胞内质网应激反应(Endoplasmic retieulum stress,ER stress)实验中,重组Reg3α蛋白能有效降低剪切型Caspase-3水平,同时增强关键分子伴侣GRP78的表达从而抑制细胞凋亡。重组Reg3α蛋白具有作为外源性药物促进胰岛β细胞增殖、抗凋亡的活性,具备根治糖尿病药物研究的潜力。
The murine Reg3α gene was constructed to express E. coli (T7), which was induced by IPTG. The recombinant Reg3α protein was identified by SDS-PAGE and HPLC. Purity was 97.6% through purification steps such as bacterial cell lysis, washing and nickel column affinity chromatography. The results of MTT assay and BrdU labeling assay showed that recombinant Reg3α protein could effectively promote the proliferation of MIN6 cells and primary islets. Western blot showed that the recombinant Reg3α protein could significantly enhance the phosphorylation of Erk1 / 2 and Akt signaling molecules and stimulate cell proliferation. In the experiment of recombinant Reg3α against Streptozotocin (STz) -induced MIN6 cell apoptosis, the decrease of the cleaved Caspase-3 level was not detected, but MTT results showed that it could maintain the number of viable cells. In the experiment of endoplasmic retieulum stress (MIN stress) induced by Thapsigargin (Tg) in MIN6 cells, recombinant Reg3α protein can effectively reduce the level of cleaved Caspase-3 and enhance the expression of key chaperone GRP78 Thus inhibiting the apoptosis. Recombinant Reg3α protein has the potential of promoting the proliferation and anti-apoptotic activity of pancreatic β-cells as an exogenous drug and has the potential to cure diabetes drugs.