论文部分内容阅读
构建重组p ET28a(+)-rReg3β表达质粒,转入T7 Expression E.coli表达菌,获得鼠源重组Reg3β表达菌株,经IPTG诱导后以包涵体形式表达重组Reg3β蛋白。重组Reg3β蛋白通过裂解菌体、镍柱亲和层析等方法进行分离纯化,纯化后透析复性,通过Western blot进行定性分析,SDS-PAGE及HPLC进行纯度分析。小鼠胰岛素瘤MIN6细胞系MTT检测显示重组Reg3β蛋白具有明显的促进细胞增殖作用,Western blot方法证明其能够显著提高Akt和Erk1/2信号分子的磷酸化水平从而刺激细胞增殖。结论:该课题成功构建和制备了重组Reg3β蛋白,并在体外实验中证实重组Reg3β具有促进胰岛β细胞系增殖的活性,并进一步探讨了其作用机制,可能在胰岛细胞的保护和再生领域具备较好前景。
Recombinant p ET28a (+) - rReg3β expression plasmid was constructed and transfected into T7 expression E. coli to obtain murine recombinant Reg3β expression strain. After induced by IPTG, the recombinant Reg3β protein was expressed as a inclusion body. The recombinant Reg3β protein was isolated and purified by means of lysing bacteria, nickel column affinity chromatography and the like. After purification, the dialyzate was refolded and qualitatively analyzed by Western blot. The purity of the recombinant Reg3β protein was analyzed by SDS-PAGE and HPLC. MTT assay showed that recombinant Reg3βprotein can significantly promote cell proliferation. Western blot showed that it can significantly increase the phosphorylation of Akt and Erk1 / 2 signaling molecules and stimulate cell proliferation. Conclusion: The recombinant Reg3β protein was successfully constructed and prepared in this study. In vitro experiments showed that recombinant Reg3β could promote the proliferation of islet β cell line and further explore its mechanism of action, which may be more in the field of islet cell protection and regeneration Good prospects.