重组胰腺再生蛋白Reg3β的制备及初步活性研究

Recombinant Reg3β from E. coli System Stimulates MIN6 Cell Proliferation

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。

Regenerating genes（ Reg） were first discovered in 90% depancreatized rats during the period of islet regeneration,as a secretory protein and shown in vitro to be antiapoptotic and anti-inflammatory. In order to study the Reg3β protein in maintaining pancreatic β cell number and function,the activity of resistance deterioration of diabetes,we construct and expressed the recombinant Reg3β protein. In this study,c DNA of Reg3β protein was obtained by reverse transcriptase-PCR with a template of the total mRNA extracted from murine pancreas tissue by Trizol method. Then we produced recombinant Reg3β protein in p ET28a（ ＋）-rReg3β T7 transformed E. coli system,and the determinated protein was purified through a series of preliminary extraction steps,Ni2 ＋affinity chromatograph and stepwise dialysis. Ni2 ＋affinity chromatograph purification column eluted with gradient increasing concentration of immidazole,and finally stepwise dialysis using reducing ureal portion and desalting processes. The final purified recombinant Reg3βprotein with a purity of 95% above was produced and identified by SDS-PAGE,HPLC and Western blot methods. MTT results suggested that recombinant Reg3β protein stimulated mouse insulinoma cells（ MIN6） proliferation which was shown dose dependence apparently,and the highest value added rate can reach 60%. Moreover,Western blot densitometric quantification indicated that the phosphorylated Akt and Erk1 /2 levels were drastically increased at 15 min and 45 min respectively after the treatment. Conclusion：recombinant Reg3β protein built and obtained successfully in this study is capable of stimulating the proliferation of insulinoma cells by activating Akt and Erk1 /2 signaling pathways in vitro test. Therefore,recombinant Reg3β protein could be potent in protection and / or regeneration of pancreatic islet β-cells to treat diabetes mellitus.