人白介素21Trx融合表达、纯化、复性以及活性检测

Human Interleukin 21 Fusion Expression, Purification, Refolding and Biological Activity Assay

全合成人IL-21基因,将IL-21编码区使用XhoI和NcoI位点插入pET32a构建原核表达载体,IPTG诱导表达,经DEAE-SepheroseFF柱和HisTrapFF柱纯化,SDS-PAGE和Western印迹检测融合蛋白的表达,MTT法检测Trx-hIL-21对Jurkat细胞的作用。获得了人IL-21的基因序列,SDS-PAGE检测可见33ku大小的融合蛋白表达,Western印迹表明其可以与抗IL-21单抗反应。Trx-hIL-21以剂量依赖的方式作用于Jurkat细胞的生长,当PHA浓度小于2μg/ml时,IL-21能刺激细胞增殖;当PHA浓度大于2μg/ml时,IL-21能抑制细胞增殖。

The gene of human IL- 21 was achieved by full synthesis and PCR walking. The coding gene was insert into pET32a vector with Nco I and Xho I sites. After being induced by IPTG, the target protein was extracted from the lysis of Rosetta-gami（DE3）in the form of inclusion bodies. Inclusion bodies were purified by DEAE-Sepherose FF and HisTrap FF respectively. The purified protein was refolded by dialysis with urea gradients. SDS-PAGE and Western blotting were used to detect Trx-hIL- 21 and determined that the fusion protein was 33kDa and reacted with anti-IL- 21 antibody. Finally, Jurkat cell was used to assay the biological acticity of refolded Trx-hIL- 21 under different concentrations of PHA. When the concentration of PHA was lower than 2μg/ml, Trx-hlL - 21 stimulated Jurkat cell proliferation; if the concentration of PHA was higher than 2μg/ml, Trx-hIL- 21 inhibited Jurkat cell proliferation.