重组人白介素21的原核表达与体外活性研究的优化

Optimization on Expression of Recombinant Human Interleukin 21 and Its Biological Activity Analysis In Vitro

为了构建重组人白介素21(rhIL-21)原核稳定表达和体外活性检测系统,采用Overlap-PCR法合成目的基因;经双酶切整合至pET28,化学转化法转入大肠杆菌BL21(DE3)构建pET-28a-rhIL-21 BL21(DE3)原核表达系统;胞内表达的包涵体经镍亲和层析色谱柱纯化后,尿素缓冲液梯度透析复性,最终溶于PBS缓冲液;双抗体夹心法ELISA、Western blotting检测复性蛋白的生物活性;细胞增殖抑制试验(MTT法)研究rhIL-21对Jurkat细胞株和HuT102细胞株的作用。实验成功建立了rhIL-21表达纯化与活性评价体系,为进一步研究IL-21的促免疫性疾病发展和抗肿瘤活性奠定了基础。

Interleukin-21 is the most recently discovered member of the type-I cytokine family.Structurally,IL-21 shows homology to IL-2,IL-4,and IL-15 proteins.IL-21 shares the common g-chain with the other three cytokines,in addition,binds to a unique IL21Ra chain,and activates the JAK / STAT pathway.IL-21 is mainly produced by activated T-cells but targets a broad range of lymphoid and myeloid cells of the immune system and therefore is able to regulate the innate and acquired immune responses.However,the studies of this cytokine have so far been hampered by the limited availability of recombinant protein preparations.This study established a system of prokaryotic expression of recombinant human interleukin-21（rhIL-21） and identified its biological activity in vitro.rhIL-21 cDNA was obtained using overlap-PCR and inserted into pET28 vector by double restriction endonuclease digesting,finally transformed into E.coli BL21（DE3） by chemical transformation to construct the pET-28a-rhIL-21 BL21（DE3） prokaryotic expression bacteria.The intracellular inclusion body was purified by nickel affinity chromatography,followed with the stage of refolding with gradient decreasing urea buffer.At last rhIL-21 was stored in PBS buffer.The biological activity of rhIL-21 was detected by double antibody enzyme linked immunosorbent assay and western blot.At the same time,the effects of rhIL-21 on Jurkat cell line and HuT102 cell line were studied by MTT method.This experiment successfully established the system of prokaryotic expression and exclaimed the biological function in vitro,which could benefit on the investigation of IL-21 in immune-diseases and cancers.The yield of rhIL-21 protein was 2.26 μg per liter medium after purification by one-step affinity chromatography and refolding.The rhIL-21 on Jurkat cell for 72h after stimulation by 0.5 μg / mL PHA and rhIL-21 on HuT102 cell for 24 h and 48 h showed that the effects of rhIL-21 on Jurkat cell and HuT102 cell were mainly stimulative.This study demonstrated that the rhIL-21 had good biological activity after refolding and could be applied for further immunology research.