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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。
In order to construct a prokaryotic expression system and in vitro activity assay system of recombinant human interleukin-21 (rhIL-21), the target gene was synthesized by Overlap-PCR and integrated into pET28 by double enzyme digestion. The recombinant plasmid was transformed into E.coli BL21 (DE3) -28a-rhIL-21 BL21 (DE3) prokaryotic expression system. The inclusion bodies expressed in the cytoplasm were purified by Ni-affinity chromatography, and were then refolded by gradient dialysis in urea buffer and finally dissolved in PBS buffer. The biological activity of renatured protein was detected by ELISA and Western blotting. The effect of rhIL-21 on Jurkat cell line and HuT102 cell line was studied by MTT assay. The successful establishment of rhIL-21 expression purification and activity evaluation system for further study of IL-21 to promote immune disease development and anti-tumor activity laid the foundation.