人白细胞介素24基因的克隆及在大肠杆菌中的高效表达

Cloning of human interleukin-24 gene and its high efficiency expression in E. coli

目的:构建人白细胞介素24(hIL-24)基因原核表达载体,在大肠杆菌中诱导表达hIL-24蛋白,并测定其生物学活性。方法:用PCR方法从含有hIL-24的质粒中扩增hIL-24cDNA序列,测序鉴定正确后,应用基因重组技术构建PQE/hIL-24,并转入大肠杆菌(E.coli)M15。IPTG诱导表达目的蛋白,镍凝胶亲和层析纯化目的蛋白,SDS-PAGE电泳和免疫印迹分析鉴定结果,应用外周血单核细胞(PBMCs),通过ELESA法分别在48和72h检测rhIL-24刺激的PBMCs中IL-6、IFN-γ及TNF-α产生情况。结果:获得与GenBank中报道一致的hIL-24基因片段。SDS-PAGE电泳和免疫印迹分析显示,获得具有hIL-24免疫原性、相对分子质量约为18400目的蛋白。Ni2+-NTAagarose纯化得到单一条带蛋白,获得rhIL-24蛋白刺激的PBMCs,PBMCs分泌IL-6、IFN-γ、TNF-α的水平显著高于未刺激前分泌水平(P<0.05),rhIL-24具有与天然hIL-24蛋白相同的生物学活性。结论成功地构建了hIL-24的原核表达载体,在E.coli中高效表达了rhIL-24蛋白,获得了与天然hIL-24蛋白具有相同生物学活性的rhIL-24。

Objective To construct a recombinant expression vector of human interleukin-24 （hIL-24） gene and express it in E. coli M15, and to evaluate the bioactivity of IL-24 fusion protein. Methods The human IL-24 cDNA fragment was amplified from plasmid by polymerase chain reaction （PCR）, and sequenced. PQE/hIL-24 was constructed by gene rearrangement, then it was transformed into E. coli M15. The expression of the target protein was induced with IPTG and purified by Ni^2＋-NTA agarose column. The expressed recombinant hIL-24 （rhIL-24） was identified by SDS-PAGE and Western blotting. Normal peripheral blood mononuclear cells （PBMCs） were cultivated with the expression protein for 48 and 72 h, the levels of IL-6, IFN-γ and TNF-α of PBMCs stimulated with rhIL-24 were detected by ELISA. Results The recombinant prokaryotic expression vector PQE/ IL-24 was constructed successfully and expressed stably in E. coli M15. At about 18 400 of molecular weight, there was an induced protein band. The levels of IFN-γ, IL-6 and TNF-α in the group of cultivated with the expression protein were obviously higher than those in the groups without the expresson protein （P〈0.05）. The rhIL-24 had a strong bioactlvity with natural IL-24 protein. Conclusion The recombinant expression vector PQE-IL-24 has been constructed successfully and has efficency expression in E. coli M15, rhIL-24 which biologic activties are same as natural IL-24 protein is obtained.