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目的:从人淋巴细胞中克隆白介素-1β(IL-1β)cD-NA,通过原核可溶性表达纯化,获得具有生物活性的可溶性IL-1β,为深入研究和利用IL-1β基因奠定基础。方法:从人PBMC中克隆出人成熟IL-1β基因,并插入到原核表达载体pSUMO中SUMO标签的下游,构建重组表达载体pSUMO-hIL-1β。将该载体转化大肠杆菌Rosetta(DE3),IPTG诱导表达hIL-1β/SUMO融合蛋白,经Ni-NTA Agarose纯化后,表达产物用SDS-PAGE和Western blot进行鉴定,用protease-1切去SUMO标签,用Real time-PCR鉴定其对人T细胞表达的细胞因子的影响。结果:SDS-PAGE分析表明表达产物的相对分子质量(Mr)为37000,成熟蛋白Mr为17000,与理论值相符。灰度扫描分析融合蛋白的表达量占菌体蛋白总量的约30%左右,主要以可溶形式存在。Western blot证实该蛋白为hIL-1β。成熟蛋白纯化产物纯度超过90%以上。通过RT-PCR检测证明其具有刺激人T细胞大量表达多种细胞因子(IL-1β,IL-4,IL-10,IL18,IFN-γ,TGF-β,TNF-α)的活性。结论:利用大肠杆菌表达系统完全可以高效可溶性表达较高纯度的具有生物活性的hIL-1β蛋白。
OBJECTIVE: To clone soluble interleukin-1β (IL-1β) cD-NA from human lymphocytes and purify it by prokaryotic soluble expression to obtain soluble IL-1β with biological activity, which lays the foundation for further study and utilization of IL-1β gene. METHODS: Human mature IL-1β gene was cloned from human PBMC and inserted into the downstream of SUMO tag in prokaryotic expression vector pSUMO to construct recombinant expression vector pSUMO-hIL-1β. The vector was transformed into Escherichia coli Rosetta (DE3). The hIL-1β / SUMO fusion protein was induced by IPTG and purified by Ni-NTA Agarose. The expressed product was identified by SDS-PAGE and Western blot. The SUMO tag was excised by protease- Real time-PCR was used to identify its effect on human T cell-expressed cytokines. Results: SDS-PAGE analysis showed that the relative molecular mass (Mr) of the expressed product was 37000, and the mature protein Mr was 17000, which was consistent with the theoretical value. Grayscale scanning analysis fusion protein expression accounted for about 30% of total bacterial protein, mainly in soluble form. Western blot confirmed that the protein hIL-1β. Mature protein purification product purity more than 90%. It has been proved by RT-PCR that it can stimulate the expression of many cytokines (IL-1β, IL-4, IL-10, IL18, IFN-γ, TGF-β, TNF-α) CONCLUSION: The highly purified and bioactive hIL-1β protein can be efficiently and solublely expressed in E. coli expression system.