人单链白细胞介素12的原核表达和生物学活性鉴定

Prokaryotic expression and bioactivity identification of single chain human interleukin-12

目的构建含人单链白细胞介素12(hscIL-12)基因的原核表达载体,在大肠杆菌中表达具有活性的hscIL-12。方法 PCR法从质粒pCA13-hscIL-12中扩增hscIL-12基因,经酶切、连接构建原核表达载体pET28a(+)-hscIL-12,转入到大肠杆菌BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,收集菌液进行蛋白质印迹分析,经镍柱亲和层析(Ni2+-NTA)纯化,体外增殖实验检测其生物学活性。结果 PCR扩增、双酶切及DNA序列测定证实pET28a(+)载体上成功插入了hscIL-12基因片段。表达产物经SDS-PAGE电泳分析,证明其相对分子质量为70×103;蛋白质印记表明重组蛋白具有hscIL-12抗原活性;表达产物纯化、复性后进行体外生物学活性实验表明,该重组蛋白能刺激外周血单核细胞(PBMC)产生IFN-γ。结论 pET28a(+)-hscIL-12原核表达载体的构建和重组hscIL-12蛋白的制备为进一步研究hscIL-12生物学功能和临床应用奠定了基础。

Objective To construct the expression vector of human single chain interleukin-12（hscIL-12）gene.It can express hscIL-12 with bioactivity in escherichia coli.Methods hscIL-12 gene was amplified from recombinant pCA13-hscIL-12.With restricted enzyme digestion,gene of interest was cloned into the prokaryotic vector pET28a（＋） to construct expressing plasmid of pET28a（＋）-hscIL-12.After pET28a（＋）-hscIL-12 was transformed into escherichia coli BL21（DE3）,the bacteria were induced by IPTG.The expression of hscIL-12 was detected by Western bloting.The hscIL-12 protein was purified by Ni-NTA affinity chromatography.The bioactivity of hscIL-12 was detected by PHA-activated peripheral blood mononuclear cell（PBMC） proliferation assay.Results PCR and restriction enzyme digestion and DNA sequencing indicated that hIL-12 gene was inserted into the procaryotic expression vector pET28a（＋）,SDS-PAGE analysis showed that molecular weight of the expressed protein was 70×103;Western blotting analysis showed that recombinant protein had the antigenicity of hscIL-12.The purified hscIL-12 protein had the bioactivities in stimulating the IFN-γ production of human PBMC.Conclusion The construction of the recombinant plasmid and the preparation of the active protein of hscIL-12 have laid a foundation for further studying the function and clinical applications of hscIL-12.