人白细胞介素-24基因的克隆、表达和纯化

目的对人白细胞介素-24(hIL-24)进行克隆、表达和纯化。方法分离人外周血单个核细胞,ConA刺激培养,提取细胞总RNA,应用RT-PCR技术从外周血单个核细胞中扩增hIL-24成熟肽编码区,定向克隆至融合表达载体pGEX-4T-1,重组载体pGEX-4T-1/hIL-24经DNA测序鉴定后,转化E.coliBL21(DE3),IPTG诱导表达,阴离子交换层析纯化融合蛋白,SDS-PAGE和Westernblot鉴定。结果所得hIL-24基因经序列分析,与GenBank公布一致。所构建融合表达载体pGEX-4T-1/hIL-24测序正确,转化BL21(DE3)后,37℃诱导表达相对分子质量约为44000的融合蛋白,主要以包涵体形式存在,占菌体总蛋白的30.63%。经纯化后,纯度达85%以上。Westernblot检测能与兔抗人IL-24的多克隆抗血清发生结合反应。结论已成功构建了hIL-24的融合表达载体pGEX-4T-1/hIL-24,并在大肠杆菌中高效表达,纯化后获得了高纯度的融合蛋白,为hIL-24的功能及活性研究奠定基础。

Gene Cloning, Fusion Expression and Purification of Human Interleukin-24

Objective To clone and express human interleukin-24 gene and purify the expressed product.Methods Isolate human peripheral blood mononuclear cells(PBMCs) and culture under stimulation of Con A.Extract the total RNA of the cultured PBMCs for amplification of the gene encoding the mature peptide of hIL-24 by RT-PCR.Directly clone the amplified gene into fusion expression vector pGEX-4T-1.Identify the recombinant plasmid pGEX-4T-1/hIL-24 by DNA sequencing,then transform to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed product by anion exchange chromatography and identify by SDS-PAGE and Western blot.Results The sequence of the cloned hIL-24 gene was consistent with that reported in GenBank.The identification by sequencing showed that fusion expression vector pGEX-4T-1/hIL-24 was correctly constructed.The fusion protein,with a relative molecular weight of 44 000,was expressed in form of inclusion body and contained 30.63% of total somatic protein.The purity of the expressed protein after purification reached more than 85%.Western blot showed specific reaction of the expressed fusion protein with rabbit anti-human IL-24 polyclonal antiserum.Conclusion The fusion expression vector pGEX-4T-1/hIL-24 was successfully constructed,and hIL-24 was highly expressed in E.coli.The fusion protein with high purity was obtained.It laid a foundation of study on function and activity of hIL-24.