缺第4外显子的IL-7剪接变异体的表达、纯化与活性研究  

Expression, purification and bioactivity study of recombinant human interleukin-7 splice variant lacking exon 4

作　　者：陈鉴华 严思[1] 李小波 金小宝 朱家勇 潘德顺[1] 

机构地区：[1]广东药学院药理系,广州510006 [2]广东省生物活性药物研究重点实验室

出　　处：《免疫学杂志》2015年第3期260-264,共5页Immunological Journal

基　　金：中国-意大利科技合作项目(200625)

摘　　要：目的构建、表达和纯化缺乏第4外显子的IL-7剪接变异体IL-7δ4(interleukin-7 splice variant lacking exon4,IL-7δ4),并对其生物活性进行初步研究。方法将TA克隆成功的IL-7δ4基因克隆至p ET-21b载体中,构建p ET-21b-IL-7δ4的原核表达载体,阳性克隆经测序正确后转化到大肠杆菌BL21(DE3)中,进行IL-7δ4重组蛋白的表达、复性、纯化、Western blot法鉴定,分析其对外周血单核细胞凋亡蛋白BCL-2表达及凋亡的影响。结果成功构建重组原核表达质粒p ET-21b-IL-7δ4,表达蛋白的相对分子质量为12 400,与预期大小一致;复性效率较高(约65%),纯化后获得的蛋白纯度大于95%,Western blot证实其为rh IL-7δ4;与对照组比较,rh IL-7δ4能明显诱导外周血单核细胞凋亡蛋白BCL-2的表达,并抑制其凋亡(P<0.01)。结论成功表达高纯度且具有生物活性的rh IL-7δ4复性蛋白,它能明显上调外周血单核细胞凋亡蛋白BCL-2的表达,起抑制PBMCs凋亡作用。This study performed to construct, express and purify recombinant human interleukin-7 splice variant lacking exon 4（IL-7δ4）, and to preliminarily study its bioactivity. IL-7δ4 gene was cloned into p ET-21 b plasmid to construct a prokaryotic expression vector after TA cloning, then positive clone was transformed into Escherichia coli（E. coli） BL21（DE3） after sequencing. Recombinant protein（rh IL-7δ4） was refolded and purified,and then identified with Western blot. The effect of rh IL-7δ4 on BCL-2 protein expression and apoptosis in peripheral blood mononuclear cells（PBMCs） was analyzed. Data showed that the recombinant p ET-21b-IL-7δ4 of the prokaryotic expression vector was constructed, the molecular weight of expressed protein was equal to anticipated 12.4 k Da, refolding efficiency of IL-7δ4 was about 65%, and its purity was ﹥ 95% after purification,besides, western blot proved that the expressed product is rh IL-7δ4 protein. Furthermore, rh IL-7δ4 could improve BCL-2 protein expression and inhibit its apoptosis, as compared with the control（P〈 0.01）. It is able to conclude that rh IL-7δ4 with high purity and bioactivity is successfully expressed, which can upgrade BCL-2 protein expression and restrain its apoptosis in PBMCs.

关 键 词：rhIL-7δ4 剪接变异体 表达 纯化 活性鉴定 

分 类 号：Q786[生物学—分子生物学]