人HMGB1真核表达载体的构建及其在单核细胞的表达

Construction of eukaryotic expression vectors of human HMGB1 and its expression in monocytes

目的构建人高迁移率族蛋白1(HMGB1)表达载体,并探讨其免疫调节作用。方法从HMGB1克隆载体酶切分离HMGB1基因片段,插入增强型绿色荧光蛋白载体pCITE-EGFP,菌落PCR和酶谱分析鉴定重组载体。重组表达质粒转染人单核细胞系(U937),梯度G418筛选,荧光和Western blot检测HMGB1的表达。结果菌落PCR和酶谱分析显示目的基因被正确插入真核表达载体,获得重组质粒pCITE-EGFP-HMGB1,转染U937细胞随培养基中G418浓度的升高而增强,转染细胞中目的蛋白HMGB1表达量明显升高。结论成功构建重组载体pCITE-EGFP-HMGB1,并获得稳定表达人HMGB1的单核细胞系,为研究HMGB1对单核巨噬细胞的作用及其机制奠定基础。

Objective To construct the eukaryotic expression vectors of human HMGB1 gene and investigate its immunomodulatory effect.Methods The HMGB1 gene fragment was isolated from cloning vector pMD-HMGB1 with digestion of Sal I and EcoR I and agarose gel separation,and inserted into the same sites of eukaryotic expression vector pCITE-EGFP.The recombinant plasmid was screened by colony PCR and restriction enzyme digestion.The human monocyte cell line U937 was transfected with recombinant expression plasmid and screened by gradient G4l8.Expression of HMGB1 was determined under an inverted fluorescence microscope and Western blotting.Results Colony PCR and the restriction endonuclease digestion showed that the HMGB1 gene was correctly inserted into the eukaryotic expression vector and the recombinant plasmid pCITE-EGFP-HMGB1 was obtained.With an increase of G418 concentration in the culture medium,the U937 cells stably expressing human HMGB1 were established.The quantity of HMGB1 in the transfected cells was increased.Conclusion The eukaryotic vector of pCITE-EGFP-HMGB1 was successfully constructed and a human monocyte cell line stably expressing HMGB1 was obtained,which established a solid foundation for further study on the effects and mechanisms of HMGB1on mononuclear-macrophages.