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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 human HMGB1 expression vector and investigate its immunomodulatory effect. Methods The HMGB1 gene fragment was isolated from the HMGB1 cloning vector and inserted into the enhanced green fluorescent protein vector pCITE-EGFP. The recombinant vector was identified by colony PCR and zymography. Recombinant plasmids were transfected into human monocytic cell line (U937). The cells were screened by gradient G418 and the expression of HMGB1 was detected by fluorescence and Western blot. Results The colony PCR and zymogram showed that the target gene was correctly inserted into the eukaryotic expression vector to obtain the recombinant plasmid pCITE-EGFP-HMGB1. The transfected U937 cells increased with the increase of G418 concentration in the culture medium. The target protein HMGB1 The expression was significantly increased. Conclusion The recombinant plasmid pCITE-EGFP-HMGB1 was successfully constructed and the monocyte-macrophage cell line stably expressing human HMGB1 was obtained, which laid the foundation for the study of the effect of HMGB1 on monocyte-macrophage and its mechanism.