HMGB1分子A-BOX的表达纯化及其促分化功能鉴定的研究

目的克隆人HMGB1 A-box的cDNA,构建高效稳定的大肠杆菌(E.coli)表达菌株并对其诱导表达纯化,同时探讨其对间充质干细胞(Mesenchymal stem cells,MSC)的促分化作用。方法根据优选合成的HMGB1基因序列设计引物,PCR扩增目的基因片段,插入克隆载体pGEM-T并进行序列测定。重组克隆载体经BamHⅠ和XhoⅠ酶切,琼脂糖凝胶电泳分离目的基因,插入含His标签的表达载体pET24a。将构建的质粒转染BL21大肠杆菌,经IPTG诱导后,SDS-PAGE观察表达量。HIS-link层析柱纯化重组人HMGB1 A-box,蛋白印迹鉴定重组蛋白。将重组蛋白加入hBMSC培养体系中培养一周后,碱性磷酸酶染色检测其促MSC成骨分化作用。结果经RT-PCR扩增得到了261 bp的DNA片段,经测序分析,与GenBank中报道的已知序列完全一致,构建了含融合蛋白的重组表达质粒。经诱导后细菌高表达A-box,表达量为总蛋白的45%。经层析柱纯化后,蛋白印迹证实目的蛋白为高纯度的A-box。将A-box加入MSC培养体系中培养后,细胞活性无明显改变,但细胞碱性磷酸酶表达明显增高。结论成功构建了重组人HMGB1 A Box的表达载体,纯化的重组蛋白能有效促进MSC向成骨细胞分化。

Expression and Purification of Human HMGB1 A-Box and Identification of Its Induction-Promoting Effects on Mesenchymal Stem Cells

Objective To investigate if human high mobility group boxl A-box （HMGB1 A-box） could promote mesenchymal stem cells （MSCs） to differentiate, A-box fragment of HMGB-1 was cloned into E. coli and the expressed products were purified. Methods HMGB1 A-box cDNA was harvested by polymerase chain reaction and inserted into a clone vector pGEM-T. The positive clone was picked out and identified by DNA sequencing. Recombinant vector was digested by restriction enzymes BamH I and Xho I, the targeted cDNA fragment was separated by agarose gel electrophoresis and was then sub-cloned into the corresponding sites of the His-tag-carrying expression vector pET24a. The recombinant plasmid was transfected into BL21 bacteria and A-box expression was induced by IPTG. The recombinant human HMGB1 A-box was further purified by a HIS-link chromatography column. The target protein was identified by SDS-PAGE and Western blotting. Human bone marrow MSCs were cultured in the presence of the purified A-box for one week and in vitro osteogenesis was revealed by alkaline phosphatase staining. Results DNA sequencing showed that human HMGB1 A-box was successfully cloned and could be expressed efficiently in the E. c01i. host, as SDS-PAGE showed that the target protein accounted for up to 45% of the total protein. After purification with chromatography, the expressed protein was found to react with an anti-human HMGB1 polyclonal antibody and an anti-His-Tag polyclonal antibody, as shown by western blotting. Further, the addition of the purified A-box into the human MSC culture was found to promote MSCs to differentiate along the osteogenic pathway. Conclusion A recombinant bacterial strain for expressing human HMGB1 A-box with biological activities was constructed successfully.