人HMGB1基因的克隆、融合表达与生物学活性

Clone, Expression of Human HMGB1 Gene and the Biological Activity of Its Recombinant Protein

采用RT-PCR,从重症肝炎病人外周血单个核细胞(peripheral blood mononuclear cellsPBMCs)的mRNA中扩增高迁移率族蛋白1(high mobility group box-1protein,HMGB1)基因,构建重组表达质粒pGEX4T-1-HMGB1,转化入大肠杆菌,测序证实其序列与基因数据库中HMGB1基因(NM_002128)一致。诱导表达的融合蛋白GST-HMGB1,用Glutathione Sepharose4B亲和层析纯化,经Thrombin酶切得到HMGB1。结果显示:经Western-blotting检测,GST-HMGB1和HMGB1均具有免疫活性;ELISA和MTT检测发现,两者均能刺激RAW264.7细胞产生大量TNF-α,明显刺激HeLa细胞增殖。GST-HMGB1具有良好的生物学活性,为今后的研究打下了基础。

HMGB-1 （ high mobility group box - 1 protein） gene was amplified by RT-PCR from the mRNA isolated from peripheral blood mononuclear cells （PMBCs） in a patient with liver failure. A recombinant expression vector of GST-HMGB1 was constructed in pGEX4T-1 ,and transformed into E. coli. It was sequenced and confirmed to be identical to the HMGB-1 gene in data bank （NM_002128）. Recombinant protein GST-HMGB1 was purified by Glutathione Sepharose 4B affinity chromatography, and HMGB1 were obtained by thrombin digestion. GST-HMGB1 and HMGB1 were binded to HMGB1＇s antibody in Western-blotting. From ELISA and MTT assays, the results showed that two proteins may stimulate RAW264.7 cells to secret TNF-α, and induc Hela cells proliferation markedly. GST-HMGB1 had good biological activity and it will be employed in further study.