人HMGB1分子的克隆、重组蛋白表达与生物学活性

采用RT-PCR,从重症肝炎病人外周血单个核细胞（peripheral blood mononuclear cells PBMCs）的mRNA中扩增高迁移率族蛋白1（high mobility group box－1 protein，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 Sepharose 4B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。

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

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 is employed in further study.