摘要 目的：克隆小鼠HMGB1基因，构建原核表达载体，在大肠杆菌中诱导表达并分离纯化获得His- HMGB1的融合蛋白。方法：脂多糖刺激后的RAW264.7细胞，提取总RNA，经RT-PCR扩增出含HMGB1的目的片段，克隆于pMD-19T载体，再亚克隆至含有pelB引导肽及His-标签肽的高效表达载体pET-26b(+)，转化大肠杆菌BL21(DE3)，经IPTG诱导后行SDS-PAGE鉴定目标蛋白表达，用镍鳌合琼脂糖凝胶亲和层析法分离纯化含His-标签肽的目的蛋白。结果：经PCR扩增出大小为648 bp的HMGB1基因片段，成功构建目标蛋白的融合表达载体，经诱导表达及分离纯化，获得了约30 kD的融合蛋白。结论：构建HMGB1的融合表达载体，获得分离纯化融合蛋白His- HMGB1，为进一步研究其生物学功能奠定了基础。

    关键词 高迁移率族蛋白1；原核表达载体；纯化

    Mouse HMGB1 Prokaryotic Expression and Purification

    Zhao Zhongfu,Yang Hui,Liu Mingshe,et al.

    Institute of Hepatology, Changzhi Medical College

    Abstract Objective:To clone mouse High mobility group box 1 (HMGB1) gene and construct the prokaryotic expression vector,to induce and purify the expressed fusion protein HMGB1.Methods:The total RNA was extracted from mouse RAW264.7 cells stimulated by LPS.The sequence including the whole length of HMGB1 was amplified by RT-PCR and inserted into pMD-19T.The combinant vector was used as a template for PCR which was cloned into vector pMD-19T,then subcloned into expression vector pET-26b(+) with pelB signal sequence and His-Taq sequence.After transforming E.coli BL21(DE3) and four hours induction by IPTG,HMGB1 expression confirmed by SDS-PAGE and the purification was performed by Ni2+-chelate affinity chromatograph.Results:The target DNA sequence of HMGB1 was obtained by PCR.The recombinant expression plasmid pET-26b(+)-HMGB1 was constructed.The Mr.30 000 fusion protein was obtained by IPTG induction and purification.Conclusion:Mouse HMGB1 prokaryotic expression vector was constructed successfully and the purified proteins were obtained. ......