鼠hmgb1蛋白的原核表达及分离纯化.docVIP

鼠HMGB1蛋白的原核表达及分离纯化 作者：赵中夫 杨慧 刘明社 张芸 杨柳絮 封江南 【关键词】 高迁移率族蛋白1；原核表达载体；纯化 　　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 　　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 SDSand 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. 　　Key words High mobility group box 1;Prokaryotic expression vector;Purification 　　高迁移率族蛋白B1(High mobility group box1，HMGB1)是广泛存在于真核细胞内的高度保守的非组蛋白染色体蛋白，基本组全长648 bp编码215个氨基酸残基［１］。已有研究表明，HMGB1可由巨噬细胞或单核细胞等主动分泌，或由坏死细胞被动释放，从而作为一种炎症晚期因子参与脓毒症等病理过程［２，３］。研究还发现经脂多糖（LPS）刺激巨噬细胞后HMGB1的分泌量较未刺激明显增多，并且在8 h～12 h达峰值。而HMGB1发挥作用的机制还不是很清楚。因此，分离纯化HMGB1蛋白将对进一步研究HMGB1的生物学功能、与其他细胞因子的相互作用关系等有重要意义。本实验以小鼠巨噬细胞RAW264.7为研