鼠HMGB1蛋白的原核表达及分离纯化

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.

Mouse HMGB 1 Prokaryotic Expression and Purification

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.