人HMGB1基因原核表达载体的构建及其在大肠杆菌中的表达和纯化

目的  构建人高迁移率族蛋白B1（HMGB1）基因的原核表达载体，观察表达水平并纯化重组蛋白。方法  根据GenBank中人HMGB1基因序列，用OptimumGeneTM行密码子优化并合成目的基因，经PCR扩增，NdeⅠ和XhoⅠ双酶切，插入原核表达载体pQE-T7-2的相应位点，构建原核表达质粒pQE-T7-2/HMGB1。经菌落PCR、酶谱分析及序列分析鉴定重组质粒，转化大肠杆菌BL21(DE3)，异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达，采用SDS-PAGE和Western blot印迹法鉴定重组蛋白，Ni NTA树脂亲和纯化目的蛋白。结果  成功构建人HMGB1克隆载体和表达载体；表达的目的蛋白约占菌体总蛋白的20%以上，Western blot法显示，重组蛋白能与抗HMGB1抗体和抗His抗体特异结合，亲和层析纯化后纯度达90%以上。结论  成功构建高效稳定的重组人HMGB1原核表达载体，获得纯化人HMGB1蛋白，为进一步研究HMGB1的功能奠定基础。

Construction of recombinant prokaryotic expression plasmid of human high mobility group box1 and its expression and purification

Objective  To construct the prokaryotic expression plasmid of human high mobility group box1 (hHMGB1) gene,  and to express and purify the His-tagged hHMGB1 fusion protein. Methods  According to the sequence reported in GenBank, we optimized codons by OptimumGeneTM and synthesized the human HMGB1 gene. To construct prokaryotic expression plasmid pQE-T7-2/HMGB1, the DNA fragment obtained from PCR was digested with NdeⅠ and XhoⅠ, and then inserted into pQE-T7-2 that was cut with the same enzymes. The recombinants were screened by colony PCR, restriction mapping and sequencing. The recombinant E.coli BL21 (DE3) was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), and the recombinant protein was identified by SDS-PAGE and Western blotting. The recombinant human HMGB1 was further purified by Ni-NTA Agarose. Results  The prokaryotic expression plasmid pQE-T7-2/HMGB1 was successfully constructed. The expressed recombinant protein accounted for about 20% of the total bacterial proteins. The recombinant protein could specifically react with anti HMGB1 antibody and anti-His antibody. The purity of recombinant human HMGB1 separated with affinity chromatography was more than 90%. Conclusion  The recombinant prokaryotic expression vector of human HMGB1 which was successfully constructed may lay the foundation for the  further study of the function of human HMGB1.