人高迁移率族蛋白B1原核表达及其高亲和噬菌体融合肽的筛选

目的 构建谷胱甘肽巯基转移酶(GST)标记的人高迁移率族蛋白B1(HMGB1)融合蛋白表达载体并在原核细胞中表达.应用噬菌体展示技术筛选HMGB1的高亲和肽.方法 用RT-PCR方法扩增HMGB1 cDNA,构建于原核表达载体pGEX4T-1并转化大肠杆菌,以异丙基-β-D-硫代半乳糖苷(IPTG)诱导GST-HMGB1蛋白表达;Ni2+-NTA和多粘菌素B亲和层析柱进行纯化重组HMGB1蛋白.以重组HMGB1蛋白为靶分子,进行4轮噬菌体展示环七肽库的筛选,从第4轮洗脱物中随机挑选20个单克隆噬菌体扩增后进行ELISA鉴定,用酶标仪测定450 nm处的吸光度值.对获得的阳性单克隆噬菌体分别进行扩增、纯化,并对DNA测序,以确定插入七肽的氨基酸序列.结果 RT-PCR扩增HMGB1 cDNA大小约648 bp,成功构建了pGEX4T-1-HMGB1重组质粒并纯化了HMGB1蛋白,其相对分子质量为65000.经过4轮筛选后,噬菌体富集了74倍(第4轮与第1轮回收量分别为5.2×108、7.0×106 pfu),随机挑取的20个噬菌体克隆中9个可与HMGB1结合,测序发现其中的6个序列一致,均为DYFVSSV.结论 筛选到1个可与HMGB1高亲和结合的噬菌体展示七肽,其对HMGB1活性的拮抗效应有待进一步阐明.

Prokaryotic expression of human high mobility group box 1 protein and screening of its high affinity binding fused - peptide

Objective To construct prokaryotic expression vector of glutathione-S-transferase ( GST) -tagged human high mobility group box 1 protein (HMGB1) and to screen its high affinity binding fused-peptide by phage display technique. Methods Human HMGB1 cDNA gene was amplified by RT-PCR and cloned into vector pGEX4T-1 and then transformed into E. coli. After induction by isopropyl P-D-1-thiogalactopyranoside (IPTG), HMGB1 protein was purified with Ni2+-NTA chromatography and polymyxin B affinity column. Using purified HMGB1 , as target molecule , four rounds of biopanning to a phage display heptapeptides library were carried out. Out of the fourth round eluted product, twenty individual clones were randomly selected and identified by ELISA assay. The absorbance of these clones at 450 nm was determined with a microplate reader. Positive clones were expanded , purified and sequenced to characterize the motifs of fused heptapeptides. Results Recombinant expression plasmid pGEX4T-1-HMGB1 was constructed ( 65 000 by MW) and the purified proteins (648 bp by cDNA) were obtained. Four rounds of biopanning resulted in 74-fold enrichment of the phages (the recovery were 5.2×108、7.0×106 pfu in fourth and first round). Of the twenty phage clones randomly selected, nine were found to bind specifically with HMGB1 proteins. The deduced amino acid sequence analysis showed that six clones displayed a common amino acid sequence (DYFVSSV). Conclusions A heptapeptide is obtained that may bind specifically to HMGB1 protein with high affinity. It remains to be determined whether this heptapeptide may block the effect of HMGB1 or not.