HMGB1 A盒与LBPK95A融合基因的构建和原核表达  

Cloning and Expression of HMGB1 A-LBPK95A Fusion Protein in E. coli

作　　者：姜南艳[1] 于文彬[2] 苏明权[2] 沈建军[1] 李立文[3] 郝晓柯[2] 张惠中[1] 

机构地区：[1]第四军医大学唐都医院临床实验,检验与输血科,西安710038 [2]第四军医大学西京医院检验科,西安710032 [3]第四军医大学西京医院全军骨科研究所,西安710032

出　　处：《现代检验医学杂志》2008年第6期9-11,共3页Journal of Modern Laboratory Medicine

基　　金：陕西省科技攻关计划项目(项目编号:2004K10-G4)

摘　　要：目的克隆、表达高迁移率族蛋白1A盒(HMGB1A box)与LBPK95A的融合基因。方法经加端-PCR获得HMGB1A盒与LBPK95A的融合基因。该基因克隆到pGEX-4T-2载体中转化TOP10感受态细胞,测序鉴定。将A-LBP质粒转化E.coliBL21,30℃诱导表达4h,超声裂解后经GSTrapFF蛋白纯化柱纯化,进行SDS-PAGE分析。结果DNA测序证明,获得了HMGB1A盒与LBPK95A的融合基因。SDS-PAGE分析表明,A-LBP融合蛋白在原核中获得高效表达,表达量约占菌体总蛋白的28,GSTrapFF纯化后获得目的蛋白。结论成功表达和纯化了HMGB1A盒与LBPK95A的融合蛋白。Objective To clone,express mouse high mobility group box chromosomal protein 1 （HMGB1） A- LBPK95A fusion protein. Methods The whole length of HMGB1 A- LBPK95A fusion gene was obtained by add-PCR. The A-LBP gene was cloned into pGEX-4T-2 vector and sequenced. Then the gene was transformed into E. colt BL21 cell line. After the recombinant bacteria was induced at 30℃ for 4 h,the expressed protein was analyzed by SDS-PAGE. Thus the protein was purified on GSTrap FF column. Results DNA sequencing result showed that A-LBP fusion gene was exactly consistent with the sequence reported in GenBank. SDS-PAGE analysis demonstrated that A-LBP fusion protein was expressed in E. coll. The protein band amounted to 28% of total bacteria protein. The production purified on GSTrap FF column. Conclusion HMGB1 A-LBPK95A gene is successfully cloned and expressed.

关 键 词：HMGB1 A盒 LBPK95A 加端-PCR 基因表达 纯化 

分 类 号：Q786[生物学—分子生物学] Q781