利用生物素-链亲和素系统筛选重症急性胰腺炎肝损伤大鼠HMGB1启动子结合蛋白

Screening HMGB1 promoter binding proteins in rat hepatic injury secondary to severe acute pancreatitis using biotinstreptavidin system

目的:筛选重症急性胰腺炎(severe acute pancreatitis,SAP)肝损伤大鼠高迁移率族蛋白B1(high mobility group protein B1,HMGB1)的启动子结合蛋白.方法:牛黄胆酸钠胆胰管逆行注射法制备SAP大鼠模型,与对照组同时处死,取肝组织提取细胞核蛋白,PCR扩增末端带生物素标记的HMGB1启动子探针,将核蛋白与HMGB1探针孵育,然后用链亲和素磁珠分离HMGB1启动子-蛋白复合物,分别用0.25 mol/L和1 mol/L NaCl洗脱探针上结合的蛋白,用SDS-PAGE电泳分离样本蛋白,SilverQuest银染试剂染色,比较SAP组和对照组的差异条带并做质谱鉴定.结果:对照组和SAP组共有14条差异条带,质谱鉴定这些差异条带得到H2A、H2B、H3、H4、S100A9转录相关蛋白.结论:该研究筛选到一些SAP肝损伤特异性的HMGB1启动子结合蛋白,对于下一步研究HMGB1在SAP肝损伤过程中的转录调控机制具有重要意义.

AIM： To screen proteins that interact with high mobility group protein B1（HMGB1） promoter in rat hepatic injury secondary to severe acute pancreatitis（SAP）.METHODS： A rat model of SAP was generated by retrograde injection of 5% sodium taurocholate into the bilio-pancreatic duct. The SAP group and control group were executed simultaneously, and the liver nuclear extracts were prepared. PCR was used to amplify the biotin labeled tail probes of the HMGB1 promoter. The probes were incubated with cell nuclear extracts, and HMGB1 promoter-protein complexes were then separated using streptavidin conjugated magnetic beads. The proteins were eluted from probes with 0.25 mol/L and 1 mol/L NaCl, resolved using SDS-PAGE electrophoresis, and visualized by silver staining, and the differential bands were identified by mass spectrometry（MS）. RESULTS： A total of 14 differential protein bands between the SAP and control groups were screened, 5 of which were identified as transcription related proteins by MS. CONCLUSION： Proteins that interact with HMGB1 promoter in SAP-associated hepatic injury were acquired and identified, which have important value for the further study of regulatory mechanism of HMGB1.