作　　者： ; ; ; ; ; ; ;

机构地区： 南方医科大学

出　　处： 《中国病理生理杂志》 2006年第6期1119-1123,共5页

摘　　要： 目的:构建带His标记的人高迁移率族蛋白1(HMGB1)融合蛋白原核细胞表达质粒,表达并纯化人HMGB1重组蛋白,观察其对人脐静脉内皮细胞(HUVEC)释放细胞因子的影响。方法:首先将目的片段HMGB1亚克隆到改造的载体pET14b上,经鉴定、测序证实序列正确后转化大肠杆菌BL21(DE3),异丙基-β-D硫代半乳糖苷(IPTG)诱导表达His-HMGB1融合蛋白,用Ni-NTA亲和树脂纯化。将纯化得到的HMGB1蛋白经透析和过滤除菌后,刺激人脐静脉内皮细胞,24h后收集培养上清,经LiquiChip液相蛋白芯片系统检测细胞因子。结果:构建的pET14b/HMGB1表达质粒经酶切和测序验证正确;表达的目的蛋白经Western blotting验证能被His抗体识别;检查了纯化的HMGB1蛋白对HUVEC产生细胞因子的影响,发现重组的HMGB1可诱导白细胞介素8(IL-8)上调,并且呈剂量依赖性关系。结论:成功构建了His-HMGB1原核细胞表达质粒并纯化得到重组的HMGB1;在HUVEC模型上证实所得到的HMGB1蛋白具有生物学活性。本实验为进一步研究HMGB1的功能提供了重要的实验材料。 AIM： To construct the prokaryotie expression plasmid of His- tagged human high mobility group box 1 fusion protein （hHMGB1） and to express the fusion protein in E. coli for the affinity purification. METHODS： The eDNA coding region of HMGB1 was amplified by PCR from pGEX4T- HMGB1 and cloned into a modified pET14b vector following the routine procedure. After identification by enzyme digestion, PCR and sequencing, the plasmid was transformed into BL21 （DE3） competent cells, and the His - HMGB1 fusion protein was induced for expression with isopropyl - beta - D - thingalactopyranoside （IPrG）, and further purified by Ni - NTA affinity chromatography. The protein was filtered for sterilization and used to stimulate human umbilical vein endothelial cells （HUVECs）. 24 hours later, the cultured supernatant of HUVECs was collected for the detection of cytokines/ehemokines with LiquiChip system. RESULTS： The His- HMGB1 fusion protein expression plasmid was identified by enzyme digestion and sequencing. The purified His - tagged fusion protein was analyzed by SDS - PAGE and Western blotting with specific anti - His antibody. It was found that the production of IL- 8 from HUVECs was highly induced in a dose - dependent manner by HMGB1. CONCLUSION： The His - tagged HMGB1 fusion protein expression plasmid was succesafully constructed, and purified. Recombinant HMGB1 protein has a high bioactivity on the induction of eytokines in HUVECs, which may significantly facilitate the future study of HMGB1 biological functions.

关 键 词： 高迁移率族蛋白质类 原核表达 细胞因子类 脐静脉内皮细胞

领　　域： [生物学]