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人杀菌渗透增强性蛋白N末端片段在大肠杆菌中的表达
引用本文:董伟,董晓慧,楚雍烈,杨娥,胡刚,郑建武. 人杀菌渗透增强性蛋白N末端片段在大肠杆菌中的表达[J]. 西安交通大学学报(医学版), 2006, 27(1): 11-14
作者姓名:董伟  董晓慧  楚雍烈  杨娥  胡刚  郑建武
作者单位:西安交通大学医学院免疫学与病原生物学系,环境与疾病相关基因教育部重点实验室,陕西,西安,710061
基金项目:陕西省科技计划;陕西省卫生厅资助项目
摘    要:目的构建表达人杀菌渗透增强性蛋白(BPI)N-端193个氨基酸的重组表达质粒,诱导表达BPI193重组蛋白。方法应用RT-PCR的方法从HL-60细胞内扩增出BPI氨基端1-193个氨基酸的基因序列,克隆入T载体。将BPI193基因片段定向克隆入原核表达载体PET-28a中,构建重组的原核表达质粒PET-BPI193,转化大肠杆菌BL21菌株,用IPTG诱导表达蛋白。应用SDS-PAGE检测蛋白表达情况,计算机薄层扫描分析蛋白占菌体总蛋白百分比;Western-blot技术检测表达蛋白的特异性。结果从HL-60细胞中扩增得到579bp的BPI193基因片段,构建了T-BPI193亚克隆和PET-BPI193重组表达质粒。转化大肠杆菌BL21,通过IPTG诱导,经SDS-PAGE检测表明,成功表达了6His-BPI193融合蛋白。计算机薄层扫描分析表达量占菌体总蛋白的12%。Western-blot检测显示表达产物具有特异性。结论成功构建了PET-BPI193重组表达质粒。在大肠杆菌BL21内,诱导获得了BPI193与组氨酸融合表达的重组蛋白。

关 键 词:杀菌/渗透增强性蛋白  基因表达  大肠杆菌
文章编号:1671-8259(2006)01-0011-04
收稿时间:2005-05-31
修稿时间:2005-10-08

Expression of human bactericidal/permeability increasing protein N-terminal fragment in E. coli
Dong Wei,Dong Xiaohui,Chu Yonglie,Yang E,Hu Gang,Zheng Jianwu. Expression of human bactericidal/permeability increasing protein N-terminal fragment in E. coli[J]. Journal of Xi‘an Jiaotong University:Medical Sciences, 2006, 27(1): 11-14
Authors:Dong Wei  Dong Xiaohui  Chu Yonglie  Yang E  Hu Gang  Zheng Jianwu
Abstract:Objective To construct a prokaryotic expression plasmid of cDNA sequence encoding first 193 amino acids of BPI N-terminal and to express it in E. coli BL21. Methods Total RNA was extracted from HL-60 cell and then was amplified by using RT-PCR. The PCR product was cloned into pMD18-T vector. BPI193 gene was directly cloned into PET-28a plasmid on the role of T4 DNA ligase. PET-BPI193 recombinant expression vector was transformed into competent E. coli BL21 and protein expression was induced by IPTG. Fusion protein was detected by SDS-PAGE and Western-blot. Results A 579 bp DNA amplicon was amplified by RT-PCR. T-BPI193 subclone and PET-BP1193 recombinant expression plasmid was constructed successfully. 6His-BPI193 fusion protein containing 193 amino acids of BPI N-terminal was expressed by IPTG induced method. Fusion protein was detected by SDS-PAGE and confirmed by Western-blot. The expressed fusion protein constituted 12% of total bacterial protein. Oonclusion PET-BPI193 recombinant expression plasmid was constructed successfully. To transform PET-BPI193 recombinant plasmid into E. coli BL21 and 6His-BPI193 fusion protein was expressed by IPTG induced manner.
Keywords:bactericidal/permeability increasing protein  gene expression  E.coli
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