【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。更多还原

【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-BPI193 recombinant expression plasmid was constructed successfully.6His-BPI193 fusion protein containing 193 amino acids of BPI Nterminal 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.Conclusion 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.更多还原