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目的 克隆杀菌 /通透性增强蛋白 (BPI) N端 c DNA,构建原核和真核表达载体 ,分别在大肠杆菌和 CHO细胞中表达 BPI蛋白 .方法 提取正常人外周血多形核粒细胞 (PMN)总 RNA,经套式 RT- PCR法扩增出 BPI N端 c DNA片段 ,将其克隆入 p GEM- T easy载体中并进行酶切鉴定和序列测定 .然后亚克隆入 p GEX- 4T- 1质粒 ,转化大肠杆菌 ,进行诱导表达 ,表达产物用 Western blot法鉴定 .同时将该基因片断克隆入 pc DNA3质粒 ,通过脂质体转染 CHO细胞 ,免疫荧光法鉴定 BPI的表达 .结果 获得 BPI N端长度为 72 6 bp的基因片断 .序列分析证实该片断中有 4个点突变 ,但均不在活性中心 .在大肠杆菌中的表达产物为相对分子质量 5 1× 10 3的GST- BPI融合蛋白 ,Western blot反应中在 5 1× 10 3处有显色区带 .G418筛选出的阳性 CHO细胞在免疫荧光反应中产生荧光反应 .结论 我们成功的在大肠杆菌和 CHO细胞中表达了 BPI2 5蛋白 ,为进一步研究 BPI生物学功能奠定了基础
Objective To clone the N-terminal c DNA of bactericidal / permeability-enhancing protein (BPI) and construct the prokaryotic and eukaryotic expression vectors to express BPI protein in Escherichia coli and CHO cells respectively.Methods Normal human peripheral blood polymorphonuclear neutrophils (PMNs) ) Total RNA was amplified by nested RT-PCR BPI N-terminal c DNA fragment, cloned into p GEM-T easy vector and digested and sequenced.And then subcloned into pGEX-4T- 1 plasmid was transformed into E.coli.After induced by Western blot, the fragment was cloned into pcDNA3 plasmid and transfected into CHO cells by lipofectamine.The expression of BPI was identified by immunofluorescence.Results BPI N terminal 72 bp gene fragment.Sequencing analysis confirmed that there are 4 point mutations in this fragment, but not in the active center.The expression product in E. coli GST-BPI fusion with a relative molecular mass of 5 1 × 10 3 Western blotting showed a band of 51 1 x 103. The positive CHO cells screened by G418 produced a fluorescence reaction in the immunofluorescence reaction.Conclusion We successfully expressed in E. coli and CHO cells BPI2 5 protein, which laid the foundation for the further study of the biological function of BPI