杀菌/通透性增强蛋白N端cDNA的克隆和表达

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

Cloning and expression for cDNA of human bactericidal/permeability-increasing protein N-terminal fragment

AIM To clone cDNA of N terminal fragment of human bactericidal/permeability increasing protein(BPI), to construct the procaryotic and eucaryotic expression vectors and to express the cDNA in E.coli and CHO cells respectively. METHODS Total RNA was extracted from human polymorphonuclear neutrophils(PMN) and then the human BPI cDNA gene was amplified by nested RT PCR. The PCR product was cloned into pGEM T easy plasmid and the sequence was confirmed by restriction enzyme digestion and dideoxy mediated chain termination. Subsequently the specific BPI N terminal fragment was subcloned into pGEX 4T 1 plasmid and transformed into E.coli . In addition, the fragment was inserted into pcDNA3 plasmid and transformed into CHO cells with liposome transfection reagent. RESULTS A 726 bp fragment was acquired. The E.coli transformed with recombinant plasmid pGEX BPI was induced by IPTG. A 51 ku expected protein of GST BPI fusion protein was synthesized. The CHO cells transformed with pcDNA3 BPI were screened by G418, and the positive clone was obtained by using immunofluorescent assay. CONCLUSION N terminal of BPI was successfully expressed in E.coli and CHO.