人糖基磷脂酰肌醇(GPI)-B7-1真核表达载体的构建及表达  

Cloning and eukaryotic expressing of GPI-B7-1 in CHO

作　　者：熊茂林[1] 宋畅[1] 罗荣城[2] 罗超权[1] 李民友[1] 李秀英[1] 朱振宇[1] 

机构地区：[1]中山大学中山医学院生化教研室,广东广州510089 [2]第一军医大学南方医院肿瘤科,广东广州510515

出　　处：《中国病理生理杂志》2005年第1期154-158,共5页Chinese Journal of Pathophysiology

基　　金：国家863课题资助项目(No.G1999053903)

摘　　要：目的在仓鼠卵巢细胞CHO细胞中高效表达糖基磷脂酰肌醇GPI-B7-1(B7-1即CD80)融合蛋白,获得大量融合蛋白以研究GPI-B7-1在肿瘤治疗中的应用潜力。方法构建真核表达载体pc31/GPI-B7-1,利用脂质体lipofectamine2000将其转染CHO细胞,G418加压筛选抗性克隆,流式细胞仪检测细胞膜上GPI-B7-1融合蛋白的表达情况,SDS-PAGE、Westernblot分析鉴定其免疫活性。结果真核表达载体转染CHO细胞经G418筛选后,流式细胞分析证实为GPI锚定蛋白,SDS-PAGE在60kD左右蛋白表达量明显高于对照组,在Westernblot在60kD左右有一条强的棕色条带,说明GPI-B7-1在CHO中得到表达。结论在CHO细胞中高效表达GPI-B7-1融合蛋白,可获得大量融合蛋白,对进一步研究GPI-B7-1在肿瘤治疗中的应用打下基础。AIM: To construct human GPI-B7-1 fusion protein and investigate the therapeutic potentials in the treatment of tumors. METHODS: The chimeric GPI anchored-B7-1 gene was obtained by overlap PCR and inserted into expressing vector pcDNA3.1, named pc3.1/GPI-B7-1. pc3.1/GPI-B7-1was transfected into CHO cells by lipofectamine ~2 000 reagent. The CHO cells, expressing GPI-B7-1 on membranes, were obtained after selecting by G418. That was confirmed by flow cytometry, SDS-PAGE and Western blot. RESULTS: Recombinant vector pc3.1/GPI-B7-1 was successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected CHO cell membrane selected by G418 was confirmed to be GPI-anchored protein by flow cytometry, and GPI-B7-1 approximately 60 kD was conformed by SDS-PAGE and Western blot. CONCLUSION: A large amount of GPI-B7-1 fusion protein was obtained and will be further studied in the treatment of tumors.2? [

关 键 词：糖基磷脂酰肌醇类 基因 B7-1 CHO细胞 真核表达 印迹法 蛋白质 

分 类 号：R363[医药卫生—病理学]