用杆状病毒-昆虫细胞系统表达、纯化重组的人源化氨基末端LBP  

Expression, Purification of Humanized Truncated LBP in Baculovirus Expression Vector System

作　　者：王长松[1] 刘友生[1] 葛晓冬[1] 李红[1] 陈燕平[2] 

机构地区：[1]第三军医大学西南医院病理学研究所,重庆400038 [2]第三军医大学研究生管理大队三队,重庆400038

出　　处：《中国生物工程杂志》2004年第12期69-73,共5页China Biotechnology

基　　金：国家自然科学基金资助项目 (3 0 2 713 42 )

摘　　要：根据已有的资料显示LBP与LPS的结合位点位于其氨基末端的第 91～ 1 0 0个氨基酸残基。在体外构建含有人LBP与LPS结合活性部分的穿梭质粒pBacmidtLBP ,并且带上 6×his的标签 ,用此穿梭质粒转染sf2 1昆虫细胞 ,获得重组病毒。然后用重组病毒液感染对数生长期的sf2 1昆虫细胞 ,72h后收获含有这种截断型人源化氨基末端LBP (NH LBP)的培养上清。用金属亲和树脂纯化后 ,采用SDS PAGE电泳以及Western Blot鉴定所得到的纯化物。成功获得相对分子质量约为 30 0 0 0的NH LBP。为进一步研究LBP在介导LPS活化靶细胞中的作用机制奠定了基础。According to the published data, the binding site of LBP to LPS is 91～100 amino acids within the amino-terminal half of human LBP. We constructed the shuttle vector pBacmidtLBP with 6×his tag, which comprised the active site of human full length LBP. After being transfected by this kind of shuttle vector with sf21 insect cells, the recombinant virus were harvested. Then infecting sf21 insect cells with recombinant virus 72h, the medium of sf21 insect cells was harvested which contained the truncated humanized amino-ends LBP (NH-LBP), then being purified with TALON metal affinity resin and identified it by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-Blot. It was observed a single band in elution of solution B, and the molecular weight is about 30 000 . It maybe used to further study the role of LBP in catalyzing the activation of LPS to target cells.

关 键 词：LBP 昆虫细胞 LPS 人源化 穿梭质粒 纯化 表达 重组病毒 生长期 收获 

分 类 号：Q78[生物学—分子生物学] R681[医药卫生—骨科学]