摘要
目的利用昆虫细胞杆状病毒系统表达人的重组可溶性L-型选择蛋白配体凝集素(human recombi-nant soluble L-selectin:sL-selectin),探索在真核细胞中高效表达人的重组可溶性L-型选择蛋白配体凝集素的新途径。方法将已经重组好的杆状病毒感染昆虫细胞,表达sL-选凝素于细胞培养上清,利用末端连接的ZZ-结构域(蛋白A来源的首尾相连的二聚化Z结构域)与IgG-琼脂糖-6的结合而分离纯化,通过SDS-PAGE鉴定分子量的大小,并用特异性抗体验证,另外通过重组的sL-选凝素与SMMC7721的黏附观察其活性。结果sL-选凝素可在昆虫细胞中表达,产物的分子量约为46 000,与人肝癌细胞SMMC7721的结合率可达70%。结论sL-选凝素可通过杆状病毒载体在昆虫细胞中有效表达,分离纯化后依然保持生理活性。
Purpose To express recombinant human sL-selectin in insect cell line via baculovirus expression system and thus to explore new way of express this protein in eukaryotic system with high efficiency. Methods Insect cells Sf9 were infected with recombinant baculovirus containing sL-selectin which were then produced and secreted to supernatant. The purification were performed by binding of sL-selectin zz-tag （head-to-tail dimerized Z domain derived from protein A） to IgG-Sepharose. The size of sL-selectin were identified by SDS-PAGE and the protein confirmed with specific antibody, the biological activity was measured by cell adhesion assay. Results The concentrated recombinant sL-selectin ran at size of 46 000 on SDS-PAGE compared with marker and approximately 70% SMMC7721 adhered to plates coated with recombinant sL-selectin. Conclusions The sL-selectin with biological activity can be efficiently expressed by baculovirus expression system.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2006年第5期647-650,661,共5页
Fudan University Journal of Medical Sciences
基金
国家自然科学基金项目(30570414
30470398)资助
