人L-选择素的原核表达及多克隆抗体的制备  

Prokaryotic expression of human L-selectin and preparation of polyclonal antibody

作　　者：高艳光[1] 费君伟[2] 费瑞[3] 曾宪录[1] 

机构地区：[1]东北师范大学遗传与细胞研究所,吉林长春130024 [2]吉林大学第一医院妇产科,吉林长春130021 [3]吉林大学基础医学院细胞生物学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2006年第1期145-149,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30370710)吉林省科技厅自然科学基金资助课题(20010553)

摘　　要：目的:构建人L-选择素原核表达载体,表达并纯化人L-选择素重组蛋白,制备兔抗人L-选择素多克隆抗体。方法:利用人L-选择素cDNA的N末端胞外结构区153个氨基酸的开放阅读框架(ORF),通过 PCR方法扩增出人L-选择素目的片段。将扩增的基因插入表达载体pQE40的BamH I和HindⅢ酶切位点之间,再经转化使其在大肠杆菌M15中表达,产生含6-His-tag的融合蛋白。融合蛋白经Ni亲合层析柱纯化和 SDS-PAGE分析后,用以免疫新西兰白兔,制备多克隆抗体。最后经Western blotting检测和斑点杂交(DIBA) 进行抗体效价测定。结果:酶切鉴定和DNA测序显示,pQE40-L-选择素重组表达载体含有人L-选择素N末端结构区153个氨基酸的cDNA序列;通过在大肠杆菌M15中的表达和纯化分析,获得了蛋白含量为 600 mg·L-1的人L-选择素蛋白;通过免疫新西兰白兔和抗体效价测定,得到抗血清效价达1:1 000的多克隆抗体。结论:人L-选择素蛋白可在M15大肠杆菌中高效表达,其多克隆抗体效价较高。Objective To construct the expression vector and express the recombinant human L-selectin in prokaryotic system, and purify the aimed protein for the preparation of rabbit anti-human L-selectin polyclonal antibody. Methods The partial eDNA of human L-selectin was amplified from the open reading frame （ORF） of N-terminus sequence of L-selectin containing 153 amino acids by PCR, then cloned into the prokaryotic expression vector pQE40 at restriction sites BamH Ⅰ and Hind Ⅲ. The recombinant expression plasmid pQE40-L-selectin was transformed into E. coli M15 for expression. The fusion protein including 6-His-tag was purified by Ni-NTA chromatographic column and analysed by SDS-PAGE. The purified protein was used to immune rabbit for preparing polyclonal antibody. Western blotting analysis and dot immunoblot assay （DIBA） were used to test the titer of the antiserum. Results The expression plasmid pQE40-L-selectin was constructed and confirmed with restriction enzyme digestion. The quantity of the purified protein from E. coli M15 was 600 mg · L^-1. By immuning the rabbit, the polyclonal antibody was successfully prepared. the antiserum had the high titcr （1 = 1 000）. Conclusion The results of dot immunoblot assay （DIBA） showed that The recombinant human L-selectin protein can express with high efficiency in E. coli M15. The prepared polyclonal antibody has a high titer.

关 键 词：L选择素 原核表达 多克隆抗体 大肠杆菌 

分 类 号：R392.11[医药卫生—免疫学]