抗人毛乳头细胞造血干细胞016基因多克隆抗体的制备  

Polyclonal antibody preparation of the human dermal papilla cell hematopoietic stem/progenitor cell (HSPC) 016 gene

作　　者：邹锋[1] 郝飞[1] 宋志强[1] 易勇[2] 翟志芳[1] 邹全明[2] 叶庆佾[1] 

机构地区：[1]第三军医大学西南医院皮肤科,重庆市皮肤性病研究所,重庆400038 [2]第三军医大学检验系临床微生物学及免疫学教研室暨重庆市生物制药工程技术研究中心,重庆400038

出　　处：《临床皮肤科杂志》2006年第9期563-565,共3页Journal of Clinical Dermatology

基　　金：国家自然科学基金资助项目(30200249)

摘　　要：目的:在大肠杆菌中重组表达人毛乳头细胞造血干细胞(HSPC)016基因功能序列,制备特异性多克隆抗体。方法:采用PCR技术从pcDNA3.1(+)/HSPC016中扩增出目的基因克隆至pMD18-T载体,酶切回收目的片段连接至表达载体pET-28a(+)。重组质粒经酶切及测序鉴定后转化工程菌E.coliBL21,异丙基β-D-硫代半乳糖苷(IPTG)诱导表达,对目的蛋白N-端氨基酸测序鉴定后免疫日本大耳白兔,制备兔抗HSPC016多抗血清,以双向免疫扩散、ELISA、Westernblotting检测抗体效价及特异性。结果:在大肠杆菌中高效表达了相对分子质量约7200的HSPC016蛋白,目的蛋白N-端氨基酸测序结果与预期序列完全一致。双向免疫扩散显示兔抗HSPC016多克隆抗体效价为1∶16;ELISA分析兔抗HSPC016血清的滴度可达320000EU;Westernblotting显示抗血清可与原核表达的HSPC016蛋白特异结合。结论:成功制备了兔抗HSPC016多抗血清,为进一步研究HSPC016蛋白的结构与生物学意义奠定了基础。Objective： To express the human dermal papilla cell hematopoietic stern/progenitor cell （HSPC） 016 in E.coli and to prepare anti-HSPC 016 polyclonal antibodies. Methods： The HSPC 016 gene was amplified from the plasmid of pcDNA3.1（＋）/HSPC 016 by PCR. The gene was subcloned into the plasmid pMD18-T and then cut out by two restriction enzymes of Nco Ⅰ and Hind Ⅲ. After purification, the HSPC 016 gene was inserted into the prokaryotic expression vector pET-28a（＋） which was treated as the same. The pET-28a （＋）/HSPC 016 was then transformed into E.coli BL21 and recombinant protein was expressed after inducted by isopropyhhiogalactoside（IPTG）. The antiserum was prepared by immunizing rabbit with HSPC 016. The titer and specificity of the antibody were detected by double immunodiffusion and ELISA and Western blot respectively. Results： The HSPC 016 protein with relative molecular mass （Mr） of 7200 was highly expressed in E.coli. The rabbit antibody against HSPC 016 was obtained. The double immunodiffusion titer of the antiserum against HSPC 016 was about 1：16. The ELISA titer of the antiserum against HSPC 016 was about 1：320 000. Western blot analysis showed that the antiserum could bind to the expressed HSPC 016 specifically. Conclusions： The rabbit polyclonal antibodies against HSPC 016 have been successfully prepared, which lays the foundation for further studying the structure and function of the protein HSPC 016.

关 键 词：人毛乳头细胞 造血干细胞016基因 原核表达 抗体 兔 

分 类 号：Q786[生物学—分子生物学]