p11 融合蛋白表达、纯化及抗血清的制备 - 中南大学学报（医学版）.pdf

2002, 27( 3) BULL HUNAN MED UNIV 189 p11 谭志平, 黄亮群, 郑 多, 房海燕, 夏 昆, 夏家辉 ( 中南大学中国医学遗传学国家重点实验室, 长沙4 10078) [ ] 目的: 表达GSTp 11 和6xHisp11 融合蛋白并制备GSTp11 特异性抗 方法: 将人p11 基因克隆入两 种原核表达载 pGEX4T2 和pQE30, 分别在大肠杆菌BL21 和M 15 中表达, 用Glu a hione Sepharose 4B 和NiNTA agar ose 亲和柱分别纯化目的蛋白利用纯化的GSTp11 蛋白制备多克隆抗 结果: 得到高表达量的融合蛋白, 经亲和 层析柱纯化获得较高纯度的GSTp11 和6xHisp11 蛋白以 GSTp11 蛋白免疫新西兰兔得到p 11 多克隆抗 , Wes ern blo ing 证实该抗 能够识别6xHisp11 蛋白, 具有较高特异性结论: 利用原核表达人p11 融合蛋白制备的p11 多克 隆抗 具有较好的特异性, 为研究p 11 蛋白在蛋白转运和定位过程中的作用提供重要的技术和材料保障 [ ] p 11( Calpac in I, ligh chain) ; 融合蛋白, GST; 亲和层析; 抗血清 [ ] R93991 [ ] A [ ] 2002) Expression and purification of p11 fusion protein in E . coli and preparation of antiserum against GSTp11 TAN Zhiping, HUANG Liangqun, ZHENG Duo, et al . ( National Laboratory of Medi cal Genetics , Central South Univers ity , Changsha 4 10078, China) Abstract: Objective To ob ain p11 fusion pro ein and prepare specif ic polyclonal an ibody agains p11. Methods A fullleng h human p11 gene was cloned in o expression vec ors, pGEX4T2 and pQE30, and ransformed in o E . coli . The expressed pro eins were purif ied from lysa es wi h Glu a hione Sepharose 4B and he NiNTA agarose column, respec ively. The purified GSTp11 was mixed wi h Freund s comple e or incomple e adjuvan and immunized rabbi s. Results A high level of expression of arge pro eins was de ec ed af er IPTG induc ion and purified pro eins were ob ained by affiniy chroma ography wi h Glu a hione Sepharose 4B and he NiNTA agarose column , respec ively. Wes ern blo ing analysis sugges ed ha he polyclonal an ibody can re