着丝粒特异性蛋白CENP-Ⅰ的原核表达、纯化及抗体制备  

Expression and Purification of Centromere-Specific Protein CENP-Ⅰ and Preparation of Its Antibody

作　　者：蔡燕[1] 翁亚光[1] 施琼[1] 徐远久[1] 刘子杰[1] 王应雄[2] 

机构地区：[1]重庆医科大学医学检验系临床检验诊断学省部共建教育部重点实验室重庆市重点实验室,重庆400016 [2]重庆医科大学公共卫生学院,重庆400016

出　　处：《西南大学学报（自然科学版）》2008年第2期143-148,共6页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(30371485);教育部博士点基金资助项目(20060631012);重庆市重点项目[渝科发计字(2004)47号]

摘　　要：用RT-PCR的方法从Hela细胞总RNA中获得编码CENP-Ⅰ蛋白第1-293位氨基酸的CENP-ⅠcDNA序列,采用基因体外重组法将其克隆到原核表达载体pET-32a(+)内;将重组质粒转化到大肠杆菌BL21(DE3)中,用IPTG诱导表达、Ni2+-NTA亲和层析纯化获得高纯度的重组蛋白质;用纯化的蛋白质免疫家兔制备抗血清,琼脂糖免疫双扩法和Western blot鉴定得到高效价的特异性抗CENP-Ⅰ抗体.Template cDNA was obtained by RT-PCR from the whole RNA of cultured HeLa cells. By gene recombination technology in vitro, the sequence encoding the amino acid 1-293 of CENP-Ⅰ was cloned into pET-32a（＋） expression vector to construct pET-CENP-Ⅰ recombinant plasmid, which was then transformed into E. coli BL21 （DE3）. The recombinant CENP-Ⅰ was induced and expressed by IPTG. The Ni^2＋-NTA affinity column was used to purify CENP-Ⅰ , which was used to immune rabbits. Specific antibody of CENP-Ⅰ with a high valence was confirmed by Western blot and agarose double immunodiffusion.

关 键 词：着丝粒特异性蛋白质 CENP-Ⅰ 染色体不稳定性 

分 类 号：R394.2[医药卫生—医学遗传学]