esprg3 多克隆抗体制备及初步功能研究.pdfVIP

16 卷 4 期 生命科学研究 灾燥造援员6 晕燥援4 圆园12 年8 月 蕴蚤枣藻 杂糟蚤藻灶糟藻 砸藻泽藻葬则糟澡 Aug. 圆园12 ESPRG3 多克隆抗体制备及初步功能研究 1, 2 1, 2* 张前军 , 卢光琇 (1. 中南大学 生殖与干细胞工程研究所, 中国湖南长沙4 10078; 2. 人类干细胞国家工程中心, 中国湖南 长沙410078) 摘 要: ESPRG 3 基因是一个与干细胞功能相关的基因, mESPRG 3 和hESPRG 3 基因被构建到原核及真核表达 载体进行表达, 原核表达蛋白用于制备多克隆抗体开展ESPRG3 基因功能研究. 真核表达载体EGFP 被用于 亚细胞定位. 构建的mESPRG 3 基因原核及hESPRG 3 真核表达载体, 成功进行了表达. 利用IPTG 诱导表达 ESPRG3 蛋白制备了多克隆抗体, 免疫印迹和免疫组化结果表明制备的抗体具有特异性; 表达的蛋白检测到体 外具有结合DNA 的能力. 染色质免疫沉淀联合芯片技术检测到ESPRG3 可以特异性结合到染色体上, 且这些 结合位点与Alu 及绝缘子序列具有关联性, 可能通过这些位点调控胚胎干细胞的功能. 关键词: ESPRG 3; 胚胎干细胞; 多克隆抗体; 染色质免疫沉淀(ChIP) 中图分类号: Q291 文献标识码: A 文章编号: 1007 7847(20 12)04 0307 07 Polyclonal Antibodies Preparation and Functional Research of ESPRG3 ZHANG Qian jun1, 2, LU Guang xiu1, 2* (1. Institute of Reproduction and Stem Cell Engineering, Central South Unive rs ity, Changsha 410078, H unan, China; 2. National Center of H uman Stem Cell Research and Engineering, Changsha 410078, H unan, China) Abstract ：ESPRG 3 is a gene related to hES cells. In order to investigate the function of ESPRG 3, the mESPRG 3 prokaryotic and hESPRG 3 eukaryotic expression vector were constructed and polyclonal antibody was prepared. Molecular cloning and IPTG inducing were used to express and purify protein of ESPRG3 for the antibodies preparation. Western blot and imunohischemistry were used to identify the specificity of the antibody of Borealin. The recombinant proteins of ESPRG3 were found to show ability of binding DNA in v ivo and in v itro with the ChIP Chip a