果蝇血液发育标记基因Dox-A3多克隆抗体的制备  

Polyclonal Antibody Preparation of Blood Development Marker Gene Dox-A3 in Drosophila

作　　者：苏勇[1] 蔡哲彦[1] 江志钢[1] 袁婺洲[1] 吴秀山[1] 

机构地区：[1]湖南师范大学蛋白质化学及鱼类发育生物学教育部重点实验室心脏发育研究中心,湖南长沙410081

出　　处：《湘南学院学报》2012年第5期33-36,共4页Journal of Xiangnan University

基　　金：国家自然科学基金项目(30671053;30871340;30930054;30900851);国家重点基础研究发展计划项目(2005CB522505)

摘　　要：从野生型成体果蝇体内提取总RNA,以cDNA作为模板进行PCR扩增,获取Dox-A3部分基因片段,将这片段连接于原核表达载体pET-28a上,成功构建重组质粒pET-28a-Dox-A3.将重组质粒转化大肠杆菌菌株Rosetta,用ITPG诱导表达出融合蛋白,然后将经Ni-IDA凝胶柱纯化的融合蛋白免疫新西兰大白兔制备Dox-A3多克隆抗体,通过Western-Blot检测效价和特异性.结果表明,实验获得了高质量的多克隆抗体.The total RNA of drosophila was extracted and reverse transcripted into cDNA. Dox - A3 partial coding sequence was obtained by PCR amplification, and inserted into pET28a vector in frame. The reconlbinant plasnmid pET- 28a - Dox - A3 was succeeded constructed. It was transformed into E. coli rosetta and then induced by IFTG to express Dox - A3 protein in E. coli rosetta expression system. The Dox - A3 protein was purified by Ni - NTA affinity chromatography under denaturing conditions. Then the New Zealand white rabbits was immuned with purified recombinant protein to get antibody. The antibody titer and specificity was identified by Western Blot. The result showed that the high quality multi- elonal antibody was obtained.

关 键 词：Dox—A3基因 果蝇 原核表达 多克隆抗体 

分 类 号：Q71[生物学—分子生物学]