果蝇心脏发育标记基因Hand多克隆抗体的制备

摘要

制备果蝇心脏标记基因Hand抗体对研究果蝇心脏发育具有重要意义.从果蝇体内提取出总RNA,反转录得到果蝇的cDNA,将其作为模板PCR得到Hand基因部分片段,将片段连接到pET-28a上,构建重组质粒pET-28a-Hand,将重组质粒转化rosetta受体菌,IPTG诱导表达,表达产物经镍柱纯化,SDS-PAGE电泳分析,结果表明Hand基因在大肠杆菌中成功表达,表达的Hand融合蛋白分子量大约为24 kD,经镍柱纯化后获得了高纯度可溶性的Hand蛋白.%Polyclonal antibody preparation of Hand gene in drosophila is important in the study of drosophila heart development. The total RNA of drosophila was extracted and reverse transcripted into cDNA. Hand partial coding sequence was obtained by PCR amplification, then was cloned into pET-28a vector to construct recombinant plasmid pET-28a-Hand. It was transformed into E. coll rosetta and then induced by IPTG to express Hand protein in E. coli rosetta expression system. The Hand protein was purified by Ni-NTA affinity chromatography under denaturing conditions. Hand protein with high purity was successfully obtained.