斑马鱼心脏发育候选基因Pygo1多克隆抗体的制备及检测

摘要

Pygol is a candidate gene for heart development,which might play an important role in Zebrafish heart development.Hydrophilic antigen region of Pygol is selected by bioinformatic method.Pygol fragment was amplified by PCR and purified.The fragment was inserted into the prokaryotic expression vector pGEMT-4T-l.This new recombined plasmid was transformed into E.Coli and expression of the GST fusion protein was induced with IPTG.After purified by GST affinity chromatography,GST-Pygol fusion protein was used to immune the New Zealand white rabbits to prepare polyclonal antibody.The antibody titer and specificity was identified by Western blot.%Pygo1是心脏发育候选基因,可能在斑马鱼心脏发育过程中起着重要的调控作用.利用生物信息学选择斑马鱼Pygo1基因抗原亲水区,将扩增出的PCR片段克隆到原核表达pGEMT-4T-1载体中,转入E.coli中后通过IPTG(Isopropyl β-D-thiogalactoside)分别诱导表达GST融合蛋白.蛋白经纯化分离后用于免疫新西兰大白兔制备多克隆抗体.用Western Blot检测抗体的效价和特异性.