鸡氨肽酶N蛋白多克隆抗体制备及其生物学功能研究

摘要

In this study, specific primers were designed according to the reference gAPN gene (NM_204861.1) of GenBank. The complete chicken amino peptidase N (gAPN) gene of totally 2 906 bp was cloned and linked with RT-PCR and SOE PCR technique. The gAPN was expressed after construction of pET-30a-gAPN and transformed E. coli Rosetta, the molecular weight of gAPN recom-binant protein is 113 ku. The purified recombinant protein was used as antigen for preparation of rabbit anti-gAPN polyclonal antibody, the titer of polyclonal antibody was 215. Western Blot showed that this polyclonal antibody had highly reactivity and specialty with recombinant protein and natural gAPN from kidney of 18-day-old of chicken. IFA test demonstrated that this polyclonal antibody could react with the HELA cell transfected with eukaryotic expression plasmid of pcDNA-gAPN.%试验参考GenBank上鸡氨肽酶N（gAPN）基因序列（登录号为NM_204861.1）设计多对特异性引物，利用RT-PCR分段克隆gAPN基因各段克隆产物并利用SOE-PCR将其进行连接，得到大小为2906 bp的全长基因。利用原核表达载体pET-30a（+）构建重组质粒pET-gAPN并转化E. coli Rosetta，经诱导表达得到大小为113 ku目的条带。以纯化gAPN重组蛋白为免疫原制备兔抗gAPN多克隆抗体，间接Elisa方法检测多抗血清效价为215。Western Blot试验证明，此多克隆抗体可以与原核表达的蛋白产生特异性条带，同时与18日龄鸡肾组织中获得的天然gAPN蛋白样品有良好的反应性。间接免疫荧光试验显示，多克隆抗体可检测到pcDNA-gAPN转染HELA细胞所表达的gAPN蛋白。上述结果可为gAPN蛋白的深入研究奠定基础。