花生学报2016，Vol.45Issue(4)：5-13,9.DOI:10.14001/j.issn.1002-4093.2016.04.002
花生iso-ARah3基因克隆及多克隆抗体制备
Gene Cloning of Peanut iso-ARah3 and Preparation of Its Polyclonal Antibody
摘要
Abstract
Iso-ARah3 is a homologue of the peanut Allergen III (ARah3).And its expression in peanut seeds was induced significantly by the invasion of Aspergillus flavus.In this study, the full-length sequence of iso-ARah3 gene was cloned through RT-PCR technology from cDNA library of peanut seeds.And the open reading frame (ORF) of iso-ARah3 with 1533bp encoded 510 amino acids.The N-terminal of this protein had a signal peptide sequence (MAKLLALSLCFCVLVLGASS).And alignment analysis showed that the cloned sequence with a deletion mutation was highly homologous as 83.6% to the trypsin inhibitor of peanut.The recombinant plasmid pET28a-isoARah3 was constructed, and the fusion protein His-isoARah3 was super-expressed in E.coli BL21 under induction of IPTG.SDS-PAGE analysis showed that the fusion protein was approximately 54kD.Then the fusion protein was purified and used as antigen to inject rabbits for four times.The prepared polyclonal antiserum was obtained and purified.And indirect ELISA analysis showed that the anti-isoARah3 polyclonal antibodies have good sensitivity and high specificity with a titer of 1:512000.Furthermore, the polyclonal antibody was used to test the expression of fusion protein in E.coli BL21 through Western Blot, and detected a specific band at 54kD.It suggested that anti-isoARah3 polyclonal antibodies have high specificity and sensitivity.Our results are useful for further functional studies of peanut iso-ARah3.关键词
花生/黄曲霉毒素/序列分析/原核表达/多克隆抗体Key words
peanut/Aspergillus flavus/sequence analysis/prokaryotic expression/polyclonal antibody分类
农业科技引用本文复制引用
姜焕焕,郝翠翠,迟晓元,王冕,陈娜,陈明娜,潘丽娟,祁佩时,王通..花生iso-ARah3基因克隆及多克隆抗体制备[J].花生学报,2016,45(4):5-13,9.基金项目
2014年国家"万人计划"青年拔尖人才 （）
国家花生产业技术体系项目(CARS-14) （CARS-14）
山东省自然科学基金项目(ZR2014YL011 （）
ZR2014YL012) （）
国家自然科学基金项目(31200211) （31200211）
山东省农业科学院青年科研基金项目(2016YQN14) （2016YQN14）
