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类花生致敏原3(iso-ARah3)是花生致敏原3的同系物,其表达活性与干旱及黄曲霉侵染相关。本研究通过RT-PCR技术从花生种子cDNA文库中克隆获得iso-ARah3的开放阅读框(ORF),全长1533bp,编码510个氨基酸,N端预测有20个氨基酸的信号肽序列。通过序列比对发现,该克隆序列有1处缺失突变,其N端210个氨基酸序列与胰蛋白酶抑制因子的同源性较高,达83.60%。通过构建原核表达载体pET28a-isoARah3,转化进大肠杆菌BL21,经IPTG诱导表达,利用SDS-PAGE检测表明,融合蛋白His-isoARah3获得高效表达,分子量约54kD。His-isoARah3经纯化和富集,对新西兰兔进行4次免疫,纯化获得的iso-ARah3多克隆抗血清,通过间接ELISA检测,表明获得了效价比(1∶512000)很好的抗体。通过对融合蛋白的诱导前和纯化后样品进行Western Blot分析,结果显示仅在纯化样品相应位置(54kD)有明显信号,表明所制备的抗体具有很高灵敏度和特异性。本研究结果为深入研究花生iso-ARah3基因的功能奠定了基础。
Iso-ARah3 is a homolog of peanut allergen 3, whose expression activity is related to drought and aflatoxin infection. In this study, the open reading frame (ORF) of iso-ARah3 was cloned by RT-PCR from a peanut seed cDNA library. The full length of the open reading frame (ORF) was 1533bp, encoding 510 amino acids and a 20 amino acid signal peptide at the N-terminus. Sequence alignment showed that there was one deletion mutation in the cloned sequence. The 210 amino acid sequence of the N-terminal sequence showed high homology with trypsin inhibitor (83.60%). The prokaryotic expression vector pET28a-isoARah3 was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE analysis showed that the fusion protein His-isoARah3 was highly expressed, with a molecular weight of about 54 kD. Purified and enriched His-isoARah3, New Zealand rabbits were immunized 4 times and the obtained iso-ARah3 polyclonal antiserum was purified by indirect ELISA. The results showed that a titer (1: 512000) antibody was obtained. Western Blot analysis of the pre-induction and post-purification samples showed that there was a clear signal only at the corresponding position of the purified sample (54 kD), indicating that the prepared antibody has high sensitivity and specificity. The results of this study laid the foundation for further study on the function of iso-ARah3 gene in peanut.