花生iso-ARah3基因克隆及多克隆抗体制备

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。

Gene Cloning of Peanut iso-ARah3 and Preparation of Its Polyclonal Antibody

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.