Foxp4为Fox基因家族的一员, 目前研究发现其与肺、肠、心脏以及中枢神经系统的发育有关。为了在斑马鱼模型中进一步研究该基因的功能, 有必要制备其多克隆抗体。按照巢式PCR原理, 经过两次PCR扩增出亲水性和特异性均好的斑马鱼Foxp4基因片段, 将其克隆入表达载体pGEX4T-1, 转化至大肠杆菌BL21中, 再通过IPTG进行诱导表达GST-Foxp4融合蛋白。融合蛋白先经谷胱甘肽琼脂糖珠亲和纯化, 再经切胶纯化回收, 然后免疫新西兰大白兔制备多克隆抗体, 分别进行Western-blot实验检测抗体效价和特异性。所得结果显示获得了较高效价和特异性好的斑马鱼Foxp4多克隆抗体, 为Foxp4功能的进一步研究奠定了基础。

As a member of Fox gene family, Foxp4 has been involved in the development of lung, intestine, heart, and the central nervous system by the current research. In order to study the further function of Foxp4 gene in Zebrafish model, it is necessary to prepare its polyclonal antibody. According to the principle of nested PCR, the hydrophilic and well specific fragment of Zebrafish Foxp4 was obtained after twice PCR amplifications, then it was cloned into the expression vector pGEX4T-1 and transformed into Escherichia coli BL21. The BL21 strain, containing Foxp4 recombinant plasmid, was induced with IPTG to acquire GST-Foxp4 fusion protein. The induced protein was purified by Glutathione Sepharose and gel cutting extraction,then it was immuned into the New Zealand white rabbit. The antibody’s titer and specification was identified by Western blot separately, and all the results showed that highly sensitivity and specificity anti-Foxp4 polyclonal antibody was generated, which provided a powerful tool for the further studies on Foxp4.