利用DNA免疫技术制备SAX蛋白多克隆抗体

Preparation of Polyclonal Antibody Against SAX Protein by DNA Immunization

骨形态发生蛋白(BMP)属于细胞因子和转化生长因子β(TGF-β)超家族,在胚胎形态发生和器官形成中起重要作用,但目前缺乏BMP信号通路I型受体SAX的各类抗体。本文利用果蝇胚胎提取出总RNA,反转录得到cDNA,将其作为模板通过PCR得到sax基因片段,将片段连接到pCAGGS-P7上,构建成重组质粒pCAGGS-P7-sax。采用质粒DNA免疫技术,免疫了BALB/C小鼠,制备了果蝇SAX蛋白多克隆抗体。进一步分析表明,构建的真核表达重组质粒pCAGGS-P7-saxDNA具有良好的免疫原性,在免疫小鼠后所获得的抗血清抗体效价达1∶100。Western blot和果蝇胚胎免疫荧光检测表明,抗血清能特异型地识别SAX蛋白。果蝇SAX抗体的成功制备为进一步研究sax基因及BMP信号在果蝇心脏发育中的作用奠定了基础。

Bone morphogenetic protein (BMP) belongs to the cytokine and transforming growth factor beta (TGF-β) superfamily and plays an important role in embryo morphogenesis and organ formation. However, it currently lacks various antibodies to the BMP signaling pathway factor SAX. In this paper, the total RNA was extracted from Drosophila embryos, and the cDNA library was obtained by reverse transcription. The SAX gene fragment was obtained by PCR using the cDNA as a template, and the fragment was ligated into pCAGGS-P7 to construct a recombinant plasmid pCAGGS-P7- sax . BALB/C mice were immunized with plasmid DNA immunoassay to prepare polyclonal antibodies against Drosophila SAX protein. Further analysis showed that the constructed eukaryotic expression recombinant plasmid pCAGGS-P7- sax DNA had good immunogenicity, and the anti-serum antibody titer obtained after immunization of mice reached 1 ∶100. Western blot and Drosophila embryo immunofluorescence assay showed that antiserum can specifically recognize SAX protein. The successful preparation of Drosophila SAX antibody laid the foundation for further study of the role of sax gene and BMP signal in Drosophila heart development.