斑马鱼Lefty1特异性多克隆抗体的制备

Preparation of Polyclonal Antibody Specially Against Zebrafish Lefty1

Lefty蛋白是TGFβ大家族的配体,通过与Nodal以及Nodal的受体结合而抑制Nodal信号转导。本文采用PCR技术扩增出斑马鱼lefty1基因(z-lefty1)的部分编码区,将其插入到pGEX-4T-1原核表达载体中。所构建的重组质粒(pGEX/z-Lefty1)转化入BL21大肠杆菌,通过IPTG诱导表达GST-z-Lefty1融合蛋白。蛋白经尿素洗涤分离后用于免疫新西兰大白兔以制备多克隆抗体。Western blotting检测结果表明获得了高效价的特异性兔抗z-Lefty1多克隆抗体。这为进一步研究斑马鱼心脏发育的分子机制创造了条件。

Lefty as the ligands of TGFβ family, inhibit Nodal signaling by competitive binding with Nodal or Nodal receptor. Here, a fraction of the encoded region in the zebrafish lefiyl gene （z-lefiy1） was obtained by PCR amplification, and then was inserted into pGEX-4T-1 vector to establish the prokaryotic expressing system. The recombinant plasmid pGEX/z-Lefiy1 was transformed into BL21 and the fusion protein was induced by IPTG. After purification by washing with urea, the fused protein of GST-z-Lefiy1 was injected into New Zealand big white rabbits to produce polyclonal anti- body. To identify the titer and specification of the antibody, Western blotting was employed and the result showed that the polyclonal antibody was of high sensitivity and specificity. The antibody will facilitate further studies on the molecular mechanism of heart development in zebrafish.