斑马鱼心脏发育模型中Nodal编码转录因子调节心脏的左右不对称发育, 为了进一步研究Nodal信号途径在心脏发育中的调控作用和心脏疾病发生的分子机制, 需要获得斑马鱼Nodal蛋白并制备其抗体。采用从斑马鱼心脏组织中提取RNA, 通过反转录得到心脏组织各种表达基因的cDNA为模板, PCR扩增得到Nodal部分编码区序列,然后将其连接到pET-28a载体上获得原核表达。经酶切及测序鉴定后, 转化Rosseta细菌, 并用IPTG诱导表达融合蛋白, Ni-IDA凝胶柱亲和纯化, 将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体, 并用Western blotting检测抗体。获得了Nodal原核表达重组融合蛋白及高效价的特异性兔抗Nodal多克隆抗体, 为Nodal功能的进一步研究奠定了基础。

In Zebrafish heart development model, Nodal encodes a transcription factors which can regulate left-right axis asymmetry of heart. The object of obtaining the Zebrafish Nodal protein and it’s antibody is to research the effect of Nodal in the development of heart and the molecular mechanism of heart disease. Using cDNA of the genes expressed in heart tissues which are gotten by reserve transcriptional PCR after isolated the total RNA from the heart of a normal Zebrafish as the template, the cDNA fragment of partial coding sequence of Nodal is obtained by PCR amplification, then was cloned into pET-28a vector and identified with restriction enzyme and sequencing. The recombinant expression plasmid containing Nodal gene was transformed into Rosseta and fusion protein was induced by IPTG. The purity protein obtained by treating the lysates with immobilized Glutathione Sepharose. The protein product was used to immunize the New Zealand white rabbits to prepare antibody. The antibody titer and specification was identified by Western bloting. We have successfully obtained His-tag fusion protein of Nodal, got high sensitivity and specificity anti-Nodal polyclonal antibody. That lays a solid foundation for the further studies of Nodal function.