人源Nodal成熟肽的原核表达及多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of human Nodal mature peptide

目的:表达人Nodal成熟肽,免疫小鼠制备其多克隆抗体。方法:构建原核表达质粒pReceiver-hNodal,转化大肠杆菌BL21(DE3),经IPTG诱导表达His6-hNodal融合蛋白,融合蛋白经Ni-NTA树脂亲和纯化后,回收融合蛋白,免疫BALB/c小鼠制备多克隆抗体。通过间接ELISA、Westernblot和免疫细胞化学染色和间接免疫荧光等方法检测抗体的效价和特异性。结果:在大肠杆菌中高效表达了与预期理论值大小相符的Mr约13 000的人Nodal成熟肽的融合蛋白,通过免疫小鼠制备的多克隆抗体效价高、特异性强。结论:成功制备了抗人Nodal成熟肽多克隆抗体,为进一步深入研究Nodal在肿瘤发生发展过程中的生物学功能奠定了基础。

AIM： To express the fusion protein of human Nodal mature peptide in prokaryocytes and to prepare mouse anti-Nodal polyclonal antibody.METHODS： The expression vector pReceiver-hNodal was constructed and transformed into BL21（DE3）.His6-hNodal fusion protein was induced by IPTG and separated on SDS-PAGE and the recovered fusion protein was used to immune BALB/c to produce polyclonal antibody.The titer of the polyclonal antibody was detected by indirect ELISA and its specificity was analyzed byWestern blot and immunochemical staining.RESULTS： The expected molecular weight of fusion protein around 15 000 was highly expressed in E.coli and specific antibody was obtained.CONCLUSION： The successful preparation of anti-Nodal mature peptide polyclonal antibody will lay the basis for further study of the biological function of Nodal on tumor progress.