小鼠B16黑色素瘤细胞核迁移蛋白基因的克隆及其在毕赤酵母中的表达

Cloning and Expression of Murine Nuclear Distribution C Protein Gene in Pichia pastoris GS115

利用RT-PCR方法从小鼠B16黑色素瘤细胞中克隆了全长核迁移蛋白C(Nuclear distribution C,NUDC)基因,构建了该基因的毕赤酵母表达质粒pPICZaA-NUDC并在受体细胞Pichia pastorisGS115中获得表达,通过制备融合表达蛋白的多克隆抗体分析其对黑色素瘤细胞活性的影响。结果表明,克隆的NUDC基因全长1 000 bp,含有完整的编码框,经SDS-PAGE和Western blotting分析表明,该基因相对分子质量约为45 kD。表达的蛋白能与兔抗NUDC抗体发生反应,制备的NUDC蛋白抗体可抑制黑色素瘤细胞生长。

The full length Nuclear Distribution C（NUDC）was cloned from mouse B16 melanoma cells using RT-PCR method,and the expression vector harboring the target gene was constructed,which was expressed in Pichia pastoris GS115.The expressed protein was then identified by SDS-PAGE and Western blotting.The effect of anti-NUDC antibody on the growth of B16 melanoma cells was also detected by MTT assay.Results showed that the full-length NUDC gene was about 1 000 bp including a complete coding frame,the relative molecular mass of recombinant expressed protein was about 45 kD.The expressed product can react to rabbit anti-NUDC antibody.Anti-NUDC antibody increased the growth of B16 cell in a dose-dependent manner.