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目的研究中国仓鼠卵巢上皮细胞是否表达蛋白激酶Cδ亚型。方法参照GeneBank数据库PKCδ亚型的cD-NA序列;通过EMBL-EBI数据库的CLUSTALW,选取种属间同源性最高的序列;利用GENERUNNER软件设计PKCδ亚型的特异引物;RT-PCR扩增CHO细胞PKCδ亚型的cDNA序列;利用AT克隆技术将PKCδ连接到pGEM-T载体上,经蓝白斑筛选,酶切鉴定后测序;Westernblotting方法检测CHO细胞中PKCδ亚型。结果运用RT-PCR和Westernblotting技术证实CHO细胞表达PKCδ亚型,并经测序证实。结论CHO细胞中发现PKCδ亚型的存在,为深入研究PKCδ亚型的结构、功能及与细胞内信号转导通路的关系奠定基础。
Objective To investigate whether Chinese hamster ovary epithelial cells express protein kinase Cδ subtypes. Methods The cD-NA sequences of the PKCδ subtype in GeneBank database were referred to. The sequences with the highest homology among them were selected by CLUSTALW in EMBL-EBI database. Specific primers of PKCδ subtype were designed by GENERUNNER software. CHO cells were amplified by RT- PKCδ subtype. The cDNA of PKCδ was linked to pGEM-T vector by AT cloning technique. The positive clones were selected by blue and white stain and identified by restriction enzyme digestion. The PKCδ subtypes in CHO cells were detected by Western blotting. Results The PKCδ subtype was confirmed by RT-PCR and Western blotting in CHO cells. Conclusion The existence of PKCδ subtype found in CHO cells lay a foundation for further study on the structure and function of PKCδ subtype and its relationship with intracellular signal transduction pathway.