摘要
蛋白激酶C家族是一类磷脂酰丝氨酸依赖丝氨酸 苏氨酸蛋白激酶。PKCδ(蛋白激酶Cδ)是蛋白激酶C家族成员。PKCδ的异常激活将会导致糖尿病和多种癌症的发生 ,因此 ,PKCδ的特异性抑制剂能成为治疗这些疾病的潜在药物 ,如能以PKCδ为分子靶点来筛选特异性抑制剂 ,就可以建立癌症及糖尿病的药物先导化合物筛选模型。通过RT_PCR的方法 ,克隆了鼠源的PKCδ,并构建了哺乳动物细胞表达载体 ,获得了可稳定表达PKCδ的COS1细胞株。通过亲和吸附的方法获得了较为纯净的PKCδ ,并对酶学活性作了进一步的分析 ,建立了以PKCδ为靶点的药物先导化合物筛选模型。
Protein kinase Cδ(PKCδ)is a member of protein kinase C family, which possess phospholipid-dependent serine and threoni ne kinase activity. PKCδ is a potential drug target of diabetes and some cancer s. The abnormal activation of PKCδ can arouse diabetes and some cancers. Theref ore the specific inhibitors of PKCδ can be applied in the research and developm ent of the drug candidate of these diseases. The present aim is to obtain active recombinant PKCδ from COS1 cells. For cloning of mouse PKCδ a pair of specifi c primers were designed based on the published sequence of this gene. The cDNA of full coding region was obtained by RT-PCR. The amplified cDNA was subsequentl y cloned into FLAG-tagged pcDNA3.0 and its sequence was confirmed by DNA sequen c ing analysis. FLAG-tagged pcDNA3.0-PKCδ was transfected into COS1 cells. A cel l strain which can stably express PKCδ was obtained by G418 screening. FLAG-tagg ed PKCδ in the supernant of COS1 cells extracts was absorbed by anti-FLAG resin and eluted by FLAG peptide. The purified protein appeared as a single band on b oth SDS-PAGE and western blotting, indicating that it was chemical and antigenic pure. By kinase assay, the recombinant PKCδ was active. Positive inhibitor, s taurosporine, was used to prove the enzyme could be greatly inhibited. Several c ompounds have been found to inhibit the enzyme, which indicates the preliminaty application in drug lead compounds screening.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第2期300-304,共5页
Chinese Journal of Biotechnology
关键词
蛋白激酶Cδ
表达纯化
激酶活性分析
protein kinase Cδ, expression and puri fication, kinase assay