人细胞周期蛋白D1及细胞周期蛋白激酶CDK4的原核表达及鉴定

【目的】构建表达人细胞周期蛋白D1及细胞周期蛋白激酶CDK4融合蛋白的重组体。【方法】用RT-PCR从HL-60细胞中扩增细胞周期蛋白D1及CDK4基因片段,克隆到pGEM-Teasy载体上。测序鉴定后切下相应的片段,连接到表达载体pET-22b(+)上,重组质粒转化大肠杆菌BL21(DL3),使之高效表达融合蛋白细胞周期蛋白D1及CDK4。SDS-PAGE电泳及Westernblotting鉴定表达蛋白。【结果】用PCR扩增得到了正确的目的基因片段并构建成pET-cyclinD1和pET-CDK4两个原核表达载体。将其转化大肠杆菌,Westernblot检测证实了转化的大肠杆菌能表达细胞周期蛋白D1及CDK4融合蛋白。【结论】本研究成功构建了细胞周期蛋白D1及CDK4原核表达载体,后两者在细胞内能正确表达细胞周期蛋白D1及CDK4融合蛋白。

Expression and Identification of Human Cyclin D1 and CDK4 Proteins

Objective] To construct recombinant expressing Cyclin D1 and CDK4 proteins. Cyclin D1 and CDK4 genes were obtained by RT PCR from HL 60 cells, then inserted into pGEM T Easy vector. Genes from T vector were inserted into expression vector pET 22b (+ ). Recombinant expression vectors were identified by restriction enzyme cutting and transformed into BL21. The fusion proteins were identified through SDS PAGE electrophoresis and Western blot. The correct Cyclin D1 and CDK4 gene fragments were obtained using PCR amplification technique from HL60 cells. Both E.Coli expression vectors of PET CyclinD1 and PET CDK4 were constructed and then transformed into E.Coli BL21. Western blot analysis confirmed that the transformedE.Coli BL21 could express Cyclin D1 and CDK4 fusion proteins.[ Conclusion] Cyclin D1 and CDK4 expression vectors which can express Cyclin D1 and CDK4 fusion proteins are successfully constructed.