人细胞周期蛋白D1/CDK4基因的真核表达及生物活性鉴定

通过生物工程获得人重组细胞周期蛋白 (cyclinD1 )及细胞周期蛋白激酶CDK4蛋白 ,作为抗癌药物筛选的分子靶点 .从人HL 6 0细胞中获得细胞周期蛋白D1 CDK4基因的cDNA ,先克隆至pGEMT Easy载体上 ,再经重组构建供体质粒pFastBac D1和pFastBac CDK4 .重组供体质粒转化感受态DH1 0Bac细胞 ,挑取确证为白色克隆的菌落振荡培养 ,分离制备高纯度杆粒DNA .以重组病毒适量感染昆虫细胞Tn 5B1 4 ,利用Bac to Bac杆状病毒表达系统在昆虫细胞Tn 5B1 4 (Hi5 )中表达相应的重组蛋白 .应用昆虫杆状病毒表达系统 (Bac to Bac)在昆虫细胞Tn 5B1 4中分别高效表达了人细胞周期蛋白D1和CDK4蛋白 .SDS PAGE分析表明 ,表达量占细胞可溶性蛋白质的 2 0 %左右 ,表达产物经Ni2 + NTA亲和层析纯化后纯度达 85 %以上 .研究表明 ,昆虫细胞表达的细胞周期蛋白D1和CDK4蛋白能促进Rb蛋白的磷酸化 ,具有生物活性 .成功构建了细胞周期蛋白D1及CDK4真核杆状病毒表达载体 ,并且在昆虫细胞中正确表达了具有生物活性的细胞周期蛋白D1及CDK4融合蛋白 .

Expression and Activity Identification of Human Cyclin D1 and CDK4 Gene in Insect Expression System

Cyclin D1 and CDK4 fusion proteins were used in antiproliferative inhibitor screening. Cyclin D1 and CDK4 genes were obtained by RT PCR from HL 60 cells and inserted into pGEM T Easy vector. The genes were released from T vector by endonuclease and insered into insect baculovirus donor plasmid. Recombinant vectors were identified by restriction enzyme cutting and homologues recombination in E coli DH10Bac. Recombinant bacmid DNA was transformed into insect cell Tn 5B1 4. The proteins were identified by SDS PAGE and Western blotting. The activity of Cyclin D1/CDK4 was detected by phosphorylation of GST Rb in kinase assay. The insect expressing vectors could express Cyclin D1 and CDK4 proteins which possessed ability of phosphorylating Rb protein, showing biological activity. Cyclin D1 and CDK4 proteins were expressed correctly in insect cells and had their biological activity.