人细胞周期蛋白依赖性激酶2(CDK2)基因克隆和原核表达

Clone and expression of the human cyclin dependent kinase 2 in E.coli

目的从人胃癌组织中克隆出人CDK2基因和原核表达CDK2蛋白。方法从胃癌组织中提取RNA,逆转录PCR扩增CDK2cDNA‘PCR产物进行TA克隆和DNA序列分析;阳性TA克隆CDK2片段亚克隆入PET28a+载体,IPTG诱导表达人CDK2。结果逆转录PCR扩增出约900bp的DNA片段,TA克隆和DNA序列分析显示重组片段是人CDK2基因序列,全长897bp;CDK2cDNA亚克隆入PET28a+载体NcoI和XhoI位点之间;IPTG诱导出约34kD的蛋白。结论从人胃癌组织中成功地扩增出人CDK2基因,并且在大肠杆菌中得到表达。

Objective To clone the human cyclin dependent kinase 2(CDK2) from human gastric cancer and express CDK2 in E.coli. Methods RNA was extracted from human gastric cancer tissue and amplified by reverse transcription PCR;PCR products were cloned into TA vector followed by DNA sequence .CDK2 cDNA fragments in TA vector were subcloned into PET28a+;and IPTG induced the CDK2 expression. Results A DNA fragment about 900 bp was amplified and the sequence result showed that the fragment was just human CDK2 cDNA(897 bp , ORF).The recombinant PET28a+ subcloned with CDK2 cDNA was induced by IPTG for 34 kD CDK2 protein Conclusions The human CDK2 cDNA was successfully cloned from gastric cancer tissue, and expressed CDK2 protein in E.coli.