摘要
目的:从人肝癌组织中克隆出人细胞分裂周期基因2(CDC2),并在大肠杆菌中表达CDC2蛋白。方法:从人肝癌组织中提取RNA,采用RT-PCR法扩增CDC2cDNA,用DNA凝胶回收试剂盒纯化并回收RT-PCR产物,将RT-PCR产物插入pET28a(+)载体NdeⅠ和XhoⅠ两酶切位点之间,转化大肠杆菌BL21(DE3);IPTG诱导表达人CDC2蛋白。结果:RT-PCR扩增出约894bp的DNA片段,与GenBank中的CDC2基因序列完全一致。经限制性内切图谱和测序鉴定,成功构建了pET28a(+)/CDC2原核表达载体。SDS-PAGE分析结果显示人CDC2蛋白在大肠杆菌中得已表达。结论:从人肝癌组织中成功地扩增出CDC2基因,并在大肠杆菌中表达。
Objective To clone human cell division cycle gene 2 （CDC2） from human liver cancer tissue and express CDC2 protein in E. coli BL-21 （DE3）. Methods The total RNA was extracted from liver cancer tissue and amplified by reverse transcription polymerase chain reaction （RT-PCR）. The PCR products were cloned into the prokaryotlc expression vector pET28a （＋） followed by DNA sequencing. The recombinant pET28a （＋） /CDC2 （rCDC2） plasmid was transformed into E. coll. The rCI）C2 was induced with IPTG and characterized by SDS- PAGE. Results The cloned CDC2 gene was composed of 894 nucleotides, and is accordance with that reported in GenBank. The prokaryotic expression vector was constructed successfully. The CDC2 protein was successfully expressed in E. coli. Conclusion The CDC2 cDNA is successfully cloned from human liver cancer tissue, and can express in E. coll.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2007年第5期834-836,共3页
Journal of Jilin University：Medicine Edition
基金
吉林省科学技术厅资助课题(20030434)
关键词
人细胞分裂周期基因2
基因克隆
原核表达
human cell division cycle gene 2
gene clone
prokaryotic expression
