人类DCC基因克隆及真核表达载体构建与鉴定

Cloning and identification of full-length DCC cDNA and construction of its eukaryotic expression vector

目的 克隆人类DCC基因并构建其真核表达载体pIRES2-AcGFPI/DCC.方法 从正常皮肤组织中提取总RNA,采用逆转录-聚合酶链反应（RT-PCR）方法扩增DCC基因全长cDNA（4351bp）,克隆人pMD18-T载体并转化大肠杆菌JM109,经PCR、酶切鉴定均为阳性的克隆,进行核苷酸测序分析,再将DCC基因定向克隆人pIRES2-AcGFP1载体中构建表达载体pIRES2-AcGFP1/DCC.结果 RT-PCR扩增后的产物在约4351 bp处出现明显的特异性条带,DCC基因的cDNA片段被成功插入真核表达载体pIRES2-AcGFP1质粒的多克隆位点,经鉴定与GenBank收录的DCC cDNA序列一致.结论 DCC基因的cDNA片段被成功克隆.

Objective To clone the full-length cDNA of human tumor suppressor DCC gene and construct its eukaryotic expression vector. Methods Total RNA was isolated from human foreskin tissue.Full-length DCC cDNA fragment （4351 bp） was amplified by reverse-transcription polymerase chain reaction （RT-PCR） and inserted into pMD18-T vector. The recombinant pMD18-T/DCC cDNA was transformed into E. coli JM109 host bacteria. The positive clones were confirmed by RT-PCR and doule-enzyme digestion assay. Orientation-based sub-cloning into pIRES2-AcGFP1 was performed as above followed by sequencing. Results Product of RT-PCR showed a clear specific band at 4341bp. pIRES2-AcGFP1/DCC was successfully constructed and transformed into E. coli JM109 host bacteria. Conclusion DCC gene cDNA has been inserted into eukaryotic expression vector pIRES2-AcGFP1 and successfully expressed.