摘要
目的:构建成骨细胞特异性转录因子Cbfa1双表达载体,以期用于成骨的体内、外研究。方法:以含有全长cDNA的pCMV/Cbfa1为模板,PCR扩增Cbfa1,T-A克隆,酶切亚克隆到pIRES2-EGFP载体上,酶切、PCR及测序鉴定正确后命名为pIRES2-EGFP,脂质体转染NIH3T3细胞,G418筛选稳定表达后,提取细胞的总蛋白,Western blot检测Cbfa1蛋白的表达,以未转染组为对照。结果:酶切、PCR及测序证实pIRES2-EGFP/Cbfa1载体序列正确,脂质体法转染NIH3T3细胞后可见绿色荧光蛋白的表达,Western blot检测到Cbfa1在蛋白水平的表达。结论:pIRES2-EGFP/Cbfa1载体构建成功,可为后续研究奠定基础。
Objective To construct the dual expression vector pIRES2-EGFP/Cbfal of osteoblast specific transcription factor for in vivo or in vitro research on bone. Methods Cbfal was amplified using PCR with pCMV/Cbfal containing a full length of cDNA and acting as a template, subsequently cloned with T-A, and then subcloned to the vector pIRES2-EGFP. After digestion by restriction endonucleases, PCR, and sequencing to confirm its exact sequences, pIRES2- EGFP then transfected cell NIH3T3 by liposome. After screening with G418, the total proteins were isolated. Western blot was used to detect the expression of Cbfal. Results The exact sequences of pIRES2-EGFP/Cbfal vector were confirmed by digestion of restriction endonucleases, PCR, and sequencing. After transfection, the expressions of green fluorescem protein were present. The protein expression of Cbfal was detectable by Western blot. Conclusion The vector pIRES2- EGFP/Cbfal has been constructed successfully, which lays the foundation for further research.
出处
《实用医学杂志》
CAS
2008年第5期704-706,共3页
The Journal of Practical Medicine
