摘要
目的获取人可溶性血管内皮生长因子受体-1(sFlt-1)基因,构建sFlt-1基因真核表达载体,为进一步研究sFlt-1抗肿瘤血管生成的基因治疗奠定基础。方法提取人胎盘组织总RNA,利用RT-PCR方法扩增获得sFlt-1基因cDNA,将其克隆到pUCm-T载体中,测序证实后,用EcoRⅠ和HindⅢ双酶切pUCm-T-sFlt-1重组载体,胶回收sFlt-1基因,再将其定向亚克隆到真核表达载体pCMV-Script中,提取质粒作双酶切和测序鉴定。结果构建的重组sFlt-1基因真核表达载体pCMV-Script-sFlt-1分别作双酶切和测序鉴定,证实其中含有sFlt-1基因,测序结果经BLAST分析与预期设计的编码区cDNA序列完全一致。结论成功克隆了人sFlt-1基因的真核表达载体。
Objective To amplify human soluble vascular endothelial growth factor receptor-1 （sFlt-1） gene and construct eukaryotic expression vector of sFlt-1 gene,which will be used for gene therapy research of anti-tumor angiogenesis. Methods The sFlt-1 cDNA coding sequences fragment which was amplified by RT-PCR methods from total RNA extracted from human placenta tissue was inserted into pUCm-T vector and confirmed by sequencing. The sFlt-1 cDNA inserted into pUCm-T vector was digested with EcoR Ⅰ and Hind Ⅲ and then subcloned into pCMV-Script eukaryotic expression vector,which was identified by double digestion with restriction endonucleases （EcoR Ⅰ and Hind Ⅲ） and sequencing. Results DNA sequence by BLAST analysis showed that the sFlt-1 gene subcloned into pCMV-Script eukaryotic expression vector was identical to cDNA coding sequences designed in Gen- Bank. Conclusion The recombinant eukaryotic expression vector of human sFlt-1 gene was successfully cloned.
出处
《重庆医学》
CAS
CSCD
2008年第5期484-486,489,共4页
Chongqing medicine
