人可溶性血管内皮生长因子受体-1基因的克隆与鉴定.pdfVIP

484 重庆医学2008年3月第37卷第5期 · 论 著 · 人可溶性血管内皮生长因子受体一1基因的克隆与鉴定 王宋平，钱桂生△，李玉英，黄桂君，安晓静 (第三军医大学新桥医院全军呼吸内科研究所，重庆 400037) 摘 要：目的 获取人可溶性血管内皮生长因子受体一1(sFlt一1)基因，构建sFlt一1基因真核表达载体，为进一步研究sFlt一1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EcoR I和HindIII 5X酶切pUCm-T-sFlt一1重组载体，胶回收sFlt一1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一1基因真核表达载体pCMV—Script—sFlt 一 1分别作双酶切和测序鉴定，证实其中含有sFlt一1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一1基因的真核表达载体。 关键词：可溶性血管内皮生长因子受体一1；基因克隆；抗肿瘤血管生成；基因治疗 中图分类号：Q785 文献标识码：A 文章编号：1671—8348(2008)05—0484—03 Cloning and identification of human soluble vascular endothelial growth factor receptor-1 gene WANG Song—ping，QIAN Gui—sheng，LJ Yu—yin，et a1． (Institute ofRespiratory Medicine ofPLA，Xinqiao Hospital，ThirdMilitary Medical University，Chongqing 400037，China) Abstract：0bjeetive To amplify human soluble vascular endothelial growth factor receptor-1(sFlt一1)gene and construct an— karyotic expression vector of sFlt一1 gene，which will be used for gene therapy research of anti—tumor angiogenesis．Methods The sFlt一1 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一1 cDNA inserted into pUCm—T vector was digested with EcoR I and Hind llI and then subcloned into pCMV-Script eukaryotic expression vector，which was identified by double diges— tion with restriction endonucleases(EcoR I and Hind llI)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 vect