人可溶性血管内皮生长因子受体1基因克隆及真核表达载体的构建

Cloning and construction of eukaryotic-expression vector carring human soluble vascular endothelial growth factor receptor-1 gene

背景:血管内皮生长因子在肿瘤新生血管的形成中发挥着关键的作用,可溶性血管内皮生长因子受体1能竞争性地抑制血管内皮生长因子诱导新生血管形成的生物学功能。目的:克隆人可溶性血管内皮生长因子受体基因1,尝试构建可溶性血管内皮生长因子受体1基因的真核表达载体。设计、时间及地点:基因表达载体构建实验,于2006-10/2007-11在中山大学附属第一医院外科实验室完成。材料:人脐静脉内皮细胞、pMD-18T载体及pcDNA3载体。方法:提取人脐静脉内皮细胞总RNA,使用反转录-聚合酶链反应的方法扩增获得到可溶性血管内皮生长因子受体1基因cDNA,并将其克隆至pMD-18T载体中,经酶切及测序证实。然后应用聚合酶链反应的方法从pMD-18T可溶性血管内皮生长因子受体1重组载体中克隆可溶性血管内皮生长因子受体1基因,再将其定向亚克隆至真核表达载体pcDNA3中,构建真核表达重组体pcDNA3-可溶性血管内皮生长因子受体1,提取质粒进行双酶切、聚合酶链反应及测序鉴定。主要观察指标:可溶性血管内皮生长因子受体1基因反转录-聚合酶链反应情况及pcDNA3-可溶性血管内皮生长因子受体1真核重组体的构建与鉴定结果。结果:构建的真核表达重组体pcDNA3-可溶性血管内皮生长因子受体1经过双酶切及聚合酶链反应鉴定,证实其中含有目的可溶性血管内皮生长因子受体1基因;测序结果经比对分析,证实与预期设计的编码区cDNA一致。结论:应用反转录-聚合酶链反应方法成功从人脐静脉内皮细胞中克隆出可溶性血管内皮生长因子受体1基因,成功构建出了可溶性血管内皮生长因子受体1基因真核表达载体。

BACKGROUND： Vascular endothelial growth factor plays a key role in the process of tumor angiogenesis, the soluble vascular endothelial growth factor receptor-1 （sFlt-1） can competitively blocking neovascularization induced by vascular endothelial growth factor. OBJECTIVE： To clone sFlt-1 gene, in addition, to construct eukaryotic expression vector of sFlt-1 gene. DESIGN, TIME AND SETTING： The construction of eukaryotic expression experiment was performed at Laboratory of Department of Surgery, the First Affiliated Hospital of Sun Yat-sen University from October 2006 to December 2007. MATERIALS： Human umbilical vein endothelia cell, pMD-18T vector and pcDNA3 vector. METHODS： The sFlt-1 gene was amplified by RT-PCR from the total RNA extracted from human umbilical vein endothelia cell （HUVEC）, and then inserted into the pMD-18T vector. Following the double enzyme digestion analysis and DNA sequencing, the sFlt-1 gene in recombinant pMD-18T was amplified by PCR and subcloned into the eukaryotic expression vector pcDNA3, which confirmed by double digestion with restriction endonucleases, PCR analysis and DNA sequencing. MAIN OUTCOME MEASURES： RT-PCR reaction of sFlt-1, and the construction and identification of expression recombination of pcDNA3- sFlt-1 was observed. RESULTS： The sFlt-1 gene was amplified from HUVECs by RT-PCR method. Double enzyme digestion analysis and sequencing showed that the fragment of sFlt-1 gene was inserted into the pMD-18T and pcDNA3 vector. Additionally, BLAST analysis showed that the DNA sequence in above vector was identical to the cDNA coding sequences in GENBANK. CONCLUSION： The sFlt-1 gene can be cloned from HUVEC by means of RT-PCR, and the recombinant eukaryotic expression vector pcDNA3-sFlt-1 of sFlt-1 gene is successfully constructed.