6HishVEGF165融合基因真核表达载体构建和鉴定.docVIP

6HishVEGF165融合基因真核表达载体构建和鉴定 　　[摘要] 目的:本实验拟构建hVEGF165基因的真核表达载体,为其应用于治疗大鼠缺血皮瓣的研究奠定基础。方法:设计引物P1和P2,在每一个引物的两端分别添加BamHⅠ、XhoⅠ酶切位点。从人外周血中提取总RNA,用RT-PCR方法得到hVEGF165的cDNA。随后再将得到的cDNA连入中间载体pMD19-T,得到pMD19-T-hVEGF165,双酶切鉴定并测定序列。将hVEGF165的cDNA亚克隆到真核表达载体pcDNA6/His A中,构建hVEGF165真核表达载体pcDNA6-hVEGF165。结果:对pMD19-T-hVEGF165的测序结果表明,本研究成功地从人外周血中扩增到hVEGF165的cDNA。将hVEGF165定向克隆入真核表达载体pcDNA6/His A,经双酶切及PCR鉴定,将阳性克隆测序,经测序证实序列正确。结论:本研究成功地构建了VEGF165真核表达载体pcDNA6-hVEGF165,并且目的基因上游融合有His标签。[关键词] VEGF165;皮瓣;载体构建;融合基因 　　[中图分类号] R34[文献标识码]A [文章编号]1673-7210(2010)03(a)-019-04 　　 　　Construction and identification of eukaryotic expression vector for 　　6His-hVEGF165 　　ZHANG Xianyu1, WANG Tao2, CHEN Weihua2, PANG Da1 　　(1.Department of Breast Surgery, the Tumor Hospital of Harbin Medical University, Harbin 150081, China; 2.Department of Reconstruction Surgery, the Fourth Clinical Hospital of Harbin Medical University, Harbin 150001, China) 　　[Abstract] Objective: To construct eukaryotic expression vector of hVEGF165 to lay the foundation for the study of the treatment of ischemic rat skin flaps. Methods: Two specific primers P1 and P2 were designed, restriction enzyme cutting site of BamHⅠ and XhoⅠwere introduced into the two ends of each primer. We isolated mononuclear cells from human venous blood and got out the total RNA and then the hVEGF165 cDNA was cloned by using RT-PCR. We cloned the cDNA into pMD19-T vector, named pMD19-T-hVEGF165. The positive clone was sent for DNA sequencing after blue-white selection and identification by double enzymes digestion. To construct the eukaryotic expression vector of hVEGF165, we subcloned the cDNA into the eukaryotic expression vector pcDNA6/His A. The PCR and identification by double enzymes digestion were performed and the positive clone was sequenced as before. Results: The result of DNA sequencing of pMD19-T-hVEGF165 showed that we successfully cloned the hVEGF165 cDNA from the human venous blood. The cDNA of hVEGF165 was directed cloned into pcDNA6