6His—hVEGF165融合基因真核表达载体的构建和鉴定

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连人中间载体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／HisA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定向克隆人真核表达载体pcDNA6／HisA，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。

Construction and identification of eukaryotic expression vector for 6His-hVEGF165

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 BamH I and Xho I were 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 bluewhite 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/His A. The positive clone was sent to sequence after PCR and identification by double enzymes. The result of DNA sequencing showed that the cDNA was cloned into pcDNA6/His A in the right direction and the sequence was right. Conclusion： We construct an eukaryotic expression vector of hVEGF165. His tag is fused with the upstream of target gene.