人血管内皮生长因子C基因真核表达载体的构建(英文)

Construction of eukaryotic expression vector for human vascular endothelial growth factor C gene

目的 :VEGF C基因的真核表达载体 ,以便进一步研究VEGF C基因在淋巴管生成中的作用。方法 :根据人VEGF C的cDNA序列 ,设计合成一对 5’端分别含有EcoRⅠ和BamHⅠ酶切位点的特异性引物 ,运用RT RCR方法扩增人乳腺癌细胞MDA MB 2 31中的VEGF CcDNA(1 2 6kb) ;回收PCR产物 (1 2 8kb) ,并将其连接至克隆载体pUMT 18中 ,重组的pUMT 18在大肠杆菌DH5α内扩增后 ,经质粒提取、EcoRⅠ和BamHⅠ酶切 ,筛选出阳性重组子 ,并进行基因测序鉴定 ;琼脂糖凝胶电泳回收含有VEGF CcDNA全长的酶切片断 (1 2 7kb) ,然后在DNA连接酶作用下将其与真核表达载体pcDNA3 1(- )连接 ,重组质粒经EcoRⅠ和BamHⅠ酶切予以鉴定。结果 :RT PCR产物含有VEGF CcDNA ,基因测序显示重组的pUMT 18中含有正确的人VEGF CcDNA全长序列 ,重组的pcDNA3.1(- )中含有人VEGF CcDNA全长序列。结论 :VEGF C pcDNA3.1(- )。

Objective:To construct eukaryotic expression vector of human vascular endothelial growth factor C(VEGF C)gene for further study on the role VEGF C gene play in lymphangiogenesis.Methods:According to human VEGF C cDNA sequence ,we designed a pair of specific primers which contained digestion site of EcoR Ⅰand BamH Ⅰ on the 5' end respectively.Then revert transcript polymorase chain reaction(RT PCR) was employed to amplify VEGF C cDNA from human breast cancer cell MDA MB 231.After being purified ,the product of RT PCR was inserted into a clone vector pUMT 18.The recombinant plasmids pUMT 18,first propagated in Esherichia coli DH5α,then extracted,purified and digested with EcoRⅠ and BamH Ⅰ.Agarose gel analysis and DNA sequence analysis showed that it contained full length of VEGF C cDNA.The obtained VEGF C cDNA was inserted into eukaryotic expression vector pcDNA3.1(-).The pcDNA3.1(-)/ VEGF C ,digested with EcoR Ⅰand BamHⅠ,was found to contain the VEGF C cDNA sequence by agarose gel electrophoresis.Results:The product of RT PCR contained the human VEGF C cDNA.The recombinant pUMT 18 contained correct nucleotide sequence of full length of human VEGF C cDNA fragment.The VEGF C cDNA fragment had been inserted into the eukaryotic expression vector pcDNA3.1(-).Conclusion:The pcDNA3.1(-)/ VEGF C,eukaryotic expression vector for human VEGF C,is constructed successfully.