【摘要】 目的:构建人血管内皮生长因子C(vascularendothelialgrowthfactorC,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-PCR方法克隆人乳腺癌细胞MDA-MB-231中的VEGF-CcDNA(1.26Kb);回收PCR产物(1.28Kb),并将其连接至克隆载体pMD18-T中;重组的pMD18-T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(1.27Kb),然后在DNA连接酶作用下将其与真核表达载体pcDNA3.1㈠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pMD18-T中含有正确的人VEGF-CcDNA全长序列,重组的pcDNA3.1㈠中含有人VEGF-CcDNA全长序列。结论:成功构建了人类VEGF-C真核表达载体VEGF-C/pcDNA3.1㈠。更多还原

【Abstract】 Objective:To construct eukaryotic expression vector for human vascular endothelial growth factor C(VEGF C)gene for further study on the role of VEGF-C gene in lymphangiogenesis.Methods:According to human VEGF-C cDNA sequence,we designed and constructed a pair of specific primers which contained digestion site of EcoRⅠand BamHⅠon the5’ end.Then reverse transcription poly-morase chain reaction(RT-PCR)was employed to clone VEGF-C cDNA from human breast cancer cell MDA-MB-231.After being purified,the product of RT-PCR(1.28Kb)was ligated into a clone vector pMD18-T.The recombinant plasmid pMD18-T,first propagated in Esherichia coli DH5α,then extracted,purified and digested with EcoRⅠand BamHⅠ,was confirmed to contain full length of VEGF-C cD-NA by agarose gel analysis and DNA sequence analysis.The resulted EcoRⅠ-BamHⅠfragment (1.27Kb)which contained the full length of human VEGF-C cDNA was ligated into eukaryotic expres-sion vector pcDNA3.1㈠digested with EcoRⅠand BamHⅠ.The pcDNA3.1㈠/VEGF-C,digested with EcoRⅠand 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 pMD18-T con-tained correct nucleotide sequence for full length of human VEGF-C cDNA fragment by DNA sequence analysis.TheVEGF-CcDNAfragmenthadbeeninsertedintotheeukaryoticexpressionvectorpcD-NA3.1㈠.Conclusion:ThepcDNA3.1㈠/VEGF-C,aeukaryoticexpressionvectorforhumanVEGF-C,isconstructedsuccessfully.更多还原