【摘要】 目的:克隆血管内皮生长因子C(VascularEndothelialGrowthFactorC)功能片段的cDNA,构建人VEGF-C基因的真核表达载体,以便进一步研究VEGF-C在宫颈癌中表达的意义及在淋巴管生长及转移中的作用。方法:根据人VEGF-CcDNA序列,设计合成二对特异性引物,第一对5’端含有EcoRⅠ及第二对3’端含有XhoⅠ酶切位点。运用RT-PCR法克隆人MDA-MB231中的VEGF-CcDNA部分编码序列(CDS);经双酶切后将其克隆入真核表达载体pcDNA3.1(+),重组质粒在处于感受态的大肠杆菌DH5α中扩增,纯化。通过PCR和双酶切鉴定阳性重组子及进行基因序列的测定。结果:用RT-PCR克隆到VEGF-CcDNA部分CDS;PCR和双酶切鉴定阳性重组子,显示有人VEGF-CcDNA编码序列,基因序列测定显示重组质粒上插入的人VEGF-C序列正确。结论:从富含VEGF-C的人MDA-MB231细胞系中克隆得到的VEGF-C基因功能片段cDNA,成功构建了人真核表达载体pcDNA3.1(+)/VEGF-C。更多还原

【Abstract】 Objective: To clone a functional human vascular endothelial growth factor C (VEGF-C) cDNA from the MDA-MB231 cell line and to construct a eukaryotic expression vector with this gene to use for further study of the role of the VEGF-C gene in lymphatic dissemination of cervical cancer at the gene level. Methods: Based on the human VEGF-C cDNA sequence, two pairs of specific primers were designed and constructed which contained an EcoRI digestion site in the first primer at the 5’ end and an XhoⅠ digestion site in the second primer at the 3’end. Reverse transcription polymerase chain reaction (RT-PCR) was employed to clone VEGF-C cDNA from the human MDA-MB231 cell line. The product of RT-PCR was digested with EcoRⅠ, SphⅠ and XhoⅠ. After purifying the product, it was ligated into the eukaryotic expression vector pcDNA3.1(+) that had been digested with EcoRⅠ and XhoⅠ. The recombinant plasmid pcDNA3.1 (+) was first propagated in Escherichia coli DH5α, and then it was extracted and purified. The recombinant plasmid was confirmed with PCR and digestion with EcoRⅠ and XhoⅠ in addition to DNA sequence analysis. Results: Human VEGF-C cDNA was amplified from MDA-MB231. The recombinant pcDNA3.1(+)/VEGF-C vector contained correct nucleotide sequence for the full length human VEGF-C cDNA fragment by PCR, restriction digestion and DNA sequence analysis. Conclusion: A eukaryotic expression vector containing full length human VEGF-C in pcDNA3.1 (+) has been successfully constructed.更多还原