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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。
OBJECTIVE: To clone the cDNA of functional fragment of vascular endothelial growth factor C (C VEGF) and construct the eukaryotic expression vector of human VEGF-C gene in order to further study the significance of VEGF-C expression in cervical cancer and its role in the growth and metastasis of lymphatic vessels effect. Methods: According to the sequence of human VEGF-C cDNA, two pairs of specific primers were designed and synthesized. The first pair contained EcoRⅠ at the 5 ’end and the second end contained Xho Ⅰ at the 3’ end. The coding sequence (CDS) of VEGF-C cDNA in human MDA-MB231 was cloned by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1 (+) by double enzyme digestion. The recombinant plasmid was expressed in the competent large intestine Bacillus DH5α amplification, purification. The positive recombinant was identified by PCR and double enzyme digestion and the gene sequence was determined. Results: The partial CDS of VEGF-C cDNA was cloned by RT-PCR. Positive recombinant was identified by PCR and restriction enzyme digestion. The coding sequence of VEGF-C cDNA was found. The sequence of human VEGF-C was confirmed by sequencing. Conclusion: The VEGF-C gene cDNA fragment was cloned from human VEGF-C-rich human MDA-MB231 cell line and successfully constructed human eukaryotic expression vector pcDNA3.1 (+) / VEGF-C.