VEGF_(165)正反义表达载体的构建及其对人胃癌细胞VEGF表达的调节

Constructions of eukaryotic expression vector of sense and antisense VEGF_(165) and its expression regulation

目的构建 VEGF_(165)(vascular endothelial growth factor,血管内皮生长因子)正、反义表达载体,将其转染人胃癌细胞,观察其对胃癌细胞 VEGF 在 mRNA 和蛋白质水平表达的调节作用.方法用限制性内切酶将 VEGF_(165) cDNA 从 pGEM-hVEGF_(165)克隆载体上切下来,以正、反方向插入质粒 pCDNA_3中,构建VEGF_(165)正、反义真核表达载体;用限制性内切酶和 Sanger 双脱氧终止 DNA 测序法证明 VEGF_(165)正、反义表达载体目的基因的序列和方向的正确性;用脂质体将 VEGF_(165)正、反义表达载体转染人胃癌细胞,用 RNA 斑点杂交及免疫荧光流式细胞仪检测 VEGF mRNA 和蛋白质的表达水平.结果酶谱分析及 DNA 测序表明 VEGF_(165)正、反义真核表达载体目的基因的序列及方向正确;转染正义表达载体后 VEGF mRNA和蛋白质表达水平增加(蛋白免疫强度为75.4%;P<0.05 vs 对照组,31.6%),转染反义表达载体后 VFGF mRNA 和蛋白表达水平降低(蛋白免疫强度为8.9%;P<0.05 vs 对照组).结论成功地构建了 VEGF_(165)正、反义真核表达载体;构建的载体能在胃癌细胞内有效表达,调节血管内皮生长因子的水平.

AIM To construct eukaryotic expression vector of sense and antisense VEGF_(165)(vascular endothelial growth factor) and transfect it into human gastric cancer cells,and to observe its regulating effect on VEGF mRNA and protein expressions of human gastric cancer cells. METHODS VEGF_(165) cDNA gene was cleaved from pGEM- hVEGF_(165) clone vector,and inserted into plasmid pCDNA_3 in both proper and reverse sequence using proper restriction enzymes.Restriction enzyme analysis and dideoxy sequencing method were used to confirm the orientation and quality of the VEGF cDNA in the pCDNA_3 vector,respectively.The lipofect amine-mediated transfection was performed in human gastric cancer cells using recombinant constructs DNA.VEGF mRNA and protein expressions of the transfectants were investigated by RNA dot blotting and immunofluorescence staining flow cytometer. RESULTS Restriction enzyme mapping and dideoxy sequencing assay performed on DNA from transformed clones demonstrated the presence of the VEGF_(165) cDNA insert cloned in the sense and antisense orientation in the pCDNA_3 vector.Introduction of VEGF_(165) sense into human gastric cancer cells resulted in a significant increase in VEGF-specific messenger RNA and protein (immunofluorescence intensity,75.4%,P<0.05 vs control 31.6% ).Conversely,stable integration of VEGF_(165) in the antisenss orientation resulted in an reduction in VEGF mRNA and protein (immunofluorescence intensity, 8.9%,P<0.05 vs control). CONCLUSION Eukaryotic expression vectors of sense and antisense VEGF_(165) were successfully constructed.The constructed vectors can highly express and regulate vascular endothelial growth factor levels in human gastric cancer cells.