靶向血管内皮生长因子和神经生长因子的shRNA真核表达载体的构建与鉴定

Construction of eukaryotic expression plasmid expressing shRNA targeting VEGF and NGF

目的利用RNA干扰(RNAi)技术,构建靶向血管内皮生长因子(VEGF)、神经生长因子(NGF)双基因的短发夹RNA(short hairpin,shRNA)真核表达载体用于胶质瘤基因治疗。方法分别设计并体外合成靶向基因VEGF、NGF的特异性编码shRNA序列的寡核苷酸,克隆到经BglⅡ-HindⅢ酶切线性化的pSUPER质粒H1启动子下游,构建shRNA重组质粒,进而脂质体介导瞬时转染胶质瘤细胞系U251,半定量RT-PCR检测VEGF mRNA、NGF mRNA表达。结果重组质粒经过酶切鉴定和测序,插入片段的序列大小、位点和方向均与已合成的寡核苷酸的序列完全一致,在瞬时转染胶质瘤细胞后下调了VEGF mRNA、NGF mRNA的表达。结论成功构建靶向基因VEGF、NGF的shRNA真核表达载体,为进一步应用RNAi技术,研究其对胶质瘤的抑制作用奠定基础。

Objective The aim of this study was to construct eukaryotic vectors expressing short hairpin RNA（shRNA） targeting vascular endothelial growth factor（VEGF） and nerve growth factor（NGF） ,which could be used in the glioma gene therapy. Methods Specific DNA oligonucleotides targeting VEGF and NGF at different sites in their open reading frames were synthesized and cloned into the downstream of the H1 promoter of pSuper plasmids linearized by Bgl Ⅱ -Hind Ⅲ to construct recombinant plasmids encoding shRNA. Results Recombinant plasmids were confirmed by restriction endonuclease digestion and sequencing. The results demonstrated that the length, site and orientation of inserted sequence were exactly correct. Conclusion The vectors expressing shRNA targeting VEGF and NGF have been constructed successfully, which may lay the foundation for co-transfecting the cells of glioma and model animals to observe their inhibiting effect on glioma.