人血管内皮细胞生长因子腺相关病毒包装质粒pSNAV-hVEGF_(165)的构建及在HEK293细胞的表达

The construction of the packaging plasmid pSNAV-hVEGF_(165) of AAV encoding human hVEGF_165 cDNA and its expression in vitro

目的构建人血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)腺相关病毒(adeno-associated virus,AAV)表达载体包装质粒pSNAV-VEGF165,并体外检测pSNAV-VEGF165表达和生物学活性。方法采用分子克隆技术,从pcDNA3(+)-VEGF165质粒获得VEGF165cDNA,并克隆至AAV包装质粒pSNAV上,构建pSNAV-VEGF165的AAV重组质粒,经酶切及测序鉴定正确,用脂质体介导pSNAV-VEGF165转染HEK293细胞和血管内皮细胞(vascular endothelial cell,VEC),荧光免疫组化方法检测VEGF165蛋白,并用MTT法测定VEGF165对VEC增殖的影响。结果经酶切鉴定及基因测序证实AAV包装质粒pSNAV-VEGF165构建成功,荧光免疫组化显示VEGF165蛋白的表达,对照组未见VEGF165蛋白的表达,MTT法显示VEGF165蛋白对VEC增殖有促进作用。结论构建的AAV包装质粒pSNAV-VEGF165在体外具有生物学活性,可作为AAV-VEGF165表达载体的包装质粒。

Purpose To construct the packaging plasmid pSNAV-VEGF165 of adeno-associated virus vector encoding hVEGF165 cDNA, and to detect the expression and bioactivity of pSNAV-VEGF165 in vitro. Methods The VEGF165cDNA obtained from plasmid pcDNA3（ ＋ ）-VEGF165 was subcloned into the packaging plasmid pSNAV of AAV by molecular clone ways. The recombinant plasmid pSNAV-VEGF1165 was identified by restriction enzymes analysis and sequencing analysis, and then transfered to the HEK293 cell and VEC by lipofectamine mediated gene transfer method. The protein VEGF165 was detected by immunocytochemistry and immunofluorescence and the influence to VEC＇s proliferation was explored by MTT method. Results The recombinant pSNAV-VEGF165 was correctly constructed and confirmed by restriction enzymes analysis and sequencing analysis. The protein VEGF165 was detected by immunocytochemistry and immunofluorescence in experimental group, but in control group the protein VEGF165 was not expressed, and VEGF165 could promote VEC＇s proliferation. Conclusions The constructed AAV-hVEGF165 packaging plasmid pSNAV-VEGF165 had the bioactivity in vitro, and could be the packaging plasmid of AAV-hVEGF165.