IRES/polyA-promoter介导的Ang-1与VEGF165双基因表达效率的对比分析

Comparison of the Co-expression Level of Ang-1/VEGF165 Mediated by IRES and PolyA-promoter

目的：构建IRES及polyA-promoter介导人血管生成素-1（Angiogenin-1,Ang-1）和人血管内皮生长因子165（vascular endothelial growth factor165,VEGF165）双基因共表达的腺病毒载体,比较IRES与polyA-promoter不同表达模式及其对位于二者前后基因的表达效率和诱导兔角膜新生血管的形成功能,为今后构建双基因或多基因高效共表达载体提供实验依据。方法：以pAdTrack-CMV-Ang-1-IRES-VEGF165质粒为模板,PCR扩增人VEGF165及Ang-1基因片段,分别将其亚克隆至改建的pAdTrack-CMV-PolyA-promoter及pAdTrack-CMV-IRES转移质粒中,构建pTrack-CMV-Ang-1-polyA-promoter-VEGF165、pTrack-CMV-VEGF165-polyA-promoter-Ang-1、pTrack-CMV-VEGF165-IRES-Ang-1基因重组转移质粒,再与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中同源重组,然后经PacI线性化后转染QBI-293A人胚肾成纤维细胞（简称293A细胞）,收获腺病毒重组病毒子Ad-Ang-1-polyA-promoter-VEGF165及Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,RT-PCR检测Ang-1和VEGF165在QBI-293A细胞中的转录,ELISA法分别检测不同腺病毒载体目的基因的表达量,比较分析Ang-1与VEGF165基因在IRES和polyA-promoter介导的不同腺病毒表达载体中的表达能力,及在同一腺病毒表达载体中前后不同位置的表达效率。并进一步于兔角膜缘注射Ad-Ang-1-polyA-promoter-VEGF165,Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,Ad-Ang-1-IRES-VEGF165,检测角膜新生血管的面积,并比较其诱导血管形成能力的差异。结果：测序显示Ang-1和VEGF165序列正确,不同重组腺病毒载体均获得成功包装,病毒效价可达2~5×1010pfu/m l,RT-PCR检测Ang-1和VEGF165均能有效转录,ELISA法检测结果表明Ang-1、VEGF165基因不仅均能在细胞中有效表达,而且IRES介导的Ang-1及VEGF165基因,无论在IRES上游或下游,其表达量均低于polyA-promoter相同位置的Ang-1及VEGF165基因表达量,大约降低60%~70%左右,同时Ang-1/VEGF165在同一载体上、下游不同位置,其下游基因的表达量均明显低于上游基因表达量,大约降低30%~40%左右。角膜血管形成动物实验的结果表明Ad-VEGF165-PolyA-promoter-Ang-1及Ad-VEGF165-IRES-Ang-1诱导角膜新生血管形成面积和血管密度的能力相对较强,且前者比后者效果更为显著。结论：在腺病毒表达载体中,由IRES/polyA-promoter介导的Ang-1与VEGF165双基因均能在细胞中成功表达,并具血管诱导性,但polyA-promoter比IRES介导的双基因表达效率高,诱导血管形成能力强;同时两者下游基因的表达量及血管诱导性能均明显低于上游基因。

Objective：Construct recombinant adenoviral vector co-expressing angiogenin-1（Ang-1）and vascular endothelial growth factor 165（VEGF165）mediated by IRES and polyA-promoter,then compare the expression of Ang-1/VEGF165 mediated by IRES and polyA-promoter as well as in the downstream and upstream of polyA-promoter/IRES likewise their vascular inducibility,to provid experimental evidence for constructing superior recombinant vector co-expressing double gene or multiple gene.Methods：The VEGF165 and Ang-1 fragments were amplified by PCR using pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmids as templates.The VEGF165 and Ang-1 fragments were subcloned into pAdTrack-CMV-IRES and pAdTrack-CMV-PolyA-promoter transfer vector,and then identified by PCR,double endonuclease digestion,and DNA sequencing.The pTrack-CMV-Ang-1-polyA-promoter-VEGF165、pTrack-CMV-VEGF165-polyA-promoter-Ang-1、pTrack-CMV-VEGF165-IRES-Ang-1 gene recombinated transfer vectors were cotransformed into the bacteria BJ5183 competent cells with pAdEasy-1 backbone vector for homologous recombination,then they were linearized with PacI digestion and transfected into the human embryonic kidney 293（293A）cells,leading to formation of the recombinant adenoviruses Ad-Ang-1-polyA-promoter-VEGF165、 Ad-VEGF165-polyA-promoter-Ang-1 and Ad-VEGF165-IRES-Ang-1.The transcription of VEGF165 and Ang-1 were identified by RT-PCR,and the expression of VEGF165 and Ang-1 in different adenoviral vector were then identified by enzyme-labeled immunosorbent assay（ELISA）,then compared the expression level of Ang-1 and VEGF165 gene in IRES and polyA-promoter mediated vector as well as in the downstream and upstream of the same adenoviral vector.Injected Ad-Ang-1-polyA-promoter-VEGF165,Ad-VEGF165-polyA-promoter-Ang-1,Ad-VEGF165-IRES-Ang-1,Ad-Ang-1-IRES-VEGF165 to the rabbit corneal limbal and detected the new vessels areae,then compared their vascular inducibility.Results：The sequencing of Ang-1 and VEGF165 were correct,three kinds of recombinant adenoviral vector were all successfully constructed and obtained,and their potency were up to 2~5×1010pfu/ml,the transcription of VEGF165 and Ang-1 were also significant by RT-PCR.Moreover VEGF165 and Ang-1 gene expressing in the WI-38 cells was confirmed by ELISA,also found that the IRES-mediated Ang-1 and VEGF165 gene,either upstream or downstream,its expression level was lower than the same location in polyA-promoter-mediated genes,degraded about 60%~70%,moreover either the IRES vector or polyA-promoter vector,the expression of downstream genes volume was significantly lower than the upstream gene volume,degraded about 30%~40%.At the same time the animal experiment of vasiformation in rabbit cornea suggestted that the vascular inducibility of Ad-VEGF165-PolyA-promoter-Ang-1 and Ad-VEGF165-IRES-Ang-1 were better,and the former was stronger than the latter.Conclusion：In the adenoviral expression vector,Ang-1 and VEGF165 gene could both successfully expressed in cell,and both had vascular inducibility.The expression level and vascular inducibility of polyA-promoter-mediated double gene was higher and strongger than the IRES-mediated double gene,at the same time the expression level and vascular inducibility of the gene volume in the downstream of polyA-promoter/IRES were lower than the genes volume in the upstream.