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[目的]构建并鉴定携带人血管内皮细胞生长因子165(VEGF165)和血管生成素-1(ANG-1)双基因共表达重组腺病毒载体pAd-VIA。[方法]采用基因克隆技术克隆目的基因VEGF165、ANG-1基因,将得到的基因通过引入内部核糖体进入位点序列(IRES),亚克隆至含有报告基因EGFP的穿梭载体pTrack-CMV中,构建携带双基因的腺病毒穿梭载体pTrack-CMV-VIA。进而使用AdEasyTM腺病毒系统重组并包装腺病毒。通过绿色荧光蛋白(GFP)表达、酶联免疫吸附分析(ELISA)方法检测制备的腺病毒pAd-VIA感染的大鼠骨髓基质干细胞中外源基因VEGF165及Ang-1的表达。[结果]该重组腺病毒质粒经测序、酶切鉴定,证明基因序列正确。转染QBI-293A细胞后,可观察到GFP明显表达。重组合腺病毒载体pAd-VIA获得成功包装,扩增后病毒滴度为2×1010PFU/ml,大鼠骨髓基质干细胞转染48 h后,绿色荧光蛋白表达阳性,ELISA结果显示转染组在感染48 h后,培养细胞上清中的VEGF165浓度为(42.5±2.082)ng/105细胞,ANG-1浓度为(16.67±2.08)ng/105细胞,未转染组几乎未检测到外源基因的表达,有统计学差异(P<0.05)。[结论]成功构建携带人VEGF165、ANG-1双基因共表达重组腺病毒载体,为组织工程人工骨血管化的研究奠定基础。
[Objective] To construct and identify the recombinant adenovirus vector pAd-VIA carrying VEGF165 gene and Angiopoietin-1 (ANG-1) double gene. [Method] The target gene VEGF165 and ANG-1 gene were cloned by gene cloning technique. The obtained gene was subcloned into the shuttle vector pTrack-CMV containing the reporter gene EGFP by introducing the internal ribosome entry site sequence (IRES) Adenovirus shuttle vector carrying double gene pTrack-CMV-VIA. The AdEasy (TM) adenovirus system was then used to recombine and package the adenovirus. The expression of VEGF165 and Ang-1 in rat bone marrow stromal cells infected with adenovirus pAd-VIA was detected by green fluorescent protein (GFP) expression and enzyme-linked immunosorbent assay (ELISA). [Result] The recombinant adenovirus plasmid was sequenced and identified by enzyme digestion, which proved that the gene sequence was correct. After QBI-293A cells were transfected, GFP expression was observed. The recombinant adenoviral vector pAd-VIA was successfully packaged. After amplification, the virus titer was 2 × 1010 PFU / ml. After 48 h transfection of rat bone marrow stromal cells, the expression of green fluorescent protein was positive. ELISA results showed that transfection group After 48 h, the concentration of VEGF165 in the culture supernatant was (42.5 ± 2.082) ng / 105 cells, and the ANG-1 concentration was (16.67 ± 2.08) ng / 105 cells. Expression, there was a statistically significant difference (P <0.05). [Conclusion] The successful construction of the recombinant adenovirus vector carrying human VEGF165 and ANG-1 double genes could lay the foundation for the study of tissue engineering artificial bone vascularization.