【摘要】 目的构建人凝血酶敏感蛋白1(thrombospondin1,TSP1)抗血管生成片段重组腺病毒。方法采用RT-PCR方法从正常人外周血单个核细胞扩增编码TSP1抗血管生成活性片段(TSP1f)的cDNA序列,并将其克隆至穿梭质粒pShuttle-CMV上,以线性化重组穿梭载体与超螺旋腺病毒骨架质粒pAdEasy-1共转化大肠杆菌BJ5183,进行同源重组并酶切鉴定筛选正确重组子,用线性化重组质粒转染人胚肾293细胞,制备重组腺病毒ADV-TSP1f,最后以PCR及Westernblot方法鉴定TSP1抗血管生成片段的表达。结果细菌内同源重组共获得43个卡那霉素抗性克隆,经酶切鉴定表明其中直径最小的10个均为正确重组子,该重组质粒转染293细胞所获得的重组腺病毒可高效表达TSP1抗血管生成片段,纯化后病毒滴度为1.0×1011pfu/mL。结论细菌内同源重组法构建重组腺病毒高效快捷,所获得的ADV-TSP1f可进一步应用于抗肿瘤血管生成的基因治疗研究。更多还原

【Abstract】 Objective To construct recombinant adenoviruses expressing antiangiogenic fragment of human thrombospondin1(TSP1 f ).Methods TSP1 f cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was subcloned into a shuttle vector pShuttle-CMV.After sequence confirmation,the resultant plasmid was linearized by the restriction endonuclease Pme Ⅰand cotransformed with the supercoiled adenoviral vector pAdEasy-1into Escherichia coli strain BJ5183.Recombinants were selected by Kanamycin resistance and screened by restriction endonuclease digestion.Then,the recombinant adenoviral construct was cleaved with PacⅠand transfected into the packaging cell line293.The adenoviral vector ADV-TSP1 f was propagated in293cells and purified by cesium chloride(CsCl)density centrifugation.PCR and Western blot analysis were performed to confirm TSP1 f expression.Results Of 43Kanamycin-resistant colonies obtained from cotransformation,all of the10smallest ones were the correct recombinants.TSP-1 f was expressed efficiently by ADV-TSP1 f .The virus stock titer after CsCl banding was1.0×10 11 pfu/mL.Conclusions Generating recombinant adenoviruses using AdEasy System results in highly efficient viral production and significantly decrease the time required to construct usable viruses.ADV-TSP1 f can be further used in in vivo gene therapy studies.更多还原