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[目的]利用细菌内同源重组法构建携带凋亡素VP3基因的重组腺病毒并观察其体外诱导结肠癌细胞凋亡的作用。[方法]设计VP3 c DNA扩增引物,从PET15b-VP3质粒中扩增VP3的DNA序列,与线性p Shuttle-IRES-hr GFP连接构建p Shuttle-VP3-hr GFP重组穿梭质粒,PCR和Eco RⅤ酶切电泳鉴定;重组穿梭质粒经Pmel酶切线性化后,转化含p Adeasy-1的超感受态BJ5183大肠杆菌,细菌内同源重组法构建重组腺病毒质粒p Ad-VP3-hr GFP,PCR、Pac I酶切电泳及测序鉴定;线性化重组腺病毒质粒经脂质体转染AD293细胞进行p Ad-VP3-hr GFP重组腺病毒的包装和扩增,Cs CI密度梯度离心法进行病毒浓缩和纯化并将其作用人结肠癌SW480细胞,观察其对细胞凋亡及周期分布的影响。[结果]p Shuttle-VP3-hr GFP重组穿梭质粒构建成功;p Ad-VP3-hr GFP重组腺病毒质粒经酶切获得一大于23kb的大片段和4.5kb的片段,PCR反应扩增出402bp的片段,测序证实VP3-hr GFP编码区成功克隆入腺病毒p Ad中,且其序列与Gene Bank中VP3序列完全一致;成功包装出携带VP3基因的腺病毒,滴度为1.738×1012opu/ml,重组腺病毒可阻滞结肠癌细胞周期于G0/G1期,诱导细胞凋亡,MOI值越大作用效应越显著(P<0.05,P<0.01)。[结论]细菌内同源重组法可快速、高效制备携带凋亡素基因的重组腺病毒,并具有较强的抗肿瘤作用,为深入研究VP3基因功能提供了选择。
[Objective] The purpose of this study was to construct recombinant adenovirus carrying apoptin VP3 gene by homologous recombination in bacteria and to observe its effect on inducing colon cancer cell apoptosis in vitro. [Method] The VP3 c DNA amplification primer was designed and the VP3 DNA sequence was amplified from the PET15b-VP3 plasmid. The p shuttle-VP3-hr GFP recombinant shuttle plasmid was constructed by ligating with the linear p Shuttle-IRES-hr GFP. PCR and Eco RV The recombinant shuttle plasmid was linearized by Pmel digestion and transformed into Escherichia coli BJ5183 containing p Adeasy-1. The recombinant adenovirus plasmid pAd-VP3-hr GFP was constructed by homologous recombination in bacteria , Pac I enzyme digestion electrophoresis and sequencing identification; linearized recombinant adenovirus plasmid was transfected AD293 cells liposome p Ad-VP3-hr GFP recombinant adenovirus packaging and amplification, Cs CI density gradient centrifugation for virus concentration And purified and applied to human colon cancer SW480 cells to observe its effect on cell cycle and apoptosis. [Results] The p Shuttle-VP3-hr GFP recombinant shuttle plasmid was successfully constructed. The recombinant plasmid p Ad-VP3-hr GFP was digested by restriction enzyme to obtain a large fragment larger than 23kb and a 4.5kb fragment. The PCR reaction amplified a 402 bp The VP3-hr GFP coding region was successfully cloned into adenoviral pAd and its sequence was identical with the VP3 sequence in Gene Bank. The recombinant adenoviruses carrying VP3 gene were successfully packaged and the titer was 1.738 × 1012opu / ml. Recombinant adenovirus can block the cell cycle of colon cancer in G0 / G1 phase and induce apoptosis. The larger the MOI, the more significant the effect (P <0.05, P <0.01). [Conclusion] Recombinant adenovirus carrying apoptin gene can be rapidly and efficiently prepared by homologous recombination in bacteria and has a strong anti - tumor effect, which provides a choice for further study on the function of VP3 gene.