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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,观察其与姜黄素单独或联合对结肠癌SW480细胞凋亡及周期分布的影响。方法设计VP3 cDNA扩增引物,从PET15b-VP3质粒中扩增VP3 DNA序列,与pShuttle-IRES-hrGFP连接构建pShuttle-VP3-hrGFP穿梭质粒,PCR和EcoRⅤ酶切电泳鉴定;线性化穿梭质粒pShuttle-VP3-hrGFP,转化含pAdeasy-1的超感受态BJ5183大肠杆菌,构建pAd-VP3重组腺病毒质粒并经PCR、电泳及测序鉴定,线性化腺病毒质粒经脂质体转染AD293细胞进行pAd-VP3重组腺病毒的包装和扩增,CsCI密度梯度离心法进行病毒浓缩和纯化并将其单独或联合姜黄素作用人结肠癌SW480细胞,观察其对细胞凋亡及周期分布的影响。结果pShuttle-VP3-hrGFP穿梭质粒构建鉴定成功,pAd-VP3重组腺病毒质粒经酶切获得一大于23 kb的大片段和4.5kb的片段,PCR反应扩增出了402 bp的片段,重组质粒测序证实VP3-hrGFP编码区成功地克隆入了腺病毒pAd中,且其序列与GeneBank中VP3 CDNA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-1,获得的重组腺病毒及姜黄素单独或合用均可阻滞细胞周期于G0/G1期,诱导细胞凋亡(P<0.05,P<0.01),二药联用效果更为显著(P<0.01)。结论细菌内同源重组法可快速、高效地制备携带凋亡素VP3基因的重组腺病毒,并能与姜黄素协同通过阻滞细胞周期于G0/G1期诱导结肠癌SW480细胞凋亡。
Objective To construct a recombinant adenovirus carrying VP3 gene by bacterial homologous recombination and observe the effect of curcumin alone or in combination on apoptosis and cycle distribution of colon cancer SW480 cells. VP3 cDNA was amplified by PCR from VP15b-VP3 plasmid and ligated with pShuttle-IRES-hrGFP to construct pShuttle-VP3-hrGFP shuttle plasmid. PCR and EcoRⅤ enzyme digestion electrophoresis were performed. The linearized shuttle plasmid pShuttle- VP3-hrGFP was transformed into Escherichia coli BJ5183 containing pAdeasy-1 to construct recombinant adenoviral plasmid pAd-VP3. The recombinant plasmid was identified by PCR, electrophoresis and sequencing. The adenovirus plasmid was transfected into AD293 cells by lipofectamine to perform pAd- VP3 recombinant adenovirus packaging and amplification, CsCI density gradient centrifugation method of virus concentration and purification and its alone or in combination with curcumin human colon cancer SW480 cells were observed the impact on cell apoptosis and cycle distribution. Results The construction of pShuttle-VP3-hrGFP shuttle plasmid was successfully identified. A large fragment of 23 kb and a 4.5 kb fragment were obtained by restriction endonuclease digestion of pAd-VP3. A 402 bp fragment was amplified by PCR. The recombinant plasmid was sequenced It was confirmed that the VP3-hrGFP coding region was successfully cloned into the adenovirus pAd and its sequence was completely consistent with the VP3 CDNA sequence in GeneBank. The adenovirus carrying VP3 gene was successfully packaged with the titer of 1.738 × 1012opu · ml-1. The recombinant adenovirus and curcumin alone or in combination blocked the cell cycle in G0 / G1 phase and induced apoptosis (P <0.05, P <0.01). The combined effect of the two drugs was more significant (P <0.01). Conclusion Recombinant adenovirus carrying ap VP3 gene can be rapidly and efficiently prepared by homologous recombination in bacteria and can induce the apoptosis of colon cancer SW480 cells through arresting the cell cycle in G0 / G1 phase with curcumin.