| 邓守恒,陈 萍,蔡晓军.携带凋亡素基因重组腺病毒的制备及诱导结肠癌细胞凋亡的实验研究[J].肿瘤学杂志,2015,21(7):572-576. |
| 携带凋亡素基因重组腺病毒的制备及诱导结肠癌细胞凋亡的实验研究 |
| Construction of Recombinant Adenovirus Containing Apoptin(VP3)Gene and Its inducing Apoptosis Effect on Human Colonic Carcinoma Cells in vitro |
| 投稿时间：2014-07-04 |
| DOI：10.11735/j.issn.1671-170X.2015.07.B009 |
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| 中文关键词: 同源重组 腺病毒 凋亡素 结肠肿瘤 |
| 英文关键词:homologous recombination adenovirus apoptin(VP3) colon neoplasms |
| 基金项目:湖北省教育厅基金（B20122413）；湖北省卫生厅基金（QJX2008-42）；湖北医药学院创新团队基金（2014CXZ01） |
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| 中文摘要: |
| 摘 要：［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 cDNA扩增引物，从PET15b-VP3质粒中扩增VP3的DNA序列，与线性pShuttle-IRES-hrGFP连接构建pShuttle-VP3-hrGFP重组穿梭质粒，PCR和EcoR Ⅴ酶切电泳鉴定；重组穿梭质粒经Pmel酶切线性化后，转化含pAdeasy-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pAd-VP3-hrGFP，PCR、PacI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pAd-VP3-hrGFP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重组穿梭质粒构建成功；pAd-VP3-hrGFP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-hrGFP编码区成功克隆入腺病毒pAd中，且其序列与GeneBank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。 |
| 英文摘要: |
| Abstract：［Purpose］ To construct recombinant adenovirus containing Apoptin (VP3)gene by using the method of homologous recombination in bacteria，and to observe its inducing apoptosis effect on human colonic carcinoma cells in vitro. ［Methods］ The VP3 DNA sequence was amplified from PET15b-VP3 and ligated into pShuttle-IRES-hrGFP to construct pShuttle-VP3-hrGFP recombinant shuttle plasmid，and it was identified with PCR and EcoR Ⅴ digestion；pShuttle-VP3-hrGFP was linealized and transformed into ultracompletent BJ5183 containing pAdeasy-1，then pAd-vp3-hrGFP recombinant adenovirus plasmid was constructed and was identified by PCR，digestion and DNA sequencing. subsequently linealized recombinant advenovirus vector pAd-VP3-hrGFP was transfected into AD293 cells by liposome to generate recombinant adenovirus particles which were purified and concentrated by CsCI density gradient centrifugation. Recombinant adenovirus carrying VP3 gene was used to infect the human colonic carcinoma cells SW480，apoptosis rate and cell phase were observed by flow-cytometry. ［Results］ Recombined adenovirus plasmid pShuttle-VP3-hrGFP was generated successfully. Linealized pShuttle-VP3-hrGFP transformed into ultra completent colibacillus BJ5183，There were two bands 4.5kb and larger than 23kb when pAd-VP3-hrGFP was digested with PacI. A 402bp VP3 cDNA fragment was amplified by PCR. The target gene VP3-hrGFP was successfully cloned into adenovirus.The sequence of VP3 segment was identical with that published in GeneBank. Recombinant advenovirus containing VP3 gene was successfully constructed and it was generated with titers of about 1.738×1012opu/L,and it could arrest human colonic carcinoma cell cycle in G0/G1 phase and induce cell apoptosis in dose-dependent manner (P<0.05，P<0.01). ［Conclusion］ Homologous recombination in bacteria can efficiently and conveniently construct high quality recombinant adenovirus containing VP3 gene，which provides a basis for the further research of VP3 gene. |
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