Ad-ING4-IRES-IL-24双基因共表达载体的构建及表达

Construction and expression of adenovirus-mediated ING4 and IL-24 co-expression

目的:构建IRES介导的携带人肿瘤生长抑制因子4(ING4)和人白介素24(IL-24)双基因的重组腺病毒共表达载体(简称为Ad-ING4-IRES-IL-24),研究其表达产物对A549肺癌细胞生长的影响。方法:将PCR扩增的IRES、ING4和IL-24基因片段分别插入pAdTrack-CMV载体中构建pAdTrack-CMV-ING4-IRES-IL-24双基因共表达重组转移载体。按腺病毒载体常规构建方法获得同源重组腺病毒质粒pAdEasy-1-pAdTrack-CMV-ING4-IRES-IL-24,并在QBI-293A包装细胞中进行包装和病毒扩增。将扩增后的Ad-ING4-IRES-IL-24腺病毒感染A549肺癌细胞,并用RT-PCR和Western blot法鉴定ING4和IL-24基因在QBI-293A细胞或A549肺癌细胞中的表达,MTT法和流式细胞仪检测其对A549肺癌细胞生长抑制和诱导凋亡的功能和抑癌增效作用。结果:DNA测序结果显示pAdTrack-CMV转移载体中插入的ING4、IRES和IL-24片段的序列与GenBank报道的完全一致,Ad-ING4-IRES-IL-24能成功介导ING4和IL-24基因在QBI-293A和A549细胞中表达,不仅能明显抑制A549肺癌细胞生长和诱导其凋亡(72小时生长抑制率为62.82%±0.65%,凋亡率为19.40%±1.29%),而且与Ad-ING4-IRES(72小时生长抑制率为42.31%±0.43%,凋亡率为13.30%±1.85%)和Ad-IRES-IL-24单基因组(72小时生长抑制率为47.44%±0.39%,凋亡率为12.40%±1.05%)相比具有显著性差异(P<0.05)。结论:成功构建了IRES介导的Ad-ING4-IL-24双基因共表达重组腺病毒载体,Ad-ING4-IL-24不仅能明显抑制A549肺癌细胞生长和诱导其凋亡,而且与Ad-ING4和AdIL-24单基因组相比具有抑癌增效作用。

Objective：To construct a recombinant adenoviral vector carrying and co-expressing human inhibitor of growth 4（ING4） and human interleukin-24（IL-24） mediated by internal ribosome entry site（IRES）（referred to as Ad-ING4-IRES-IL-24） and explore its effect on the growth of A549 human lung carcinoma cells in vitro.Methods：The IRES,ING4,and IL-24 fragments were amplified by PCR using pGEZ-Term,pcDNA3.0-IL-24,and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-IRES-IL-24,respectively.The pAdTrack-CMV-ING4-IRES-IL-24 transfer vector linearized with Pme Ⅰ digestion and pAdEasy-1 backbone vector were further cotransformed into the bacteria BJ5183 competent cells for homologous recombination.The resultant pAdEasy-1-pAdTrack-CMV-ING4-IRES-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰ digestion and transfected into the human embryonic kidney 293（QBI-293A） cells by liposome,leading to formation of the recombinant adenoviruses Ad-ING4-IRES-IL-24 co-expressing ING4 and IL-24.Infected the A549 cells by the expanded adenoviruses Ad-ING4-IRES-IL-24,Adenovirus-mediated ING4 and IL-24 expression in QBI-293A and A549 cells was examined by RT-PCR and Western blot.The growth-suppressing and apoptosis-inducing effect of Ad-ING4-IRES IL-24 co-expressing ING4 and IL-24 on A549 human lung carcinoma cells were assessed by MTT assay and FCM,respectively.Results：DNA sequencing showed that the ING4,IRES,and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in QBI-293A and A549 cells.Adenovirus-mediated ING4 and IL-24 co-expression significantly suppressed A549 lung carcinoma cell growth and induced cell apoptosis.The adenoviral vector co-expressing ING4 and IL-24 mediated by IRES,Ad-ING4-IRES-IL-24,was successfully constructed.Adenovirus-mediated ING4 and IL-24 co-expression had marked anti-tumor effect in suppressing A549 human lung carcinoma cell growth and inducing cell apoptosis in vitro.Compared with Ad-ING4-IRES（growth inhibition ratio at 72 h was 42.31%±0.43%,apoptosis rate was 13.30%±1.85%） and Ad-IRES-IL-24（growth inhibition ratio at 72 h was 47.44%±0.39%,apoptosis rate was 12.40%±1.05%）,Ad-ING4-IRES IL-24（growth inhibition ratio at 72 h is 62.82%±0.65%,apoptosis rate is 19.40%±1.29%） presented significant difference（P0.05）.Conclusion：The adenoviral vector co-expressing ING4 and IL-24 mediated by IRES（Ad-ING4-IL-24） was successfully constructed.Ad-ING4-IL-24 had marked anti-tumor effect in suppressing A549 human lung carcinoma cells growth and inducing cell apoptosis in vitro.Compared with Ad-ING4 and Ad-IL-24,Ad-ING4-IL-24 enhanced anti-tumor effect.