中国免疫学杂志2011，Vol.27Issue(8)：701-708,8.DOI:10.3969/j.issn.1000-484X.2011.08.006
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
摘要
Abstract
Objective:To construct a recombinant adenoviral vector canying and co-expressing human inhibitor of growth 4(ING 4) 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,pcDNA 3.0-IL-24,and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pA dTrack-CMV-ING4-IRES-IL-24,respectively.The pAdTrack-CMV-ING4-IRES-IL-24 transfer vector linearized with Pme Ⅰdigestion 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 Ⅰdigestion 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,A denovirus-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-ERES IL-24 co-expressing ING 4 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 A 549 cells.A denovirus-mediated ING4 and IL-24 co-expression significantly suppressed A549 lung carcinom a cell growth and induced cell apoptosis.The adenoviral vector co-expressing ING 4 and IL-24 mediated by IRES,Ad-ING4-IRES-IL-24,was successfully constructed.Adenovirus-mediated ING 4 and IL-24 co-expression had marked anti-tum or effect in suppressin A549 human lung carcinom a cell growth and inducing cell apoptosis in vitro.Compared with Ad-ING 4 -IRES(growth inhibition ratio at 72h was 42.31％±0.43％,apoptosis rate was 13.30％±1.85％)and AdIRES-IL-24(growth inhibition ratio at 72h was 47.44％±0.39％,apoptosis rate was 12.40％±1.05％),Ad-ING4-IRES IL-24(growth inhibition ratio at 72h is 62.82％±0.65％,apoptosis rate is 19.40％±1.29％)presented significant difference(P＜0.05).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-tum or effect in suppressing A 549 human lung carcinoma cells growth and inducing cell apoptosis in vitro.Compared with Ad-ING4 and AdIL-24,Ad-ING4-IL-24 enhanced antitum or effect.关键词
腺病毒/ING4/IL-24/肺癌/肿瘤基因治疗Key words
A denovirus/Inhibitor of growth 4/Interleukin-24/Lung carcinma/Cancer gene therapy分类
医药卫生引用本文复制引用
盛伟华,谢宇锋,缪竞诚,顾范博,单云波,朱晔涵,陈华昕,杜贤荣,杨吉成..Ad-ING4-IRES-IL-24双基因共表达载体的构建及表达[J].中国免疫学杂志,2011,27(8):701-708,8.基金项目
本文为国家自然科学基金资助项目(81001016)和江苏省卫生厅医学科研基金资助项目(No.H200914) （81001016）
