人IFN-β重组慢病毒的制备及对肺癌细胞增殖的影响  

Construction of recombinant encoding Hu IFN-β gene and its effect on A549 cells proliferation

作　　者：郭亮[1] 谢晓[1] 胡丰庆[1] 梅举[1] 丁芳宝[1] 肖海波[1] 

机构地区：[1]上海交通大学医学院附属新华医院心胸外科,上海200092

出　　处：《临床肺科杂志》2013年第4期683-686,共4页Journal of Clinical Pulmonary Medicine

基　　金：国家自然科学基金(30901476)

摘　　要：目的构建人β干扰素(IFN-β)基因重组慢病毒,体外转染人肺癌A549细胞,观察IFN-β基因的表达及其对A549细胞的增殖抑制作用。方法通过聚合酶链反应(PCR)获得人IFN-β基因,克隆到慢病毒载体,通过转染293细胞包装制备慢病毒液,并体外感染A549细胞,采用RT-PCR及Western Blot检测IFN-β基因在A549细胞中的表达情况。应用MTS法检测转染后对A549细胞增殖的影响。结果 IFN-β基因重组质粒经酶切后电泳结果显示能得到与理论大小相符的片段,基因测序结果与GenBank序列完全一致,重组质粒经鉴定正确,转染293细胞获得病毒滴度为3×107PFU/ml,体外感染A549细胞后,RT-PCR、Western Blot检测IFN-β表达水平明显增高,并可明显抑制A549细胞增殖。结论本研究成功构建IFN-β基因重组慢病毒,体外感染后能够稳定表达并抑制A549细胞增殖,为应用IFN-β基因治疗肺癌奠定初步基础。Objective To construct an HIV-based lentivial vector encoding HuIFN-β and to evaluate its effect on infected A549 cells through expression. Methods The Hu IFN-β specific fragment was amplified by PCR and cloned into lentiviral vector. The recombi- nant plasmid was transfected into 293 cells with packing plasmids to obtain recombinant lentivial. A549 cells were infected with recombi- nant lentiviral vector. The expression level of Hu IFN-β was detected by RT-PCR and Westem-blot. MTS assay was used to test the effect of recombinant lentiviral on the proliferation of A549 cells. Results A 518-bp strap was obtained from positive clone of colony PCR am- plification after enzyme incision and gel eleetrophoresis, while DNA sequencing confirmed that the positive clone was consistent with Gen- Bank. The concentration of recombinant lentivial after 293T packing was 2×107 PFU/mL. The levels of mIINA and protein of Hu IFN-β in infected A549 cells were elevated and the A549 cells proliferation was suppressed. Conclusion Lentivial vector encoding Hu IFN-β is successfully constructed and the Hu IFN-β gene can be stably expressed in infected A549 cells and suppress the cell proliferation. This is a fundamental work for IFNβ-based gene therapy of lung cancer.

关 键 词：人β干扰素 慢病毒载体 基因转染 A549细胞 

分 类 号：R734.2[医药卫生—肿瘤]