摘要
目的构建人类mir-7-3基因慢病毒表达载体，为进一步研究mir-7-3基因的功能及其在肿瘤治疗中的应用奠定基础。方法采用反转录-聚合酶链（RT—PCR）技术从含有mir-7-3基因的质粒pENTR-MIRNAVECTOR扩增目的基因mir-7-3，并将基因克隆到慢病毒载体表达质粒Lenti-GFP-RNAiVECTOR[含增强型绿色荧光蛋白（EGFP）基因]中，构建慢病毒载体表达质粒Lenti-GFP-mir-7—3，酶切、测序验证mir-7-3基因后，将Lenti-GFP-mir-7-3质粒和包装质粒pRsv—REV、pMDlg-pRRE、pMD2G共同转染人类胚胎肾上皮细胞系293T细胞，获得携带mir-7-3基因和EGFP基因的重组慢病毒FIV-CMV．EGFP-mir-7-3，取浓缩纯化后的病毒上清感染293T细胞和人类胶质瘤细胞U251，荧光显微镜观察293T细胞的荧光表达，RT—PCR鉴定U251细胞中mir-7-3基因的表达水平。结果Lenti-GFP-mir-7-3共转染包装细胞293T能产生高浓度的重组慢病毒FIV—CMV-EGFP-mir-7-3，荧光显微镜下能直接观察到EGFP，FIV—CMV-EGFP-mir-7-3中携有正确的mir-7-3基因，目的基因mir-7．3能被重组慢病毒高效地转导入U251。结论成功构建了携带mir-7-3基因的重组慢病毒载体；为进一步从分子水平探讨mir-7-3基因治疗胶质瘤奠定了基础。
Objective To construct a lentiviral vector containing mir-7-3 gene and green fluorescent protein （GFP） gene, and to detect the expression of mir-7-3 gene in U251 cells. Methods The fragments containing all the mir-7-3 gene were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP, which was transfected together with the packaging plasmids into 293T cells by CaCI2. The supernatant was collected, concentrated, identified, and was transfected to U251 cells of gliomas. Fluorescent microscopy was used to observe the fluorescence in the 293T cell, and real time RT-PCR was used to examine the relative contents of mir-7-3 in U251 cells. Results Electrophores was shown that the sequence of the RT-PCR product was consistent with the data of mir-7-3 by DNA sequence analysis, indicating that the mir-7-3 gene was successfully cloned, and strong green fluorescence was observed by fluorescent microscopy. The supernatant of lentivirus-transfected 293T cells effectively infected U251 cells and the relative content of mir-7-3 was observed in the transfected U251 cells. Conclusion It is concluded that the lentiviral vector containing mir-7-3 gene was constructed successfully, which provides a basis for further study of mir-7-3 function.
出处
《肿瘤研究与临床》
CAS
2011年第4期220-222,共3页
Cancer Research and Clinic
基金
扬州大学临床医学院基金
