Fractalkine基因真核表达质粒的构建及其在小鼠肝癌细胞中的表达

Construction of Eukaryotic Expression Vector Containing Mouse Fractalkine Gene and Its Expression in Mouse Hepatocellular Carcinoma Cell Line

为克隆小鼠趋化因子Fractalkine(FK)基因, 构建真核表达质粒, 并在小鼠肝癌细胞中表达, 用以进行肿瘤的基因治疗, 用RT PCR法, 从小鼠乳腺癌细胞D2F2扩增FK的cDNA, 插入pCR2. 1 TOPO载体, 测序证实后,将其亚克隆至质粒pIRES中构建FK真核表达载体; 用脂质体将重组质粒转染小鼠肝癌MM45T Li细胞, 经G418筛选获得抗性细胞克隆, 用RT PCR和免疫化学方法鉴定转染细胞中 FK基因的表达. 结果表明: 经限制性内切酶酶切图谱分析和 DNA 序列测定证实目的基因已插入重组质粒, RT PCR 和免疫化学方法证明转基因MM45T .Li细胞克隆中存在小鼠FK基因的表达.

Objective: To clone mouse fractalkine cDNA, construct its eukaryotic expression vector and to express it in mouse hepatocellular carcinoma cell line. Methods: The total RNA was extracted from mouse breast cancer cell line D2F2. The full-length cDNA encoding mouse FK gene was obtained by RT-PCR method and inserted into pCR2. 1 TOPO cloning vector. After the sequencing was confirmed, the gene was subcloned to pIRES to construct recombinant eukaryotic expression vector pIRES-FK. The recombinant plasmid was transfected into MM45T. Li cells by lipofectamine method and positive cell clones were screened with G418. Expression of mouse FK gene in the transfected cells was confirmed with RT-PCR and immunocytochemistry. Results: Enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant vector. Expression of mouse FK gene in the transfected MM45T.Li cells was identified with RT-PCR and immunocytochemistry. Conclusion: The eukaryotic expression plasmid containing mouse FK gene is successfully constructed. The positive MM45T.Li cell clones expressing mouse FK gene are obtained, which may be a promising cell model for studying.