改建型STAT1-CC基因慢病毒载体的构建及其在肺癌A549细胞中的表达

目的:构建携带STAT1基因和突变型STAT1-CC基因的慢病毒表达载体,转染肺癌A549细胞并检测STAT1和STAT1-CC基因在肺癌A549细胞内的表达.方法:采用PCR技术从质粒模板中扩增得到目的基因STAT1和STAT1-CC,将其接入含有CMV启动子和绿色荧光蛋白(EGFP)的慢病毒载体中,构建慢病毒载体表达载体Lenti-STAT1和Lenti-STAT1-CC,双酶切和测序鉴定.分别将重组子Lenti-STAT1、LentiSTAT1-CC和包装质粒共转染293T细胞,包装产生慢病毒颗粒,感染非小细胞肺癌细胞株A549.荧光显微镜观察A549细胞EGFP表达,Western Blotting验证STAT1在A549细胞株中的表达.结果:酶切及测序结果显示成功构建重组慢病毒表达载体Lenti-STAT1和Lenti-STAT1-CC,将慢病毒颗粒感染A549细胞48 h后,观察到宿主细胞内EGFP表达,Western Blotting证实STAT1蛋白在A549细胞中过表达.结论:本实验成功构建了携带STAT1基因和STAT1-CC基因的慢病毒表达载体,并在A549细胞中过表达STAT1基因.

Construction of lentiviral vector encoding modified stat1-cc gene and its expression in A549 lung cancer cell

Objective: To construct the lentiviral vectors carrying stat1,stat1-cc genes and investigate their expression in A549 lung cancer cells.Methods: stat1,stat1-cc genes were acquired from clone plasmids by PCR,and then subcloned into the expression plasmid of lentiviral vector which contained CMV promoter and EGFP to generate the lentiviral vectors Lenti-STAT1 and Lenti-STAT1-CC.The lentiviral vectors Lenti-STAT1 and Lenti-STAT1-CC were identified by restriction enzyme and DNA sequencing.Recombinant lentiviruse was produced by 293T cells following cotransfection of Lenti-STAT1 and Lenti-STAT1-CC with the packaging plasmids.The resulting recombinant lentiviruses were then used to infect Nonsmall-cell carcinoma A549 cells.EGFP expression in A549 cells was observed under fluorescence microscope,STAT1 expression in A549 cells was detected by Western blotting analysis.Results: The recombinant lentiviruses Lenti-STAT1 and Lenti-STAT1-CC carried the STAT1,STAT1-CC gene,The recombinant lentiviruses Lenti-STAT1 and Lenti-STAT1-CC could efficiently infect and deliver STAT1,STAT1-CC and EGFP genes to A549 cells.The EGFP was expressed efficiently.And STAT1 gene can be expressed efficiently in A549 cells by Western blotting analysis.Conclusion: The recombinant lentiviruses Lenti-STAT1 and Lenti-STAT1-CC are pro-duced successfully and they can deliver target gene STAT1,STAT1-CC to A549 cells.