携带人Gfi1基因重组慢病毒载体稳定转染32D细胞株的建立

Establishment of a Stable Transfectant 32D/Gfi1 Cell Line by Recombinant Lentiviral Expression Vector

本研究建立真核细胞表达的Gfi1基因重组慢病毒载体包装系统,并实现Gfi1在32D细胞中长期、稳定的表达,为进一步研究Gfi1基因在恶性血液病中的发生发展建立一个有效的平台。制备完整的重组慢病毒载体三质粒系统:转移质粒(pLOX-Gfi1/pLOX)、包装质粒(pCMVΔR8.2)及包膜蛋白质粒(pMD.G)。用脂质体法将三质粒共转染包装细胞293T,48小时后收集病毒上清,并转染靶细胞32D;用Western-blot法检测293T及32D细胞中Gfi1的整合及表达。结果表明:慢病毒的3种质粒可以高效转染293T细胞,并成功包装出慢病毒。目的基因Gfi1能被重组慢病毒高效导入靶细胞32D,并稳定表达,在荧光显微镜下可直接观察到GFP。Western-blot能检测到Gfi1蛋白在包装细胞293T及32D细胞中的表达。结论:慢病毒介导的Gfi1基因可以高效稳定转染32D细胞并持续表达,证明本研究建立了一种有效的基因转移系统。

The aim of study was to establish the packaging system of the recombinant lentiviral vector encoding Gill gene for eukaryotic expression and to realize the efficient, stable expression of Gill 32D cells so as to provide effective platform for further studying the development of Gfil gene in hematologic malignancies. The three-plasmid recombinant lentiviral vector consisting of transfer plasmid （ pLOX-Gfi1 /pLOX）, the packaging plasmid （ pCMVAR8.2 ） and the envelop plasmid（ pMD. G） was prepared and purified. Human embryonic kidney 293T cells were cotransfected with the three plasmids by lipofectamine 2000. After transfection for 48 hours, the viral supernatant was collected and the target cell 32D was transfected with the recombinant lentivirus; the Gill integration and expression in 293T and 32D cells were detected by Western-blot. The results showed that the three plasmids of lentivirus could be transfected into 293T with high efficiency and packaged successfully, and the Gfil protein could be detected by fluorescent microscopy. The recombinant lentiviruses carrying Gfil could transfer and deliver Gfi1 gene to 32D cells, and the Gfi1 expression in 293T and 32D cell could be detected by Western blot. It is concluded that the recombinant lentivirus carrying Gfil can deliver target gene to 32D cells with high efficiency, and the expression of Gfi1 protein is stable in 32D.