Tiam1基因慢病毒表达载体构建及其在HT29细胞中的表达

Construction of recombinant lentiviral vector carrying Tiam1 gene and Tiam1 gene expression in HT29 cells

目的:构建Tiam1与绿色荧光蛋白(GFP)融合基因慢病毒表达载体,观察其在人结直肠癌HT29细胞中的表达。方法:通过酶切Tiam1/C1199HA质粒获得Tiam1cDNA片段,并将其克隆到慢病毒载体表达质粒pCDF1-copGFP中,经酶切、测序鉴定。慢病毒表达质粒和包装质粒共转染293FT细胞,获得携带Tiam1和GFP融合基因的重组慢病毒。取病毒上清感染人结直肠HT29细胞株,荧光显微镜检测绿色荧光蛋白及免疫细胞化学检测Tiam1在靶细胞中的表达。结果:重组慢病毒质粒pCDF1-Tiam1-copGFP酶切鉴定结果与目的基因条带吻合,克隆测序结果与NCBI收录的Tiam1基因序列(NM_003253)完全一致。重组慢病毒质粒可高效转染293FT细胞,病毒包装效率为88.34%。收获慢病毒上清可高效感染HT29细胞,荧光显微镜下可观察到大量绿色荧光,感染效率为55.89%,感染后靶细胞中Tiam1稳定高表达。结论:成功构建了携带Tiam1与GFP融合基因的慢病毒表达载体pCDF1-Tiam1-copGFP,并使目的基因在靶细胞中得到稳定表达,为进一步研究Tiam1基因的相关功能提供了优质的稳定转染载体。

Objective To construct a recombinant lentiviral vector carrying both Tiaml gene and green fluorescent protein （GFP） gene and investigate Tiaml gene expression in colorectal carcinoma cell line HT29. Methods Tiaml gene fragments were separated from Tiaml/Cl199HA plasmid and cloned into the lentiviral expression vector pCDFI-copGFP. The correct Tiaml gene was identified by endonucleases digestion and sequencing. Recombinant lentiviruses were produced by 293FT cells following the co-transfection of pCDF1-Tiaml-copGFP and packaging plasmids. The supernatant of virus-producing cells containing Tiaml and GFP genes were then used to transfect HT29. Labelling protein GFP was detected by fluorescent microscopy. The expression of interest protein Tiaml was detected by immunocytochemistry. Results The pCDF1-Tiaml-copGFP matched the strips of the target gene, and the sequences of the Tiaml were identical to those recorded in NCBI. The recombinant plasmid effectively transfected 293FT cells, with a transfection rate of 88.34%. The superuatant of lentivirus effectively infected HT29 cells with a infection rate of 55.89%. Abundant green fluorescence was observed by fluorescent microscopy. Tiaml protein highly expressed in the target cells. Conclusions The recombinant lentiviral vector pCDF1-Tiaml-copGFP was constructed successfully, and the target protein was expressed in HT29 cells. This study provides high quality transfection vectors for further study on the relevant function of Tiaml gene.