人FGF-9慢病毒表达载体的构建、包装及鉴定

目的构建人成纤维细胞生长因子-9（FGF-9）慢病毒表达载体，鉴定其表达，并对慢病毒载体进行包装，为在体及体外研究FGF-9的功能及其与肿瘤等疾病的关系提供基础。方法PCR扩增人FGF-9基因。全长序列交换入线性表达载体，连接产物转化感受态细胞，PCR及基因测序鉴定阳性克隆。重组质粒转染293T细胞，荧光显微镜观察增强型绿色荧光蛋白（eGFP）表达，Western—blot鉴定FGF-9表达。重组病毒质粒在脂质体介导下与结构质粒和包膜质粒共转染293T细胞包装成人FGF-9过表达慢病毒，RT—qPCR测定滴度。结果PCR及测序证实人FGF-9基因正确克隆至病毒质粒；感染293T细胞后，荧光显微镜观察到eGFP，Western—blot检测到FGF一9融合蛋白表达；三质粒共转染293T细胞获得慢病毒，测其浓度为2×10^8TU／mL。结论成功构建人FGF-9慢病毒表达载体．可在293T细胞中表达，并包装成慢病毒，为在体及体外研究FGF-9的功能及其与肿瘤等疾病的关系提供基础。

Construction and identification of human FGF-9 lentiviral expression vector

Objective To construct a lentiviral vector carrying human FGF-9 gene, detect its expression, and pack the lentivi- ral vector, in order to provide the basis for studying the function of FGF-9 in vivo and in vitro and its relationship with tu-mor. Methods Full-length of FGF-9 gene was amplified by PCR, and cloned into the plasmid pGC-FU-3FLAG-SV40-ECFP, PCR and gene sequencing were used to identify positive clones. 293T cells were transfected by the recombinant plas-mid pGC-FU-3FLAG-SV40-EGFP, and a fluorescence microscope was used to observe the expression of eGFP, and West-era-blot was used to detect the expression of FGF-9. 293T cells were then co-transfected with recombinant plasmid pGC-FU-3FLAG-SV40-EGFP, pHelper 1.0 and pHelper 2.0, and the titer of lentivirus was determined by RT-qPCR. Results The recombinant expression vector was successfully constructed, eGFP and FGF-9 can express successfully in 293T cells, and lentiviruses were successfully packed by the 293T cells, the titer was 2×10^8 TU/mL. Conclusion The recombinant expression vector is successfully constructed, and lentiviruses are successfully packed, it＇s the foundation for further exploration on FGF-9 and its relationship with tumor.