人神经生长因子重组慢病毒载体构建及检测

Construction and Identification of Lentiviral Vector Carrying Nerve Growth Factor Gene

目的:构建含神经生长因子(NGF)基因的重组慢病毒载体,并在293T细胞中验证NGF是否可以过表达。方法:将NGF基因克隆至慢病毒穿梭质粒Ubi-MCS-3FLAG-SV40-EGFP,然后利用酶切鉴定、PCR鉴定及测序验证后,同两个辅助质粒共转染到293T细胞中,构建重组慢病毒载体。重组慢病毒感染293T细胞,通过荧光检测慢病毒的转染效果,用Western Blot检测NGF基因的表达。结果:酶切鉴定、PCR扩增鉴定和对重组慢病毒进行测序,提示重组慢病毒构建正确。荧光显微镜观察重组慢病毒感染的293T细胞,可见强荧光。Western Blot结果提示目的基因可正常表达。RT-PCR测得病毒滴度达到可利用标准。结论:人NGF重组慢病毒载体构建成功。

Objective：To construct the recombinant lentiviral vector containing nerve growth factor （NGF） gene and test the overexpression of NGF gene in 293T cells. Methods：The NGF gene was cloned into lentiviral shuttle plasmid（Ubi-MCS-3FLAG and-SV40-EGFP）, and identified by the enzyme cut assay, PCR and gene sequencing. It was then transfected into 293T cells to construct the recombinant lentiviral vector plasmid. The infection efficiency of lentivrius transfection was detected by fluorescence microscope and the protein expression of NGF was tested by Western blot. Results：The construction of lentiviral vector carrying NGF gene was correct which was verified by the enzyme cut assay, PCR and gene sequencing. Strong fluorescence was observed in 293T cells after lentiviral vectors transfection. Western Blot analysis showed a characteristic band of NGF.Conclusion：Recombinant lentiviral vector carrying NGF gene was constructed successfully.