SUMF2基因过表达慢病毒载体的构建及鉴定

Construction and Identification of Lentiviral Vector Over-expressing Sulfatase-modifying Factor 2

目的构建硫酸酯酶修饰因子2(SUMF2)基因过表达慢病毒载体,以便进一步研究SUMF2功能。方法针对人SUMF2 DNA序列设计带酶切位点的引物,PCR法获得目的基因片段;用AgeⅠ和NheⅠ酶双酶切SUMF2基因片段及PLJM1-EGFP载体。用T4连接酶对两种酶切产物进行连接、PCR鉴定、测序。将阳性克隆质粒与包装质粒及包膜蛋白质粒共转染293T细胞,并荧光显微镜观察EGFP的表达。结果 PCR和测序证实成功构建了SUMF2的慢病毒表达载体,病毒效价为1×108TU/ml。结论成功构建SUMF2-PLJM1-EGFP慢病毒载体,并在293T细胞中得到表达。

Objective To construct a human sulfatase - modifying factor 2 （SUMF2） over - expressing lentiviral vector for further functional study. Methods The primers of SUMF2 with restriction endonuclease sites were designed,and gene were amplified by PCR. Purified SUMF2 fragments and PLJM1 - EGFP plasmid were linked by T4 ligase after they were digested by AgeⅠ and Nhe Ⅰ. Positive clones of SUMF2 - PLJMI - EGFP vectors were confirmed by PCR and sequencing. The lentiviral vectors containing SUMF2 gene were transfected into 293T packaging cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. The recombinant lenti- viruses were transfected in to 293 Tcells and the expression of EGFP observed by fluorescence microscopy. Results PCR and DNA se- quencing demonstrated that the lentiviral vector was constructed successfully. The titer of concentrated virus was 1 × 10^8 TU/ml. Conclu- sion The lentiviral vector over - expressing SUMF2 gene was success fully constructed,which can be expressed in 293T cells.