构建含肿瘤坏死因子相关凋亡诱导配体基因慢病毒载体

Construction of a lentivirus vector for Trail gene in rats

背景:腺病毒的表达时间有限,会限制目的基因的表达时间和表达量,不利于实验的持续进行,故文章选择慢病毒作为载体,与目的基因大鼠肿瘤坏死因子相关凋亡诱导配体基因片段进行基因重组。目的:探讨构建含有大鼠肿瘤坏死因子相关凋亡诱导配体基因慢病毒载体的方法。方法:根据GenBank中大鼠肿瘤坏死因子相关凋亡诱导配体序列(NM 145681.1),设计出该基因的特异性引物Trail-AgeⅠ-F和Trail-AgeⅠ-R,用PCR法从大鼠cDNA文库中扩增出目的基因,AgeⅠ酶切将该基因克隆入真核表达载体GV218中,产生目的重组质粒,用脂质体Lipofeetamine 2000包裹构建的重组质粒和辅助包装载体,3个质粒共同转染293T细胞,产生含有表达肿瘤坏死因子相关凋亡诱导配体蛋白的慢病毒颗粒,收集病毒进行滴度鉴定,收集细胞提取蛋白进行基因表达情况的检测。结果与结论:筛选出的克隆阳性菌测序获得肿瘤坏死因子相关凋亡诱导配体基因,全长861 bp,与GenBank中发表的肿瘤坏死因子相关凋亡诱导配体核苷酸序列完全一致。包装后的病毒LV-mTrail在转染2 d后收集病毒液,经PCR和Western blot方法鉴定,从基因和蛋白的层面均证实携带有目的基因肿瘤坏死因子相关凋亡诱导配体基因。孔稀释法及实时荧光定量PCR病毒滴度测定法测定结果为2×109 TU/mL。提示大肠杆菌同源重组法能有效和方便地构建出含有肿瘤坏死因子相关凋亡诱导配体基因的慢病毒载体。

BACKGROUND： Adenovirus, expressing within a limited period, can limit the expression time and amount of target genes that is not conducive to ongoing experiments. Here, we select adenovirus as vectors for genetic recombination with tumor necrosis factor （TNF）-related apoptosis-inducing ligand （Trail） gene fragment. OBJECTIVE： To explore the construction of recombinant lentiviral vector carrying Trail in rats METHODS： The Trail gene was obtained： according to GenBank in rat Trail gene sequence （NM_145681.1）, we designed the gene specific primers of Trail-Age I-F and Trail-AgeI-R, and used AgeI as enzyme cutting site. PCR was applied to amplify Trail gene from rat cDNA Library and construct recombinant plasmids after cutting Trail gene to be cloned into expression vector GV218 by AgeI. Recombinant plasmids were transfected into 293T cells by Lipofeetamine2000 encapsulated recombinant plasmid and auxiliary packaging carrier. The Trail protein of lentiviral plasmids was expressed. Following virus collection, we identified virus titer and extracted protein from cells to detect Trail expression by western blot assay. RESULTS AND CONCLUSION： Screened positive Escherichia coli DH5a competent cells were sequenced with 861 bp, which was consistent with Trail nucleotide sequence in GenBank. After transfection 2 days, virus liquid was collected and confirmed as recombinant plasmid including Trail gene by PCR and Trail proteins expressed in 293T cells by western blot assay. Hole dilution method and real-time fluorescent quantitative PCR determination showed that the virus titer was 2×109 TU/mL. In this study, recombinant lentiviral vector carrying Trail is successfully constructed by homologous recombination in Escherichia coli.