Jurkat新基因J441与FLAG融合基因慢病毒载体的构建

Construction of lentivirus carrying FLAG and novel gene J441 from Jurkat

目的 作者前期实验经“高通量测序”得到T淋巴细胞白血病细胞株基因表达数据,经生物信息学分析得一新序列,暂命名为J441.为了对这一新序列进行进一步深入研究,本实验构建了T淋巴细胞白血病新基因J441与FLAG标签肽融合基因慢病毒载体.方法 引物上引入FLAG标签,实时荧光定量聚合酶链反应（RT-PCR）从T淋巴细胞白血病细胞系Jurkat细胞中钓取J441的开放阅读框（ORF）片段,形成J441-FLAG融合基因,并将之克隆到带GFP荧光报告基因的慢病毒表达载体质粒pHAGE-CMV-MCS-IzsGreen中构建成pHAGE-J441-FLAG质粒,利用脂质体将其与包装质粒psPAX2和包膜质粒pMD2.G共转染293T细胞.收集含有病毒的上清,浓缩,鉴定.取浓缩纯化后的病毒上清感染293T细胞和宿主Jurkat细胞.观察荧光及测序鉴定293T细胞中J441的表达,细胞免疫荧光检测293T细胞中FLAG的表达及细胞定位,预测J441的细胞定位.结果 核酸序列测序证实成功构建了含J441-FLAG基因的重组慢病毒表达载体,病毒滴度为5×1010 ifu/L.重组慢病毒感染293T细胞可以检测到J441与FLAG标签融合基因的表达,初步预测J441在胞质中表达.结论 成功构建了J441-FLAG融合基因慢病毒表达载体,为进一步研究J441基因的相关功能提供了稳定转染载体,为揭示J441基因在T淋巴细胞白血病的发生发展过程中的重要作用奠定基础.

Objective The gene expression data of T lymphoblastic leukemia cell lines were obtained by ＂ high-throughput sequencing＂,a new sequence was found,which called J441.The aim of this study was to establish a recombinant lentivirus harboring J441 gene and FLAG-tagged peptide.Methods We got gene expression data from Jurkat by ＂high-throughput sequencing＂.A new gene was found by bioinformatic analysis,we called it J441.For further study,a FLAG-J441 fusion gene was constructed by real-time polymerase chain reaction（RT-PCR） and packaged into pHAGE-CMV-MCS-Izs-Green lentivirus vector.The recombinant plasmid pHAGE-J441-FLAG was identified by PCR and sequencing.The recombinant plasmid,packaging vector psPAX2 and envelope vector pMD2.G were co-transfected into 293T cells and the resulting lentivirus was collected.After infection of the recombinant lentivirus,the expression of J441 in 293T was investigated by the expression of ZsGreen and sequencing.The expression of FLAG was investigated by immunofluorescence.Results A recombinant lentivirus plasmid pHAGE-J441-FLAG was successfully constructed.The viral titer was 5 × 1010 ifu/L.The expression of J441 in 293T could be detected by the expression of ZsGreen and sequencing.Immunofluorescence showed that FLAG was expressed in cytoplasm.Conclusions The lentivirus-based delivery system has been successfully constructed.Exogenous J441-FLAG can be stably expressed.The result of immunofluorescence suggest that the J441 gene might be expressed in cytoplasm.