Construction of lentivirus carrying FLAG and novel gene J441 from Jurkat / 中华实用儿科临床杂志

ABSTRACT

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.