Construction and identification of eukaryotic expression vector of rat Delta1 gene / 中国组织工程研究

ABSTRACT

BACKGROUND: Recent data suggested that Notch signal pathway plays important regulatory effects in peripheral transplantation immunological response, promotes differentiation of regulatory T cells, induces antigen specific immune tolerance. We proposed that Notch/Notch ligand may play important roles in MHC/TCR interface.OBJECTIVE: To construct the eukaryotic expression vector of rat Deltai gene (Notch ligand), and to examine its expression in dendritic cells.METHODS: The complete encoding cDNA of rat-Delta1 was isolated from bone marrow cells by reverse transcription-polymerase chain reaction (RT-PCR) and this gene was recombined into pcDNA3.1(+) plasmid vector.pcDNA3.1/Delta1 plasmid was transfected into rat dendritic cells with lipofectamine gene transfection method.RESULTS AND CONCLUSION: Double enzyme digestion detection demonstrated that Delta 1 had been successfully constructed in Hindilll and xbal of pcDNA3.1. A positive clone pcDNA3.1/Delta1 was delivered to Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. for sequencing. Sequencing results were identical to Delta1 gene sequence in Genebank, with correct reading frame. The Delta 1 gene-transfected dendritic cells showed similar morphology as their parent cells. Western blotting assay detected that Delta 1 expression was significantly increased in cells. The eukaryotic expression vector pcDNA3.1/Delta1 was constructed, and significant increase of Delta 1 expression was detected after transfection.