Genetically modified cell of coexpression mouse membranous type interleukin 15 and retinoic acid early transcript 1 epsilon (Rae-1 epsilon) and preparation method thereof

Abstract

The invention relates to a genetically modified cell of coexpression mouse membranous type interleukin 15 and retinoic acid early transcript 1 epsilon (Rae-1 epsilon) and a preparation method thereof. The genetically modified cell is a BaF3 cell which can stabilize the genetically modified cell of the coexpression membranous type IL-15 and Rae-1 epsilon protein. The genetically modified cell can be prepared by a method comprising the following steps: amplifying a mouse Rae-1 epsilon gene and a mouse membrane expression type IL-15 gene by a polymerase chain reaction (PCR) technology; respectively inserting the amplified mouse Rae-1 epsilon gene and the amplified mouse membrane expression type IL-15 gene into two polyclone sites of an eukaryotic expression vector pVITRO2-mcs to acquire a recombined vector; transfecting the recombined vector into a mouse tumor cell strain BaF3; and acquiring the genetically modified cell by antibiotics screening and flow cytometry sorting. The genetically modified cell can provide IFN-gamma producing killer dendritic cells (IKDC) with triple stimulating signals as an instrument for efficiently amplifying and activating the IKDC in vitro. In addition, the genetically modified cell can also be used for antineoplastic immunotherapy research.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a transgenic cell co-expressing mouse membrane-type interleukin-15 and mouse retinoic acid early transcription factor 1ε (retinoic acid early transcript 1ε, Rae-1ε) genes and a preparation method thereof . Background technique [0002] A recently discovered new type of dendritic cell subpopulation (IFN-γ producing killer dendritic cells, IKDC) that can secrete IFN-γ and has killing function has both NK cells and dendritic cells (dendritic cells, DC) on the surface mark and function. Existing studies have shown that after IKDC directly kills tumor cells, it can further present tumor antigens derived from tumor cells to T cells, thereby initiating an anti-tumor adaptive immune response. Therefore, IKDC has a good application prospect in tumor immunotherapy. At present, there are two main methods to obtain IKDC. One is the direct sorting method: take the corresponding lymphoid tissue of...

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Problems solved by technology

However, the content of IKDC in mice is very small, only accounting for spleen CD11c + 1-2% of cells (about 5000) and bone marrow CD11c + 2% of the cells, the number of IKDCs obtained by direct sorting is far from meeting the number of cells required for adoptive immunotherapy

The other is to use interleukin 15 (IL-15) to expand IKDCs in vitro: firstly, bone marrow-derived DCs are routinely cultured, and then CD49b is enriched by magnetic bead sorting + cells, again from CD49b + CD11c was sorted by FACS in cells dim B220 + NK1.1 + Finally, IKDC was co-cultured with feeder cells and recombinant IL-15, and IL-15 was trans-presented by feeder cells to amplify IKDC. The presentation of IL-15 only acts on adjacent cells and lacks the targeting of presentation. Experimental data also show that the expansion efficiency of IKDC is relatively low (only 10-30 times the number of growth)

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