The purpose of ccl5

Abstract

The invention relates to the field of biotechnology, and the invention discloses a use of CCL5. The present invention utilizes the chemotactic binding ability of CCL5 and immune cell surface receptors such as DC cells to transport different antigenic proteins to the surface of DC cells, improves the efficiency of phagocytosis, processing and presentation of various antigenic proteins by DC cells, and improves the efficiency of DC cells. The effect of preventing and treating related diseases. The present invention also adds T2 sequence to the antigen so as to enhance the immune effect.

Description

technical field

[0001] The present invention relates to the field of biotechnology, in particular to the application of CCL5. Background technique

[0002] Dendritic cells (DC) are professional antigen-presenting cells (Antigen presenting cells, APCs) with the strongest function in the body, which can efficiently ingest, process and present antigens, and immature DCs have a strong ability to migrate Mature DC cells can bind to T cells with a variety of cell surface proteins and activate them, and finally cross-present the epitope peptides of antigenic proteins captured by DC cells themselves to T cells and promote their differentiation into antigen-specific cells Toxic T lymphocytes. Finally, the process of cellular immunity is used to recognize and degrade antigenic substances.

[0003] Tumor vaccines induce the function of effector T cells in patients by enhancing the existing anti-tumor response or activating naive T cells. Antigen-specific CD8+ cytotoxic T lymphocytes ...

Examples

Embodiment 1

[0088] Monocytes and mononuclear cells, T cell subsets, eosinophils, and basophils were isolated from mouse bone marrow and peripheral blood, respectively. The bone marrow mononuclear cells were added with M-CSF, GM-CSF, and IL4 to induce differentiation into macrophages and DC cells, and then chemotaxis experiments were performed. Put the above-mentioned isolated or induced differentiation cells in the upper chamber of the chemotaxis chamber (transwell chamber with carbonate membrane: 5 μm; Costar, Cat: 3422), and the number of cells added is 1×10 based on the previous work in the laboratory. 6 / 100 μl / well. At the same time, the spontaneous migration control group and the CCL5 cytokine group were set up, and the number of cells added was the same. CCL5 factor adoption E. coli Purify the recombinant mouse CCL5 protein, according to the previous work in the laboratory, 100ng / ml is the best chemotactic efficiency dose. After 4 hours, the cells in the lower chemotaxis chambe...

Embodiment 2

[0090] Antigen design scheme of fusion gene or protein vaccine and construction and preparation of mammalian expression plasmid

[0091] Construction of pVR-CCL5-E6E7-T2 plasmid: Construct fusion protein CCL5-E6E7-T2 according to E6 and E7 proteins of human papillomavirus subtype HPV16, human CCL5 protein and T2 polypeptide sequence. An IgE signal peptide with an amino acid sequence of MDWTWILFLVAAATRVHS is connected to the N-terminus of the fusion protein CCL5-E6E7-T2; a Flag tag consisting of 8 amino acids of DYKDDDDK is connected to the C-terminus of the fusion protein CCL5-E6E7-T2.

[0092] The resulting fusion protein, from N-terminal to C-terminal, includes: IgE signal peptide, human CCL5 protein sequence, linker sequence (GGGGGSGGGGG), E6 protein sequence, E7 protein sequence, T2 protein sequence, and Flag tag sequence.

[0093] The amino acid sequence of the fusion protein was optimized for the codon expression preference of mammalian cells, and its fusion gene sequenc...

Embodiment 3

[0101] The in vitro cell transfection experiment of constructing plasmid (the antigen that is constructed with embodiment 2 is the carrier of HPV16 E6 and E7 protein is example, the mode to the vector transfection that contains other antigens is the same):

[0102] 24 hours before transfection, inoculate 2.5×10 5 HEK293T cells, when the cell density grows to 60%-70%, start the transfection experiment. Before transfection, preheat cell culture medium and serum-free Opti-MEM medium in a 37°C water bath. During transfection, add 5 μg of empty vector (Vector), pVR-CCL5-E6E7-T2 expression vector, pVR-CCL5-E6E7 expression vector, pVR-E6E7 expression vector and 20 μL PEI transfection reagent to 200 μL serum-free Opti -MEM, after mixing evenly, let stand at room temperature for 20 minutes. Replace the cells to be transfected with fresh medium, gently add the above transfection system, and shake gently. Return the cells to the cell culture incubator for 6 hours before changing the m...

technical field

[0001] The present invention relates to the field of biotechnology, in particular to the application of CCL5. Background technique

[0002] Dendritic cells (DC) are professional antigen-presenting cells (Antigen presenting cells, APCs) with the strongest function in the body, which can efficiently ingest, process and present antigens, and immature DCs have a strong ability to migrate Mature DC cells can bind to T cells with a variety of cell surface proteins and activate them, and finally cross-present the epitope peptides of antigenic proteins captured by DC cells themselves to T cells and promote their differentiation into antigen-specific cells Toxic T lymphocytes. Finally, the process of cellular immunity is used to recognize and degrade antigenic substances.

[0003] Tumor vaccines induce the function of effector T cells in patients by enhancing the existing anti-tumor response or activating naive T cells. Antigen-specific CD8+ cytotoxic T lymphocytes ...

Examples

Embodiment 1

[0088] Monocytes and mononuclear cells, T cell subsets, eosinophils, and basophils were isolated from mouse bone marrow and peripheral blood, respectively. The bone marrow mononuclear cells were added with M-CSF, GM-CSF, and IL4 to induce differentiation into macrophages and DC cells, and then chemotaxis experiments were performed. Put the above-mentioned isolated or induced differentiation cells in the upper chamber of the chemotaxis chamber (transwell chamber with carbonate membrane: 5 μm; Costar, Cat: 3422), and the number of cells added is 1×10 based on the previous work in the laboratory. 6 / 100 μl / well. At the same time, the spontaneous migration control group and the CCL5 cytokine group were set up, and the number of cells added was the same. CCL5 factor adoption E. coli Purify the recombinant mouse CCL5 protein, according to the previous work in the laboratory, 100ng / ml is the best chemotactic efficiency dose. After 4 hours, the cells in the lower chemotaxis chambe...

Embodiment 2

[0090] Antigen design scheme of fusion gene or protein vaccine and construction and preparation of mammalian expression plasmid

[0091] Construction of pVR-CCL5-E6E7-T2 plasmid: Construct fusion protein CCL5-E6E7-T2 according to E6 and E7 proteins of human papillomavirus subtype HPV16, human CCL5 protein and T2 polypeptide sequence. An IgE signal peptide with an amino acid sequence of MDWTWILFLVAAATRVHS is connected to the N-terminus of the fusion protein CCL5-E6E7-T2; a Flag tag consisting of 8 amino acids of DYKDDDDK is connected to the C-terminus of the fusion protein CCL5-E6E7-T2.

[0092] The resulting fusion protein, from N-terminal to C-terminal, includes: IgE signal peptide, human CCL5 protein sequence, linker sequence (GGGGGSGGGGG), E6 protein sequence, E7 protein sequence, T2 protein sequence, and Flag tag sequence.

[0093] The amino acid sequence of the fusion protein was optimized for the codon expression preference of mammalian cells, and its fusion gene sequenc...

Embodiment 3

[0101] The in vitro cell transfection experiment of constructing plasmid (the antigen that is constructed with embodiment 2 is the carrier of HPV16 E6 and E7 protein is example, the mode to the vector transfection that contains other antigens is the same):

[0102] 24 hours before transfection, inoculate 2.5×10 5 HEK293T cells, when the cell density grows to 60%-70%, start the transfection experiment. Before transfection, preheat cell culture medium and serum-free Opti-MEM medium in a 37°C water bath. During transfection, add 5 μg of empty vector (Vector), pVR-CCL5-E6E7-T2 expression vector, pVR-CCL5-E6E7 expression vector, pVR-E6E7 expression vector and 20 μL PEI transfection reagent to 200 μL serum-free Opti -MEM, after mixing evenly, let stand at room temperature for 20 minutes. Replace the cells to be transfected with fresh medium, gently add the above transfection system, and shake gently. Return the cells to the cell culture incubator for 6 hours before changing the m...

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