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Fusion protein containing CCL1, preparation method and application

A technology of fusion protein and protein, which is applied in the field of preparation of fusion protein containing CCL1, to achieve the effect of improving the prevention and treatment of related diseases, strong immune enhancement effect, and the effect of inhibiting tumor growth

Inactive Publication Date: 2022-06-03
保定诺未科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the molecules that have been proven to have a strong effect on the presentation of DC cells include XCL-1, but it is still urgent to find out more DC cell surface molecules that can promote the presentation

Method used

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  • Fusion protein containing CCL1, preparation method and application
  • Fusion protein containing CCL1, preparation method and application
  • Fusion protein containing CCL1, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

Embodiment 2

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

Construction of pVR-CCL1-E6E7-T2 plasmid: The fusion protein CCL1-E6E7-T2 was constructed according to the E6 and E7 proteins of human papillomavirus subtype HPV16, human CCL1 protein and T2 polypeptide sequences. An IgE signal peptide with an amino acid sequence of MDWTWILFLVAAATRVHS was connected to the N-terminal of the fusion protein CCL1-E6E7-T2; a Flag tag consisting of 8 amino acids of DYKDDDDK was connected to the C-terminal of the fusion protein CCL1-E6E7-T2.

[0054] The final fusion protein, from N-terminal to C-terminal, includes: IgE signal peptide, human CCL1 protein sequence, linker sequence (GGGGGSGGGGG), E6 protein sequence, E7 protein sequence, T2 protein sequence, and Flag tag sequence.

[0055] The amino acid sequence of the fusion protein is optimized by codons preferred for mammalian cell expression, and the fusion gene sequence is deter...

Embodiment 3

The in vitro cell transfection experiment of constructing the plasmid (taking the antigenM constructed in Example 2 as the carrier of HPV16 E6 and E7 proteins as an example, the mode of transfection of vectors containing other antigens is the same as this):

24 hours before transfection, inoculate 2.5 × 10 cells in a 6-well cell culture plate 5 1 HEK293T cells, and the transfection experiment was started when the cell density reached 60%-70%. Before transfection, pre-warm cell culture medium and serum-free Opti-MEM medium in a 37°C water bath. During transfection, 5 μg of empty vector (Vector), pVR-CCL1-E6E7-T2 expression vector, pVR-CCL1-E6E7 expression vector, pVR-E6E7 expression vector and 20 μL PEI transfection reagent were added to 200 μL serum-free Opti. -MEM, after mixing well, 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. Change the medium after placing ...

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Abstract

The invention discloses a CCL1-containing fusion protein, a preparation method and application, the fusion protein sequentially comprises an IgE signal peptide, CCL1, a linker and an antigen from an N terminal to a C terminal, or the IgE signal peptide, CCL1, the linker, the antigen and a T2 fragment, and the amino acid sequence of the T2 fragment is shown as SEQ ID NO: 4. According to the invention, different antigen proteins are transferred and crossly presented to the surface of the DC cell by utilizing the chemotactic binding capacity of CCL1 and surface receptors of immune cells such as the DC cell, so that the efficiency of phagocytosis, processing and presentation of various antigen proteins by the DC cell is improved, and the effect of preventing and treating related diseases is improved. Experiments prove that the T2 sequence disclosed by the invention has a very strong immune enhancement effect, and can further excite body fluid and cellular immune response in the process of promoting antigen presentation, so that the effect of inhibiting growth of related tumors is finally achieved.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular, to a fusion protein containing CCL1, a preparation method and an application. Background technique [0002] Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APCs) in the body. They can efficiently take up, process and present antigens. Immature DCs have strong migration Ability, mature DC cells can bind to T cells with a variety of cell surface proteins and activate them, and finally cross-present the antigen protein epitope peptides captured by DC cells themselves to T cells and promote their differentiation into antigen-specific cells Toxic T lymphocytes. Finally, the cellular immune process is used to recognize and degrade antigenic substances. [0003] Tumor vaccines induce effector T cell function in patients by enhancing the existing antitumor response or initiating naive T cells by immunization. Antigen-specific CD8+ cytotoxic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/385A61P35/00A61P31/20
CPCC07K14/523C07K14/005A61K39/385A61P35/00A61P31/20C07K2319/02C07K2319/33C07K2319/74C12N2710/20022C12N2710/20033C12N2710/20034A61K2039/6031A61K2039/6068A61K2039/575A61K2039/57
Inventor 谢皇帆孙志娟许红岩王凤
Owner 保定诺未科技有限公司
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