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VNSAK polypeptide and application thereof

An N-terminal and epitope technology, applied in the field of VNSAK polypeptides, can solve the problems of weak immunogenicity and immune tolerance, and achieve the effects of enhancing immunogenicity, overcoming weak immunogenicity, and overcoming immune tolerance

Active Publication Date: 2015-02-11
CHONGQING QINLIAN BIOMEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, weak immunogenicity and the existence of immune tolerance are two major obstacles to the application of CTL epitopes as tumor therapeutic polypeptide vaccines

Method used

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  • VNSAK polypeptide and application thereof
  • VNSAK polypeptide and application thereof
  • VNSAK polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Preparation of DC cells loaded with VNSAK polypeptide and effector cells specific for VNSAK polypeptide

[0032] 1. Preparation of DC cells loaded with VNSAK polypeptide

[0033] Principle: PBMC induces DC cells, DC cell phagocytosis, can swallow VNSAK polypeptide to form DC cells loaded with VNSAK polypeptide.

[0034] The cell culture in this step is performed at 37℃, 5% CO 2 In an incubator.

[0035] 1. Synthesize the polypeptide shown in sequence 1 of the sequence listing (named VNSAK polypeptide).

[0036] Sequence 1 of the sequence listing: VYDYNCHVDLNVLHFFNAPLSLLMWITQCAAYKDEL.

[0037] 2. Take human peripheral blood, isolate and obtain peripheral blood mononuclear cells (PBMC).

[0038] 3. Take the peripheral blood mononuclear cells obtained in step 2 and prepare 5×10 with RPMI-1640 medium 6 Cells / ml of cell suspension.

[0039] 4. Add the cell suspension obtained in step 3 to the cell culture flask and culture for 2 hours (to make DC cells adhere to the wall), the...

Embodiment 2

[0060] Example 2. The ability of effector cells to produce IFN-γ

[0061] ELISPOT detection kit (Human IFN-γELISPOT Kit): Shenzhen Juying Biotechnology Co., Ltd., item number "856 051 001", IFN-γ capture antibody, biotin-labeled anti-IFN-γ antibody and streptavidin label The alkaline phosphatase is a component of the kit. The effector cell suspension refers to the VNSAK-CTL system, VAK-CTL system, NAK-CTL system, or SAK-CTL system prepared in Example 1.

[0062] Take the effector cell suspension, use the ELISPOT detection kit and operate according to the kit instructions to detect the ability of various effector cells to produce IFN-γ. The specific steps are as follows: add 70% (volume ratio) ethanol aqueous solution to a 96-well plate at room temperature After 10 minutes of incubation, wash with PBS buffer, then add 100μl of IFN-γ capture antibody diluted to 100 times volume with PBS buffer to each well, incubate at 4°C for 12 hours, then wash with PBS buffer; then add effector c...

Embodiment 3

[0064] Example 3. The killing effect of effector cells on target cells

[0065] T2 cells: ATCC, ATCC number is " CRL-1992 TM ".

[0066] 1. Preparation of T2 cells loaded with antigen peptides

[0067] 1. Preparation of T2 cells loaded with VAK polypeptide

[0068] In RPMI-1640 culture medium, the artificially synthesized VAK polypeptide and T2 cells were heated at 37℃, 5% CO 2 Incubate for 24 hours under conditions (at the initial time of incubation, the concentration of VAK polypeptide is 50μg / ml, and the concentration of T2 cells is 1×1 0 6 cells / ml), and then collect the cells, which are T2 cells loaded with VAK polypeptide.

[0069] 2. Preparation of T2 cells loaded with NAK polypeptide

[0070] In RPMI-1640 culture medium, the artificially synthesized NAK polypeptide and T2 cells are heated at 37℃, 5% CO 2 Incubate for 24 hours under conditions (at the initial time of incubation, the concentration of NAK polypeptide is 50μg / ml, and the concentration of T2 cells is 1×10 6 Cells / m...

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Abstract

The invention discloses a VNSAK polypeptide and an application thereof. The VNSAK polypeptide sequentially comprises an epitope zone and an endoplasmic reticulum retention base sequence zone from an end N to an end C; the epitope zone comprises an SART3-109 epi-position, an HNRPL-501 epi-position and an NY-ESO-1 epi-position; the SART3-109 epi-position is indicated as amino acid residues from the first position to the tenth position of the N end of a sequence 1 of the sequence table, the HNRPL-501 epi-position is indicated as the amino acid residues from the eleventh position to the twentieth position of the N end of the sequence 1 of the sequence table, the NY-ESO-1 epi-position is indicated as the amino acid residues of the twenty-first position and the twenty-ninth position of the N end of the sequence 1 of the sequence table, and the endoplasmic reticulum retention base sequence zone is indicated as the amino acid residues of the thirty-third position to the thirty-sixth position of the N end of the sequence 1 of the sequence table. The invention also protects the application of the VNSAK polypeptide in preparing tumor treating medicines. The VNSAK polypeptide and the application thereof have an important significance on treating the tumor.

Description

Technical field [0001] The present invention relates to VNSAK polypeptide and its application. Background technique [0002] Tumor biotherapy is the fourth mode of treatment after surgery, radiotherapy, and chemotherapy. Tumor cell immunotherapy is one of the most practical methods in the field of tumor biotherapy. Due to the polymorphism of tumor antigens, it is impossible to cure tumors by only one method. Tumor immunotherapy mainly uses therapeutic vaccines to stimulate and enhance the body's immune function to achieve the purpose of controlling and killing tumor cells. [0003] Dendritic cell (dendritic cell, DC), as a bone marrow-derived antigen-presenting cell (APC), has a strong antigen-presenting ability, which can express antigen peptides to dendritic cells. It presents molecules (MHC I and MHC II), and activates CD4 and CD8 T cells respectively, which can induce the production of cytotoxic T lymphocytes (CTL cells), which is very suitable for immunotherapy of various tu...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/0784A61K39/00A61K35/15A61P35/00
Inventor 周亮高
Owner CHONGQING QINLIAN BIOMEDICAL TECH
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