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vnsak polypeptide and its application

A technology of HLA-A and HLA-A24, which is applied in the field of VNSAK polypeptides, can solve the problems of weak immunogenicity and immune tolerance, and achieve the effects of enhanced immunogenicity, stable binding, and accelerated activation

Active Publication Date: 2017-05-10
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 its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0033] Principle: PBMC induces DC cells, and DC cells can phagocytize VNSAK polypeptides to form DC cells loaded with VNSAK polypeptides.

[0034] Cell culture in this step was carried out at 37°C, 5% CO 2 performed in the 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 and separate peripheral blood mononuclear cells (PBMC).

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

[0039] 4. Add the cell suspension obtained in step 3 into the cell culture flask and incubate for 2 hours (to allow D...

Embodiment 2

[0060] Example 2, the ability of effector cells to produce IFN-γ

[0061] ELISPOT Detection Kit (Human IFN-γ ELISPOT Kit): Shenzhen Juying Biotechnology Co., Ltd., catalog number "856 051 001", IFN-γ capture antibody, biotin-labeled anti-IFN-γ antibody and streptavidin labeling 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 instructions of the kit, and 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 the 96-well plate, room After incubation for 10 minutes, wash with PBS buffer, then add 100 μl 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 ...

Embodiment 3

[0064] Example 3, 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 antigenic peptides

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

[0068] In RPMI-1640 culture medium, artificially synthesized VAK polypeptide was mixed with T2 cells at 37°C, 5% CO 2 Incubate under the same conditions for 24 hours (at the initial moment of incubation, the concentration of VAK polypeptide is 50 μg / ml, 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, artificially synthesized NAK polypeptide was mixed with T2 cells at 37°C, 5% CO 2 Incubate for 24 hours under the same conditions (at the initial moment of incubation, the concentration of NAK polypeptide is 50 μg / ml, the concentration of T2 ...

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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 polypeptides and applications thereof. Background technique [0002] Tumor biological therapy is the fourth treatment mode 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 rely on only one method to cure tumors. Tumor immunotherapy mainly stimulates and enhances the immune function of the body through therapeutic vaccines, so as to achieve the purpose of controlling and killing tumor cells. [0003] Dendritic cells (dendritic cells, DCs), as a bone marrow-derived antigen-presenting cell (APC), have a strong ability to present antigens, and can express antigenic peptides to dendritic cells for antigen presentation. It presents molecules (MHC I and MHC II), and activates CD4 and CD8 T cells respectively, and can induce the generation of cytotoxic T lymphocytes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N5/0784A61K39/00A61K35/15A61P35/00
Inventor 周亮高
Owner CHONGQING QINLIAN BIOMEDICAL TECH
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