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Antituberculous CTL epitope peptide and application thereof

A tuberculosis and drug technology, applied in the field of anti-tuberculosis CTL epitope peptides, can solve the problems of large differences in the protective effect of adults

Active Publication Date: 2019-12-31
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In terms of tuberculosis vaccines, BCG has a century-old history of preventing tuberculosis and is currently the only clinically available vaccine, but it only shows a certain preventive effect on tuberculosis that occurs in children, and the protective effect on adults varies greatly

Method used

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  • Antituberculous CTL epitope peptide and application thereof
  • Antituberculous CTL epitope peptide and application thereof
  • Antituberculous CTL epitope peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 binding force and stability experiment

[0022] The binding force experiment scheme is as follows;

[0023] (1) Dissolve the prepared candidate peptides in sterilized PBS (pH 7.2), and make 1 mg / mL aliquots for use;

[0024] (2) T2A2 cells in good growth state were obtained, centrifuged at 2000 rpm at 4°C for 5 min, and washed twice with serum-free IMDM medium. Adjust density to 1×10 6 cells / mL, spread in 24-well plate, 500 μL / well.

[0025] (3) Take out the dissolved and subpackaged peptides in advance and put them in a 4°C refrigerator. After the cells are plated, add human β 2 Microglobulin (β 2 -M) (0.5 μg / mL), and then add the dissolved antigen peptide (50 μg / mL). According to the experimental arrangement, set up: experimental group (epitope peptide), negative control group (PBS), positive control group (COX-2 321-329 ) and the background group (T2A2 cells). Mix the cell suspension with pinch plate, put it in the cell culture incubator at 37°C,...

Embodiment 2

[0048] Example 2 Separation and induction of human peripheral blood mononuclear cells (PBMCs)

[0049] (1) According to the amount of 30U heparin sodium / mL peripheral blood, add a certain amount of heparin sodium into a 40mL centrifuge tube according to the amount of peripheral blood drawn. First recruited volunteers were tested for peripheral blood HLA type, and some HLA-A2+ volunteers underwent tuberculin (PPD) intradermal test. 40 mL of peripheral blood was drawn from each volunteer obtained from the screening. The blood in the syringe should be injected into the wall when it is transferred into a 50mL centrifuge tube, then shake it slowly several times and place it on ice.

[0050] (2) Dilute the retrieved anticoagulated peripheral blood with PBS of pH 7.2 at a ratio of 1:1, and gently pipette to mix. Take 10 15mL centrifuge tubes, open the human peripheral blood lymphocyte separation medium in the ultra-clean workbench, and add 4mL of the separation medium to each 15mL ...

Embodiment 3

[0053] Embodiment 3 ELISPOT experiment (enzyme-linked immunospot experiment)

[0054] ELISPOT experiments were performed using human IFN-γ pre-coating kits.

[0055] (1) Collection of target cells: T2A2 cells in culture were observed under an inverted optical microscope, and cells in a better state were selected. Gently blow and aspirate the medium in the culture flask with an electric pipette to make a uniform cell suspension. Transfer to a 15mL centrifuge tube, centrifuge in a horizontal centrifuge: 1000rpm, 8min. After discarding the supernatant, add serum-free IMDM medium and wash once. Adjust the cell density to 2 x 10 6 cells / mL, spread into a 24-well plate, 1 mL / well. Set epitope peptide group, PBS group, blank group.

[0056] (2) Target cell-loaded peptide: add epitope peptide / PBS (pH 7.2) to the cell suspension in each well at an amount of 50 μg / mL, and add human β at an amount of 3 μg / mL 2 -M. Gently blow the cells in the well plate with a pipette, and incubat...

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Abstract

The invention discloses a polypeptide. An amino acid sequence of the polypeptide is shown as SEQ ID No. (sequence identifier number) 1 or 2. The invention further provides a drug composition, food ora health care product containing the polypeptide and a corresponding application of the polypeptide. An epitope peptide is obtained through screening and identified by in-vitro ELISPOT (enzyme-linkedimmunospot assay), a cell killing experiment and the like; a theoretical basis is provided for subsequent development of a tuberculosis vaccine and a diagnostic agent based on a drug-resistant mutation antigen; and more options are provided for a design of a multi-epitope tuberculosis vaccine based on a mixed T cell epitope.

Description

technical field [0001] The invention specifically relates to an anti-tuberculosis CTL epitope peptide and its application. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that seriously endangers human health. The continuous emergence and spread of drug-resistant Mycobacterium tuberculosis (including multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB)) has made tuberculosis control face great challenges. [0003] For the treatment of tuberculosis, the current WHO-recommended therapy for drug-sensitive tuberculosis infection requires the combination of first-line drugs rifampicin (RFP), isoniazid (INH), (EMB) and (PZA). This therapy is effective, but the treatment time is longer. WHO recommends at least 20 months of continuous second-line drug therapy for MDR-TB. In short, multi-drug combination chemotherapy takes a long time, has obvious side effects, and many proble...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C12N15/31A61K39/04A61P31/06A23L33/18G01N33/569G01N33/68
CPCA23V2002/00A61K39/04A23L33/18A61P31/06C07K14/35G01N33/5695G01N33/68G01N2333/35A23V2200/30A23V2250/55
Inventor 祁元明吴亚红高艳锋冉令董钰时冉冉李玉冰
Owner ZHENGZHOU UNIV
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