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Inha-derived anti-tuberculosis ctl epitope peptide and its application

A tuberculosis and DNA molecule technology, applied in the field of anti-tuberculosis CTL epitope peptides, can solve the problem of large differences in the protection effect of adults

Active Publication Date: 2021-04-13
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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  • Inha-derived anti-tuberculosis ctl epitope peptide and its application
  • Inha-derived anti-tuberculosis ctl epitope peptide and its application
  • Inha-derived anti-tuberculosis ctl epitope peptide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1 binding force and stability experiment

[0021] The binding experiment scheme is as follows:

[0022] (1) Dissolve the prepared candidate peptides in sterilized PBS (pH 7.2), and make 1 mg / mL aliquots for use;

[0023] (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.

[0024] (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, and i...

Embodiment 2

[0047] Example 2 Isolation and induction of human peripheral blood mononuclear cells (PBMCs)

[0048] Firstly, volunteers were recruited to determine their peripheral blood HLA types, and some HLA-A2+ volunteers underwent tuberculin (PPD) intradermal test to screen suitable volunteers.

[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 for later use. 40 mL of peripheral blood was drawn from each volunteer. 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 anticoagulated peripheral blood with PBS of pH 7.2 at a ratio of 1:1, and gently pipette to mix. According to the ratio of peripheral blood:PBS:separation solution=1:1:1, slowly add to the centrifuge tube in the ultra-clean workbench. Then gently transfer the centrifuge tube containing the s...

Embodiment 3

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

[0054] On the 21st day, the induced T lymphocytes in each experimental group were collected, and the ELISPOT experiment was performed using a human IFN-γ precoating kit.

[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 a...

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Abstract

The invention discloses a polypeptide whose amino acid sequence is shown as SEQ ID No.1 or SEQ ID No.2. The present invention also provides pharmaceutical compositions, food or health products containing the polypeptide and corresponding uses of the polypeptide. The invention screens and obtains epitope peptides, and identifies the epitope peptides through in vitro ELISPOT and cell killing experiments, etc., which provides a theoretical basis for the subsequent development of tuberculosis vaccines and diagnostic preparations based on drug-resistant mutant antigens, and provides a basis for the design of T-cell expression-based vaccines. The TB multi-epitope vaccine provides more choices.

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 Patents(China)
IPC IPC(8): C07K14/35C12N15/31A23L33/18G01N33/569G01N33/68A61K39/04A61P31/06
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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