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Method for screening and identifying helicobacter pylori epitope peptides

A technology of Helicobacter pylori and antigen, applied in the field of medicine and biology, can solve the problems of missed screening, treatment failure and low accuracy, and achieve the effects of reducing the risk of use, high immunogenicity and high accuracy

Active Publication Date: 2013-11-20
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it can achieve an eradication rate of 85%, it has the following disadvantages: 1. Drug therapy has large toxic and side effects; 2. Complicated drug combination leads to poor patient compliance; 3. Drugs cannot prevent the potential infection of H. pylori; 4. Antibiotic treatment is easy to produce Drug dependence leads to treatment failure; 5. For patients in developing countries, drug therapy is also economically difficult
[0006] At present, the reported Hp antigen CD4+ T cell epitopes are all mouse H-2 restricted Th epitopes. Due to the difference between mouse and human MHC molecules, H-2 restricted epitopes may not be suitable for human vaccine design , so it is necessary to screen for the identification of HLA-restricted epitopes
In addition, currently reported Hp epitopes are mostly predicted by bioinformatics software, and the accuracy rate is not high. Although it can be verified through experiments, the phenomenon of epitope leakage cannot be avoided.

Method used

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  • Method for screening and identifying helicobacter pylori epitope peptides
  • Method for screening and identifying helicobacter pylori epitope peptides
  • Method for screening and identifying helicobacter pylori epitope peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 : Synthesis of 18 amino acid short peptides with overlapping steps and preparation of mixed peptide library

[0043] HpaA is a subunit of Helicobacter pylori flagellin protein A, and its sequence is conserved among various strains, with a total length of 260 amino acids, of which 1-27 amino acids are signal peptides. The recombinantly constructed rHpaA is a 28-260 polypeptide derived from the 11637 international standard strain. Therefore, the HpaA protein sequence (No. P55969) derived from Helicobacter pylori 11637 was retrieved in the UniProt protein database, and a short peptide with 18 amino acid steps overlapping was synthesized from the 28th amino acid (synthesized with the assistance of Shanghai Gil Biochemical Company), a total of 37 ( The last one is a 17 amino acid short peptide). The purity is greater than 90%. The synthetic peptide information is shown in Table 1. The synthesized peptides were dissolved in DMSO to a storage concentration of 5 ...

Embodiment 2

[0051] Example 2 : Detection of the frequency of antigen-specific CD4+ T cells in PBMC of Hp infection positive patients

[0052] 2.1 Collection and storage of PBMC from positive Hp infection patients

[0053] The peripheral blood of Hp positive patients was obtained from the Chongqing Blood Station of the People’s Liberation Army. 13 For those positive for Helicobacter pylori infection screened by urease breath test, buffy coat cells were collected after blood collection, and then PBMCs were separated with Ficoll-diatrizoate lymphocyte separation medium (Tianjin TBD Company) according to the actual instructions. The isolated PBMCs were resuspended to a cell density of 1×10 7 / mL, add 1mL / tube into a cryopreservation tube, put it in a freezer box at -70°C for overnight, and then transfer it to liquid nitrogen for cryopreservation.

[0054] 2.2 ICS method to detect the frequency of HpaA-specific CD4+ T cells in PBMC of Hp positive patients

[0055] Resuscitate a tube of PB...

Embodiment 3

[0059] Example 3 : Effective in vitro expansion of antigen-specific CD4+ T cells in peripheral blood of Hp-infected patients

[0060] 3.1 Methods for expanding antigen-specific T cells in vitro

[0061] Adjust the concentration of PBMC cells in patients with positive Hp infection to 2.5×10 6 / ml, inoculate in 48-well cell culture plate (1ml / well), add appropriate amount of HpaA antigen, mix well and incubate at 37°C, 5% CO 2 cultivated under conditions. On the fifth day, a low dose of recombinant human IL-2 (rhIL-2) (final concentration 25 U / ml) was added. On the 8th day, the culture medium began to turn yellow, and half of the medium was changed (the culture medium replaced contained 25 U / ml rhIL-2, and the cells were subcultured in different wells in due course.

[0062] 3.2 Detection of antigen-specific T cells in vitro

[0063] After the effective expansion of antigen-specific CD4+ T cells in vitro, the frequency has reached the detection range of ICS, and the freque...

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Abstract

The invention discloses a method for screening and identifying helicobacter pylori (Hp) antigen HLA restricted immunodominant epitope peptides. The method provided by the invention comprises a method for amplifying and culturing Hp antigenic specific T cells in vitro, a detection method of antigenic specific T cell in vitro response, a method for screening immunodominant epitope by using superposition peptides synthesized from antigenic specific T cells and with a walking method, and a method for determining epitope HLA restriction through HLA-II molecular antibody inhibition experiments and epitope presentation experiments of different HLA-II molecular subtype EBV used for converting B lymphoid cell series (BLCL). With the method provided by the invention, the obtained HpaA antigen immunodominant epitope peptides have amino acid sequences represented by SEQ ID No: 8, 22, 30, and 42. With the method provided by the invention, missed screening and mis-screening can be avoided. The epitope peptide obtained by screening can be naturally presented by antigen presenting cells.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and relates to a method for screening and identifying antigenic epitope peptides, in particular to a method for screening and identifying Helicobacter pylori antigenic epitope peptides. Background technique [0002] Helicobacter pylori (Hp) was discovered in 1982 by Australian scholars Warren and Marshall. In the past nearly 30 years, studies have confirmed that Hp is the main cause of gastritis, gastric ulcer, and duodenal ulcer, and is closely related to the occurrence of gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma (MALT). Listed as a class I carcinogen for gastric cancer. Moreover, the infection rate of Hp is very high. Epidemiological surveys show that 50% of the population in the world carries this bacterium, especially in some developing countries, the infection rate is even as high as 80%. Therefore, effective treatment of Hp infection is very impor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08
Inventor 吴超邹全明陈立罗萍石云赵卓余抒
Owner ARMY MEDICAL UNIV
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