Combination and screening method of dominant antigens of Helicobacter pylori based on CD4+ T cell immunization

A technology of Helicobacter pylori and dominant antigen, applied in the field of medicine and biology, can solve the problem that a new type of Helicobacter pylori vaccine has not been developed, etc.

Inactive Publication Date: 2019-09-10
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the immunodominant antigens of th1 and th17 have never been systematically screened, and no new H. pylori vaccine based on them has been developed

Method used

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  • Combination and screening method of dominant antigens of Helicobacter pylori based on CD4+ T cell immunization
  • Combination and screening method of dominant antigens of Helicobacter pylori based on CD4+ T cell immunization
  • Combination and screening method of dominant antigens of Helicobacter pylori based on CD4+ T cell immunization

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Evaluation of CD4+ T cell response in mice immunized with whole bacteria of H.pylori

[0056] 1. Experimental protocol for animal immunization and challenge

[0057] Experimental animals: BALB / c female mice aged 6-8 weeks.

[0058] antigen:

[0059] (1) Immune / challenge group (I / C): H.pylori inactivated whole bacteria. 100μg / piece.

[0060] (2) Unimmunized / challenged group (U / C): phosphate buffered saline (PBS).

[0061] Adjuvant: Freund's adjuvant. 100μl / piece.

[0062] Immunization method: subcutaneous injection.

[0063] Immunization volume: 200μl / monkey.

[0064] Immunization scheme: subcutaneous immunization 3 times (week 0, 2, 4). Complete Freund's adjuvant was used for the first time, incomplete Freund's adjuvant was used for the second time, and no adjuvant was added for the third time.

[0065] One week after the last immunization, 1.0×10 9 CFU Helicobacter pylori orally, once a day, for 4 consecutive days. Mice were sacrificed at 4 weeks after chall...

Embodiment 2

[0083] Molecular Sieve Chromatography Divides H.pylori Whole Bacterial Antigens into Different Fractions According to Molecular Size

[0084] First, Helicobacter pylori strain B0 was dissolved in 8mol / L urea, containing 10mmol / L dithiothreitol (DTT) 1g / 6ml, and stirred gently at 4°C for 18 hours. Next, centrifuge at 12,000 g, collect the supernatant, and filter with a 0.2 μM filter. Again, through molecular sieve chromatography, proteins with different molecular weights were divided into 30 groups, and the mixed protein components were named PC01-PC30 ( figure 2 ). Molecular screening uses a 10 / 300GL Superdex 200 chromatographic column, the loading volume is 1ml, the flow rate is set at 0.5mL / min, and 1ml / tube is collected. The obtained protein components were analyzed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis, and the concentration of each protein component was determined by a bicinchoninic acid (BCA) detection kit. see results figure 2 . ...

Embodiment 3

[0086] 3 H-TdR incorporation method to detect the degree of proliferation of CD4+ T cells stimulated by different components

[0087] BALB / c mouse peritoneal macrophages were used as antigen presenting cells (APC). Add 1×10 per well in a 96-well round bottom plate 5 APCs and 200 μL complete RPMI1640 medium. Combine it with H. pylori strain B0 whole antigen or each protein component PC01~PC30 at 37°C 5% CO 2 Co-cultivate in an incubator, the final concentration of each antigen is 50 μg / mL, and three replicate wells are set for each group. Ten hours later, spleen CD4+ T lymphocytes were sorted with magnetic beads from H. pylori-immunized / infected and H. pylori-immunized / uninfected mice 4 weeks later. 1×10 5 Splenic CD4+ T lymphocytes were co-cultured with the above APCs for 96 hours. joined in the last 18 hours 3 H-TdR 1 μCi / well. Afterwards, the CPM value of each well was detected with a liquid scintillation counter, and expressed as a stimulation index (SI), and SI was...

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Abstract

The invention relates to a helicobacter pylori dominant antigen assembly based on CD4+T cell immunity and a preparation method. The dominant antigen assembly comprises the following three components and homologous protein of the three components: inosine monophosphate dehydrogenase, type II citrate synthase and urease B subunit, wherein the amino acid sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The screened dominant antigen assembly based on CD4+T cell immunity has the obvious immune protection effect, has the protection effect superior to that of H.pylori holoprotein antigen, has strong capacity of scavenging helicobacter pylori, and causes extremely slight pathological injuries. The three immunity dominant antigens provided by the invention can induce the body to generate strong immune response reaction aiming at antigens, therefore, through the means of inducing the body to generate the response aiming at the immunoprotecive dominant antigens, or directly immunizing the body by adopting the protective antigens, the effective immune protection effect can be achieved on the helicobacter pylori infection, and the scheme can be used for the further study on the preventive and therapeutic polyvaccines of helicobacter pylori.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and relates to a Helicobacter pylori dominant antigen combination, in particular to a Helicobacter pylori dominant antigen combination and a screening method based on CD4+T cell immunity. Background technique [0002] Helicobacter pylori (H. pylori) colonizes the gastric mucosa and is a class I pathogenic factor of gastric cancer. main pathogenic factor. With a global infection rate of over 50%, there is an urgent need for an effective H. pylori vaccine. [0003] The screening of candidate antigens is the core of vaccine development. Unlike viruses with a limited number of antigens, bacteria contain hundreds or thousands of antigens, making it difficult to systematically screen them to determine their immunodominant protective antigens. This has hindered the development of vaccines against bacteria. [0004] Studies have shown that local th1 and th17 cells in the gastric mucosa h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/80C12N9/04C12N9/10C12Q1/689C12Q1/02A61K39/02A61P31/04C12R1/01
Inventor 吴超邹全明孙合强袁寒梅赵卓谭燃景李滨郭刚章金勇敬海明秦溢
Owner ARMY MEDICAL UNIV
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