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Helicobacter Pylori urease B subunit Th epitope peptide, its coding DNA, vaccine and uses

A technology of Helicobacter pylori and urease, which is applied in the field of medicine and biology, can solve the problems of economic difficulties of drug therapy, large toxic and side effects of drug therapy, treatment failure, etc., and achieves good application prospects, good immunogenicity, and good specificity.

Inactive Publication Date: 2006-07-26
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, multi-drug therapy of antibiotics is mainly used to treat Hp infection clinically. Although the eradication rate can reach 85%, it has the following disadvantages: 1. Drug therapy has high toxicity and side effects; 2. Complicated drug combination leads to poor compliance of patients; Prevent Hp reinfection; 4. Antibiotic treatment is prone to drug resistance and lead to treatment failure; 5. For patients in developing countries, drug therapy is also economically difficult
However, current studies have shown that the immune protection against Hp infection is caused by CD4 + It is mediated by T lymphocytes, so the use of immune means to prevent and treat Hp infection must stimulate the body's CD4 + T lymphocyte immune response, but unfortunately, no one has yet identified the Th cell epitope of Hp, so the present invention starts with identifying the Th epitope of the urease B subunit of Hp, and studies the method for preventing and treating Hp infection

Method used

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  • Helicobacter Pylori urease B subunit Th epitope peptide, its coding DNA, vaccine and uses
  • Helicobacter Pylori urease B subunit Th epitope peptide, its coding DNA, vaccine and uses
  • Helicobacter Pylori urease B subunit Th epitope peptide, its coding DNA, vaccine and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1, screening identification of Th epitope:

[0099] 1.1 Prediction and synthesis of epitope peptides

[0100] First, the UreB sequence of Hp was searched in the NCBI protein database. UreB is conserved in various strains. The UreB amino acid sequence of one of the strains, Helicobacter pylori 26695 (No. NP 206872) was selected for prediction. The UreB sequence has a total of 569 amino acids. The online software RANKPEP software was used to analyze the amino acid sequence of UreB of Hp, and seven possible Th epitope peptides were screened, and seven peptides were prepared by conventional peptide total artificial synthesis (assisted by Beijing Zhongke Yaguang Co., Ltd.). The purity of the polypeptides that may induce the body to produce Th response is above 85%. See Table 1 for information on the synthesized peptides.

[0101] NCBI's protein database search URL:

[0102] http: / / www.ncbi.nlm.nih.gov / entrez / querv.fcgi? db=Protein&itool=toolbar

[0103] RAN...

Embodiment 2

[0111] Example 2: Detection of epitope peptide-induced polarization direction of Th cells by ELISA method:

[0112] Spleen cells from mice immunized with rUreB were collected, and spleen CD4 + For T lymphocytes, resuspend the cells with RPMI-1640 complete medium, place in a 24-well flat-bottomed culture plate, and add CD4 to each well + T lymphocytes 5×10 5 ,Co 60Irradiated (20Gy) splenic mononuclear cells (as antigen-presenting cells) 5×10 5 , while adding the diluted synthetic peptide (final concentration 1.25 μg / ml), no synthetic peptide was added to negative control wells, the final volume was 0.5 ml / well, and 3 parallel wells were made for each group. 72 hours after the mouse spleen lymphocytes were stimulated by the synthetic epitope peptide, 400ul of the culture supernatant was collected and stored at -70°C for later use. The Th1 / Th2 typing kit from eBIOSCIENCE was used to detect cytokines. The specific steps are as follows: Coat the microtiter plate with gamma int...

Embodiment 3

[0114] Example 3: Co-stimulatory effect of epitope peptides

[0115] The specific operation is the same as in Example 1, the difference is that in addition to using a separate synthetic epitope peptide to stimulate lymphocytes, the three selected epitope peptides are also mixed in pairs or all as stimulators to stimulate lymphocyte proliferation. The final total concentration of the peptides was 1.25 μg / ml, and the rest of the steps were the same to compare whether there was any difference between the stimulation effect of the combination of epitope peptides and the stimulation effect of individual epitope peptides.

[0116] Results: The stimulating effects of the combination of three epitope peptides in pairs and the three mixtures were higher than those of individual epitope peptide stimulation (see Table 2), suggesting that the three epitopes can be combined in the subsequent vaccine design.

[0117] epitope peptide

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Abstract

The invention provides three Th cell epitope peptide and code DNA of pylorus spirillum urease B subunit, also providing a bacterin of B cell epitope which contains the triepitope peptide and an additional urease B subunit. Among them, the epitope peptide is the polypeptide which has one of the amino acid residue sequences as follows: 1) hasing amino acid sequence of sequence 2, sequence3 and sequence 7 in sequence table; 2) replacing, deleting or appending the amino acid sequence of sequence 2, sequence3 and sequence 7 in sequence table by one or several amino acid residue to form derivanting polypeptide. The coded DNA is one of the following sequences: 1) hasing the ribonucleotide sequence of sequence 8, sequence 9 and sequence sequence 10 table. 2) Hasing the ribonucleotide sequence of same coded product with sequence 8, sequence 9 and sequence10 in sequence table. The epitope vaccine which contains the polypeptide of this invention has the protein vaccine of amino acid sequence in sequence 12 of sequence table. The vaccine or medication which is made by using the polypeptide of the invention as active component can clean out the infection caused by pylorus spirillum and has wide application prospect in medical realm.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and relates to a polypeptide from Helicobacter pylori (Hp), its coding DNA and application. Among them, the preferred three polypeptides are helper T lymphocyte (Th) epitopes from urease B subunit (UreB), and the present invention relates to compositions containing these epitope peptides, which can be used for treatment and (or ) to prevent Helicobacter pylori infection. The present invention provides an epitope vaccine comprising 3 preferred Th epitopes and a B cell epitope (SIKEDVQF). In addition, the present invention also relates to synthetic peptide vaccines, gene recombinant subunit vaccines and nucleic acid vaccines etc. comprising these Th epitopes. Background technique [0002] In 1982, Australian scholars Warren and Marshall first isolated and cultured Helicobacter pylori (Hp) from human gastric mucosa. Their discovery made great changes in the understanding and treatment of ulcer ...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/52C12N15/63G01N33/53A61K38/16A61P31/04
Inventor 邹全明吴超石云周维英
Owner ARMY MEDICAL UNIV
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