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Helicobacter pylori urease B subunit B cell antigen epitope polypeptide, identification method and application

A technology of Helicobacter pylori and antigenic epitopes, applied in the directions of bacterial antigenic components, applications, bacterial peptides, etc., can solve the problems that drugs cannot prevent Hp reinfection, the accuracy rate is only about 50%, and the drug therapy has large toxic and side effects Achieve the effect of clearing Helicobacter pylori infection, fast method and good immunogenicity

Inactive Publication Date: 2007-09-12
ARMY MEDICAL UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] 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
In recent years, many scholars have used some bioinformatics software to analyze the amino acid sequence of some proteins and predict their T cell or B cell epitopes, but the prediction accuracy is only about 50%. Therefore, an accurate and effective screening method for B cells has been established. Cell epitope method is particularly important

Method used

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  • Helicobacter pylori urease B subunit B cell antigen epitope polypeptide, identification method and application
  • Helicobacter pylori urease B subunit B cell antigen epitope polypeptide, identification method and application
  • Helicobacter pylori urease B subunit B cell antigen epitope polypeptide, identification method and application

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Embodiment 1

[0143] Example 1. Cloning of 5 segmented antigens U12, U13, U47, U15, U16 encoding genes of Helicobacter pylori urease B subunit (UreB)

[0144] 1. Cultivation of HP

[0145] The HP strain NCTC11637 was inoculated on 20ml of HP-specific liquid medium at 37°C, containing 5% O. 2 , 85%N 2 , 10%CO 2 cultured under microaerophilic conditions for 24 hours.

[0146] 2. Preparation of Helicobacter pylori (Hp) NCTC11637 genomic DNA: (operate according to the instructions of the bacterial genomic DNA extraction kit)

[0147] 1) Take 1-5ml of bacterial culture solution. Centrifuge at 10000rpm for 1 minute, and try to suck up the supernatant.

[0148] 2) Add 200 ul of buffer GA to the cell precipitation, and shake until the cells are completely suspended.

[0149] 3) Add 20ul of proteinase K (20mg / ml) solution to the tube and mix well.

[0150] 4) Add 220ul of buffer GB, shake for 15 seconds, place at 70°C for 10 minutes, the solution becomes clear, and briefly centrifuge to remove...

Embodiment 2

[0180] Example 2 Construction of U12, U13, U47, U15, U16 expression plasmids of Helicobacter pylori HP urease and construction and screening of high-efficiency expression engineered bacteria

[0181] 1. Construction of recombinant plasmids

[0182] The U12, U13, U47, U15, U16 gene amplification (PCR) products were subjected to 1.0% agarose gel electrophoresis, gel recovery and purification, and then connected to the vector pMD-18T, transformed into E. coli DH5α, and the plasmids were extracted, using NdeI and BamHI respectively. Double-enzyme digestion, 1.0% agarose gel electrophoresis identification.

[0183] The pMD-18T vector containing U12, U13, U47, U15, U16 target genes and pET-28a(+) were double digested with NdeI and BamHI, and the digested products were subjected to 1.0% agarose gel electrophoresis, and the target fragment was recovered and purified. After ligation with T4 ligase, E. coli DH5α was transformed, the plasmid was extracted, double digested with NdeI and ...

Embodiment 3

[0238] Example 3 Preliminary Identification of Antigen Recognition Epitope of Anti-Helicobacter Pylori Urease B Subunit Monoclonal Antibody 6E6

[0239] 1. Identify the region where the epitope recognized by the 6E6 monoclonal antibody is located by immunoblotting.

[0240] The specific operation steps of Western blot are as follows:

[0241] 1) SDS-PAGE electrophoresis

[0242] Take the processed samples of recombinant bacteria pET11c-U12, pET11c-U13, pET11c-U47, pET11c-U15 and pET11c-U16, use the empty vector engineered bacteria pET11c and irrelevant proteins as negative controls, and use the purified recombinant protein UreB as positive controls , and set the protein molecular weight standard, conduct vertical SDS-PAGE on 5% stacking gel, 15% separation gel, run 60V electrophoresis first, after the sample runs through the stacking gel, pressurize to 120V, 2-3 hours, until the sample runs to the bottom of the separating gel.

[0243] 2) Transfer film

[0244] (1) After e...

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Abstract

This invention provides a B cell epitope peptide and its identification method to neutralize helicobacterpylori urease B subunit, determines the antigen epitope corresponding with the monoclonal antibody 6E6 of helicobacterpylori urease B subunit, and confirms this B cell epitope is specific for the 6E6. The invention also provides a chimeric epitope vaccine and its preparation method containing B cell epitope and carrier protein BSA, and the B cell epitope has strong antigenicity.

Description

technical field [0001] The present invention relates to the fields of biopharmaceuticals and genetic engineering, in particular to a neutralizing B cell antigen epitope polypeptide and its encoded DNA from Helicobacter pylori (Hp) urease B subunit protein, and identification of the neutralizing B cell The method of cell antigen epitope and its application in the field of medical biotechnology. 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 disease in the past 20 years . On October 3, 2005, the Nobel Prize Review Committee announced that the 2005 Nobel Prize in Physiology and Medicine would be awarded to these two Australian scientists for their discovery of Helicobacter pylori (Hp) and the role of this bacterium in gastritis and gastric ulcers. role in diseases. Studies have confirmed...

Claims

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Application Information

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IPC IPC(8): C12N15/13C07K14/195G01N33/53G01N33/68G01N33/573C12Q1/68A61K39/02
Inventor 毛旭虎邹全明李海侠吴亚男石云罗萍张卫军余抒陈洪章郭刚童文德吴超周维英鲁东水刘开云
Owner ARMY MEDICAL UNIV
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