Helicobacter pylori multivalent epitope vaccine and preparation method thereof

A technology of Helicobacter pylori and epitope vaccine, applied in the field of biomedicine, can solve problems such as gastric mucosal damage

Active Publication Date: 2015-12-23
NINGXIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult for bacteria to survive in the high-acid environment of the stomach, but Ure can hydrolyze urea to produce ammonia to neutralize gastric acid and cause pathological damage to gastric mucosa

Method used

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  • Helicobacter pylori multivalent epitope vaccine and preparation method thereof
  • Helicobacter pylori multivalent epitope vaccine and preparation method thereof
  • Helicobacter pylori multivalent epitope vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0060] Example 1: Molecular structure design of Helicobacter pylori multivalent epitope vaccine CWAE

[0061] According to "the body's immune protection mechanism against Hp" and "the immunological properties of key virulence factors and adhesion factor epitopes such as Hp urease A and B double subunits, HSP60 and HpaA", UreA was screened by bioinformatics 27-53 、UreA 183-203 , HpaA 132-141 、HSP60 189-203 , UreB 185-225 , UreB 327-385 Antigenic epitopes or segments and intramolecular Th1-type cellular immune adjuvant NAP and mucosal immune adjuvant CTB were used in the construction of a new type of Hp multivalent epitope vaccine CWAE. Then, through the construction theory of the epitope vaccine and the analysis of bioinformatics, the connection sequence, spacer sequence and antigen epitope copy number of the selected antigenic epitope or segment are analyzed and determined, and finally a scientifically reasonable vaccine is designed. Structure of the Hp multivalent vaccin...

example 2

[0063] Example 2: Construction of recombinant expression vector pETCWAE (containing fusion gene CWAE)

[0064] (1) Gene synthesis of polyepitope peptide WAE nucleotide sequence

[0065] The amino acid sequence of the previously designed multi-epitope peptide WAE was transformed into the corresponding nucleotide sequence according to the codon preference principle of Escherichia coli, and Nanjing Jinsirui Biotechnology Co., Ltd. was entrusted to carry out gene synthesis.

[0066] (3) Connection of multi-epitope peptide WAE gene and pETC expression vector

[0067] The recombinant plasmids pUCWAE and pETC (constructed in the laboratory) were extracted by the plasmid mini-extraction kit (Tiangen), and double-digested with KpnI / XhoI respectively. The reaction system is as follows:

[0068]

[0069] 37°C enzyme digestion reaction for 2h, then electrophoresis with 1% agarose gel, and observe the electrophoresis results. The multi-epitope peptide WAE gene and pETC double digestio...

example 3

[0073] Example 3: Prokaryotic expression of multivalent epitope peptide fusion protein CWAE

[0074] Transform the correct recombinant expression plasmid pETCWAE into E.coliBL21(DE3) strain. On the pre-prepared LB plate containing 50 μg / mL Amp, inoculate the loop-streaked genetically engineered strain pETCWAE / BL21(DE3), place it upside down in a 37°C incubator, and after cultivating overnight for 12-16 hours, pick a single colony and inoculate it on a plate containing In LB medium with 50 μg / mL Amp, culture at 37°C, 180 rpm, for 12-16 hours. Inoculate the recombinant bacteria with 2% inoculum in LB medium containing 50 μg / mL Amp, shake overnight at 37°C and 180 rpm, and inoculate the bacterial solution in LB liquid medium containing 50 μg / mL Amp in the next morning with 1% inoculum After shaking the flask at 37°C and 180rpm for 3h, add IPTG to make the final concentration reach 1mmol / L, induce expression at 37°C and 180rpm, and use the empty vector strain pET22b / BL21(DE3) as a ...

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Abstract

The present invention provides a Helicobacter pylori multivalent epitope vaccine, wherein the activity is a polypeptide, and the polypeptide comprises urease subunit A, urease subunit B, adhesin HpaA, heat shock protein HSP60 advantage Th, B cell epitope or segment, neutrophil activating protein NAP and cholera toxin subunit B. According to the present invention, the artificial gene is synthesized through the gene synthesis technology, and comprises the gene sequences of urease subunit A, urease subunit B, adhesin HpaA, heat shock protein HSP60 advantage Th, B cell epitope or segment and neutrophil activating protein NAP, the artificial gene is coupled to cholera toxin subunit B gene to form a fusion gene, the fusion gene is expressed through escherichia coli, and protein purification is performed to obtain the multivalent epitope vaccine; and the multivalent epitope vaccine can stimulate the body to produce the T cell immune response and the antibody humoral immunoresponse against urease, adhesin HpaA, heat shock protein HSP60 and neutrophil activating protein NAP, and can be used for prevention and treatment of Helicobacter pylori infection-related diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a novel multi-epitope vaccine of Helicobacter pylori and its preparation method and application. Background technique [0002] Helicobacter pylori (Hp) is an important pathogenic factor of chronic gastritis, peptic ulcer and duodenal ulcer, and is closely related to gastric cancer. The World Health Organization (WHO) has listed Hp as a class I carcinogen. The infection rate of Helicobacter pylori is very high. The Hp infection rate of the world population exceeds 50%, and the Hp infection rate of the Chinese population is 58.07%. It presents family aggregation and the situation is even more severe. The current methods of treating Hp infection are mainly based on the "triple" drug therapy of antibiotics, which has disadvantages such as high cost, poor patient compliance, increase of drug-resistant bacteria, and intestinal flora imbalance, so it is not suitable for large-scal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/106A61K39/39C12N15/70C12N1/21A61P1/04
Inventor 郭乐刘昆梅徐广贤汤锋廖国玲杨华
Owner NINGXIA MEDICAL UNIV
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