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Helicobacter pylori tetravalent virulence factor multi-epitope vaccine and preparation method thereof

A technology of Helicobacter pylori and virulence factor, which is applied in the field of biomedicine, can solve the problems of increasing the risk of gastrointestinal diseases of duodenal ulcer and gastric cancer, poor patient compliance, high price, etc. Avoid biological toxicity, easy to express effects

Active Publication Date: 2015-12-02
NINGXIA MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current method for the treatment of Hp infection is mainly based on "triple" drug therapy (ingredients: proton pump inhibitors, antibiotics and bismuth), which has many disadvantages: (1) The widespread use of antibiotics has led to an increasing number of Hp drug-resistant strains, and the efficacy of drugs is changing. Under the sun, clinical use of 2 or 3 antibiotics at the same time is still difficult to completely cure it
(2) There are toxic and side effects of therapeutic drugs
(3) Drug treatment is difficult to avoid the embarrassing situation of repeated treatment and repeated infection
(4) The drug is expensive, the treatment cycle is long, and the patient's compliance is poor
Epidemiological studies have found that infection with CagA-positive Hp strains is more likely to increase the risk of serious gastrointestinal diseases such as duodenal ulcer and gastric cancer than CagA-negative Hp strains

Method used

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  • Helicobacter pylori tetravalent virulence factor multi-epitope vaccine and preparation method thereof
  • Helicobacter pylori tetravalent virulence factor multi-epitope vaccine and preparation method thereof
  • Helicobacter pylori tetravalent virulence factor multi-epitope vaccine and preparation method thereof

Examples

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example 1

[0053] Example 1: Molecular structure design of Helicobacter pylori tetravalent virulence factor multi-epitope vaccine FVpE

[0054] According to "the body's immune protection mechanism against Hp" and "the immunological properties of key adhesion factor epitopes or segments such as Hp urease A and B double subunits, CagA and VacA", CagA was screened by bioinformatics 302-437 , VacA 34-79 , VacA 365-527 、UreA 74-94 , UreB 229-251 、UreA 183-203 , UreB 327-334 The isoantigen epitopes or segments and the Th1 cell immune adjuvant NAP are used in the construction of the tetravalent virulence factor multi-epitope vaccine FVpE of Helicobacter pylori. 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 Helicobacte...

example 2

[0056] Example 2: Construction of recombinant expression plasmid pET-FVpE (containing fusion gene FVpE)

[0057] (1) Gene synthesis of adhesion factor polyepitope peptide FAdE nucleotide sequence

[0058] The pre-screened and designed CagA dominant antigen epitope segment (CagA E ), VacA dominant epitope segment complex (VacA E ), the amino acid sequence of the urease dominant epitope complex (UE) was converted into the corresponding nucleotide sequence according to the codon preference principle of Escherichia coli, and Zhongding Biotechnology Company was entrusted to carry out gene synthesis.

[0059] (2) Construction of recombinant expression vector pET-FVpE

[0060] The synthetic gene fragment (CagA E , VacA E and UE) and neutrophil activating protein NAP gene according to NAP-CagA E -VacA E -UE sequence is spliced ​​into a fusion gene, which is the FVpE fusion gene; then, FVpE is cloned between the NcoI and XhoI sites of pET28a to obtain the recombinant expression v...

example 3

[0062] Example 3: Prokaryotic expression of recombinant protein FVpE

[0063] Transform the correct recombinant expression plasmid pET-FVpE into the ArcticExpress strain. On the pre-prepared LB plate containing 50 μg / ml Kan, inoculate the loop-streaked genetically engineered strain ArcticExpress / pET-FVpE, 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 50 μg In a test tube of 3ml LB culture solution containing Kan / mlKan, shake overnight at 37°C at 220rpm; inoculate 1:100 the next day in 30ml LB culture solution containing 50μg / mlKan, shake at 37°C at 220rpm until the cell OD600 is 0.6-0.8 (about 2h ). Take out 1ml of the culture, centrifuge at 10000g for 2min at room temperature, discard the supernatant, and resuspend the bacterial pellet with 100μl of 1× loading buffer. Add IPTG to the remaining culture to a final concentration of 0.5 mM, shake at 220 rpm at 37°C for 4 hou...

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Abstract

The invention provides a helicobacter pylori tetravalent virulence factor multi-epitope vaccine and a preparation method thereof. The active constituent of the vaccine is polypeptide. The vaccine mainly comprises the dominant Th and B cell epitopes or sections of a urease A subunit, a urease B subunit, cytotoxin associated protein A and vacuolating cytotoxin associated protein A, and neutrophil activating protein. The gene synthesis and molecular cloning technology is used for building a fused gene containing the dominant Th and B cell epitopes or sections of ureases, the cytotoxin associated protein A and the vacuolating cytotoxin associated protein A, and the neutrophil activating protein. Escherichia coli is used for expressing the fused gene, and the tetravalent virulence factor multi-epitope vaccine is obtained after protein purification. The vaccine can stimulate a body to generate T cell immunity response and specific antibody humoral immunity response aiming at the ureases, the cytotoxin associated protein A, the vacuolating cytotoxin associated protein A and the neutrophil activating protein and can be used for preventing and treating diseases related to helicobacter pylori infection.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a novel helicobacter pylori quadrivalent virulence factor multi-epitope vaccine 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. Helicobacter pylori infection is distributed worldwide. In developed countries, the Hp infection rate is 30% to 50%. In my country, the Hp infection rate is even more serious, as high as 58.07% (more than 600 million people), and presents obvious family clustering. According to statistics, the medical expenses for Hp infection-related diseases in my country are as high as 10 billion RMB every year, and the occurrence of Hp infection-related diseases is still unable to be effectively controlled. my country is a high-incidence area of ​​gastric cancer. The annual prevale...

Claims

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Application Information

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IPC IPC(8): A61K39/106A61P1/04C07K19/00C12N15/62C12N15/70
Inventor 刘昆梅郭乐汤锋廖国玲徐广贤刘宏鹏
Owner NINGXIA MEDICAL UNIV
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