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Helicobacter pylori tetravalent adhesion multi-epitope vaccine and preparation method thereof

A technology of Helicobacter pylori and adhesion factor, which is applied in the field of biomedicine, can solve the problems of vaccine difficulties, poor patient compliance, and increased Hp drug resistance, and achieves prevention of immune pathological injury, biological toxicity, and high immune specificity. Effect

Active Publication Date: 2015-12-09
NINGXIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the treatment of Hp infection is mainly based on antibiotic drug therapy. Although it has been proved to be an effective method for eradicating Hp infection in clinical practice, antibiotic drug therapy has many disadvantages: (1) The dosage of drugs is large and the treatment cycle is long, and the patient poor compliance
(2) Antibiotic drug therapy has toxic and side effects
(3) Antibiotic drug therapy leads to enhanced drug resistance of Hp
(4) It is difficult to avoid Hp superinfection after antibiotic drug therapy
Lpp20 is another outer membrane protein of Hp. Studies have shown that Lpp20 has good immunogenicity and immunoprotection. The separation and purification of natural Lpp20 protein is difficult, which brings difficulties to the preparation of Lpp20 subunit vaccines.

Method used

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

Examples

Experimental program
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Effect test

example 1

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

[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, outer membrane protein Lpp20, HpaA and CagL", through biological information UreA 27-90 、UreA 183-225 , UreB 327-385 , Lpp20 58-97 , Lpp20 mimotope (SWPLYSDASGLG), HpaA 52-194 , CagL 21-139 The isoantigen epitopes or segments and the intramolecular mucosal immune adjuvant CTB were used in the construction of the Helicobacter pylori tetravalent adhesion factor multi-epitope vaccine CFAdE. 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 rea...

example 2

[0056] Example 2: Construction of recombinant expression vector pCzn1-CFAdE (containing fusion gene CFAdE)

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

[0058] The amino acid sequence of the previously designed adhesion factor multi-epitope peptide FAdE was transformed 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] (3) Connection of adhesion factor multi-epitope peptide FAdE gene and pCzn1-C expression vector

[0060] The synthesized adhesion factor multi-epitope peptide FAdE gene and pCzn1-C (containing CTB gene) were double digested with BamHI / Xbal respectively, and the reaction system was as follows:

[0061]

[0062] 37°C enzyme digestion reaction for 2h, then electrophoresis with 1% agarose gel, and observe the electrophoresis results. The adhesion factor multi-epitope pept...

example 3

[0066] Example 3: Prokaryotic expression of recombinant protein CFAdE

[0067] Transform the correct recombinant expression plasmid pCzn1-CFAdE into the ArcticExpress strain. On the pre-prepared LB plate containing 50 μg / mL Amp, inoculate the loop-streaked genetically engineered strain ArcticExpress / pCzn1-CFAdE, 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 AMP / mlAMP, shake overnight at 37°C at 220rpm; inoculate at 1:100 the next day in 30ml LB culture solution containing 50μg / mlAMP, 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...

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Abstract

The invention provides a multi-epitope vaccine for a helicobacter pylori tetravalent adhesion, wherein the activity of the multi-epitope vaccine is presented as a polypeptide which mainly consists of urease A and B subunits, superior Th and B cell epitopes or fragments of three outer membrane proteins (Lpp20, HpaA and CagL) as well as cholera toxin B subunit. According to the invention, an artificial gene is synthesized by virtue of gene synthesis technology, wherein the synthesized artificial gene consists of urease A and B subunits, and superior Th and B cell epitopes or fragments of three outer membrane proteins (Lpp20, HpaA and CagL), and the artificial gene is coupled with gene sequence of the cholera toxin B subunit, so as to form a fusion gene. The fusion gene is expressed by escherichia coli, and upon protein purification, the tetravalent adhesion multi-epitope vaccine is obtained. The vaccine can be used for inducing a body to generate T cellullar immunologic response and specific antibody humoral immune response in accordance with the urease A and B subunits and the three outer membrane proteins (Lpp20, HpaA and CagL); and the vaccine is suitable for preventing and controlling helicobacter pylori infection related diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a novel Helicobacter pylori quadrivalent adhesion factor multi-epitope vaccine and its preparation method and application. Background technique [0002] Helicobacter pylori (Hp) infection is globally distributed, and it is associated with gastric diseases such as chronic gastritis, peptic ulcer, gastric cancer and gastric mucosa-associated tissue lymphoma (MALT), and is classified as a class I carcinogen by the World Health Organization factor. Among different countries, between different regions of the same country, and even between different ethnic groups, there are obvious differences in the Hp infection rate. In developed countries, the Hp infection rate is 30%-50%. my country is a region with a high infection rate in the world. The infection rate was as high as 58.07%, and it showed obvious family aggregation. At present, the treatment of Hp infection is mainly based ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/106A61P31/04C12N15/70C12N15/62C07K19/00C07K1/18
Inventor 郭乐刘昆梅汤锋贾伟李永红秦玉红
Owner NINGXIA MEDICAL UNIV
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