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Pyloric spiral bacillus antigen recombinant vaccine

A technology of Helicobacter pylori and recombinant vaccines, applied in the directions of bacterial antigen components, antibacterial drugs, etc., can solve the problems of difficult large-scale cultivation, cumbersome methods, and difficult, and achieve easy separation and purification, high expression efficiency, and high yield. Effect

Inactive Publication Date: 2010-01-13
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] On the other hand, Hp bacterial culture conditions are harsh, and it is difficult to carry out large-scale cultivation
Bacterial antigen components are complex and the content is low. It is difficult to directly isolate and purify protective antigens from whole bacteria, and the method is cumbersome, which is not conducive to the industrial production of vaccines.

Method used

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  • Pyloric spiral bacillus antigen recombinant vaccine
  • Pyloric spiral bacillus antigen recombinant vaccine
  • Pyloric spiral bacillus antigen recombinant vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] HpaA of embodiment 1 Helicobacter pylori, UreB414, the cloning of NapA coding gene

[0058] 1. Helicobacter pylori was purchased from ATCC and kept in our laboratory. The strain is Helicobacter pylori 26695 (ATCC Number700392)

[0059] 2. Take out the preserved Hp strain from the liquid nitrogen tank and spread it on the special solid medium for Hp, at 37°C, containing 5% O 2 , 85%N 2 , 10% CO 2 Conditioned for 72h. The Genome Extraction Kit was used to extract the Hp genome.

[0060] 3. The coding genes of NapA, HpaA and UreB414 were respectively amplified from the Hp genome by PCR method.

[0061] 1) The primers were designed and synthesized as follows (the restriction site and linker base sequence are underlined)

[0062] According to the gene sequence published by GenBank and the principles of primer design, the corresponding primers were designed and restriction sites were introduced. Introduce the base sequence of the designed linker in the middle.

[0063]...

Embodiment 2

[0092] The acquisition of embodiment 2 fusion gene NapA-LTB

[0093] 1. NapA and LTB gene amplification

[0094] 1) Primer design and synthesis

[0095] NapA P7 5'-GCGGG CATATG AAAACATTTGAAA-3'

[0096] Nd

[0097] P8 5'- CGGAGGATCCTGCGG AGCTAAATGGGC-3'

[0098] linker

[0099] LTB P9 5'- GGAGGCGGAAGTGGAGGAGGTAGC GCTCCCCAGTCTATT-3

[0100] '

[0101] linker

[0102] P9' 5'- CCGCAGGATCCTCCG GCTCCCCAGTCTATT-3'

[0103] linker

[0104] P10 5'- CTCGAG GTTTTCCATGCTGATTGC-3'

[0105] wxya

[0106] Using Helicobacter pylori SS1 and wild-type toxin-producing E. coli H44815 genomic DNA as templates, use P7 and P8, P9' and P10 to amplify NapA and LTB genes, and extract bacterial genomes by days. Kit I performed. The PCR amplification system is: 10 μL of amplification buffer without magnesium ions, 10 μL of MgCl 2 (25mmol / L) 10μL, dNTPs (25mmol / L each) 8μL, upstream and do...

Embodiment 3

[0111] The acquisition of embodiment 3 fusion gene NapA-CTB

[0112] 1. NapA and CTB gene amplification

[0113] 1) Primer design and synthesis

[0114] NapA P7 5'-GCGGG CATATG AAAACATTTGAAA-3'

[0115] Nd

[0116] P8 5'- CGGAGGATCCTGCGG AGCTAAATGGGC-3'

[0117] linker

[0118] CTB P11'5'- CCGCAGGATCCTCCG ATTAAATTAAAATTTGGT-3'

[0119] linker

[0120] P12 5'- CTCGAG ATTTGCCATACTAATTGCG-3'

[0121] wxya

[0122] With P7 and P8, P11 ' and P12 amplified NapA, CTB gene, bacterial genome extraction carries out by day for the times centrifugal column type bacterial genome DNA extraction kit 1. The PCR amplification system is: 10 μL of amplification buffer without magnesium ions, 10 μL of MgCl 2 (25mmol / L) 10μL, dNTPs (25mmol / L each) 8μL, upstream and downstream primers 2μL each, the above NapA or CTB genome each 1μL, Ex-Taq DNA polymerase (5 units / μL) 1μL, add steri...

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Abstract

The present invention discloses recombinant vaccine based on the Helicobacter pylori neutral granulocyte activated protein A (NapA) antigen and its preparation process and application in inducing the protecting immune reaction resisting Helicobacter pylori infection. The vaccine consists of independent Helicobacter pylori NapA as the basic active component, or the fusion protein comprising Helicobacter pylori NapA, adhesion HpaA and urase B subunit active segment (UreB414), and one or several kinds of pharmaceutically acceptable adjuvant and excipient.

Description

technical field [0001] The invention relates to a recombinant protein vaccine, in particular to a recombinant vaccine based on Helicobacter pylori neutrophil activating protein (Hp-NAP) antigen, its preparation method and its application in preventing and treating Helicobacter pylori infectious diseases. Background technique [0002] Helicobacter pylori (Hp) is a Gram-negative Helicobacter that colonizes the human gastric mucosa, and about 50% of people in the world are infected with Helicobacter pylori. People have done a lot of research work on Helicobacter pylori, including genetics, physiology and pathogenic factors, and it has also been confirmed that it is the main pathogenic factor of gastric diseases. For different individuals, different strains of Helicobacter pylori can cause different lesions, ranging from clinical asymptomatic to chronic gastritis, peptic ulcer and even gastric cancer. In 1998, Japanese scholar Watanadbe et al. first reported that Hp-infected Mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/02A61P31/04
Inventor 邹全明高原杨珺张卫军毛旭虎郭刚吴超石云
Owner ARMY MEDICAL UNIV
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