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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 cumbersome methods, high difficulty, and difficulty in large-scale cultivation, and achieves high expression efficiency, high yield, and easy separation and purification. Effect

Inactive Publication Date: 2007-01-24
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] Example 1 Cloning of HpaA, UreB414, NapA encoding genes of Helicobacter pylori

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

[0059] 2. The Hp strain taken out and stored in the liquid nitrogen tank is spread on the Hp special solid medium, at 37℃, containing 5% O 2 , 85% N 2 , 10% CO 2 Cultivate 72h under the conditions. The genome extraction kit extracts the Hp genome.

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

[0061] 1) The primer design and synthesis are as follows (underline shows the restriction site and linker base sequence)

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

[0063] HpaA P1 5’-C CCTGCTGTACCACCACCT AATTACCATCCA-3’

[0064] ...

Embodiment 2

[0092] Example 2 Obtaining the 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] NdeI

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

[0098] linker

[0099] LTB P9 5’- GGAGGCGGAAGTGGAGGAGGTAGC GCTCCCCAGTCTAT-3

[0100] linker

[0101] P9’ 5’- CCGCAGGATCCTCCG GCTCCCCAGTCTATT-3’

[0102] linker

[0103] P10 5’- CTCGAG GTTTTCCATGCTGATTGC-3’

[0104] XhoI

[0105] Using Helicobacter pylori SS1 and wild-type toxin-producing Escherichia coli H44815 genomic DNA as templates, NapA and LTB genes were amplified with P7 and P8, P9' and P10. Bacterial genome extraction was based on the age of the day. Kit I was carried out. The PCR amplification system is: 10×10μL of magnesium ion-free amplification buffer, MgCl 2 (25mmol / L) 1...

Embodiment 3

[0110] Example 3 Obtaining the fusion gene NapA-CTB

[0111] 1. NapA and CTB gene amplification

[0112] 1) Primer design and synthesis

[0113] NapA P7 5’-GCGGG CATATG AAAACATTTGAAA-3’

[0114] NdeI

[0115] P8 5’- CGGAGGATCCTGCGG AGCTAAATGGGC-3’

[0116] linker

[0117] CTB P11’ 5’- CCGCAGGATCCTCCG ATTAAATTAAAATTTGGT-3’

[0118] linker

[0119] P12 5’- CTCGAG ATTTGCCATACTAATTGCG-3’

[0120] XhoI

[0121] P7 and P8, P11' and P12 were used to amplify the NapA and CTB genes, and the bacterial genome extraction was performed according to the day-time spin column type bacterial genomic DNA extraction kit I. The PCR amplification system is: 10×10μL of magnesium ion-free amplification buffer, MgCl 2 (25mmol / L) 10μL, dNTPs (25mmol / L each) 8μL, upstream and downstream primers each 2μL, the above NapA or CTB genome each 1μL, Ex-Taq DNA polymerase (5 units / μL) 1μL, add st...

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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, a preparation method thereof, and application in the prevention and treatment of Helicobacter pylori infectious diseases. Background technique [0002] Helicobacter pylori (Hp) is a gram-negative pylori that colonizes human gastric mucosa. About 50% of people in the world are infected with Helicobacter pylori. People have done a lot of research 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 Helicobacter pylori strains can cause different pathologies, from clinical asymptomatic to chronic gastritis, peptic ulcer and even gastric cancer. In 1998, Japanese scholar Watanadbe et al. first reported that Mongolian gerbils infected by Hp c...

Claims

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Application Information

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Patent Type & Authority Applications(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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