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Gene engineered poly-valence subunit vaccine of Hp AhpC and preparation method thereof

A genetically engineered vaccine, Helicobacter pylori technology, applied in chemical instruments and methods, bacterial antigen components, drug combinations, etc., can solve problems such as difficult large-scale cultivation, unfavorable vaccine industrialization preparation, and harsh conditions for Hp bacterial culture

Inactive Publication Date: 2006-11-22
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the conditions for culturing Hp bacteria 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.
There is no report on the use of the Helicobacter pylori outer membrane antigen AhpC as an immunogenic material, especially the use of the fusion protein of AhpC and NapA as an immunogenic material to prepare a multivalent vaccine for Helicobacter pylori

Method used

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  • Gene engineered poly-valence subunit vaccine of Hp AhpC and preparation method thereof
  • Gene engineered poly-valence subunit vaccine of Hp AhpC and preparation method thereof
  • Gene engineered poly-valence subunit vaccine of Hp AhpC and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1 Cloning of Helicobacter pylori AhpC, NapA coding genes and extramolecular adjuvant LTB, CTB genes

[0117] 1. Helicobacter pylori is derived from clinical isolates from patients in the Gastroenterology Department of Southwest Hospital (strain number: CQ9803)

[0118] 2. Take out the preserved Hp strain (CQ 9803) 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 (shown in Figure 1).

[0119] 3. The coding genes of AhpC and NapA were respectively amplified from the Hp genome by PCR method.

[0120] 1) The primers were designed and synthesized as follows (the restriction site and linker base sequence are underlined)

[0121] According to the gene sequence published by GenBank and the principles of primer design, the corresponding primers were designed and restriction sites were introduced. The followin...

Embodiment 2

[0210] Example 2 Acquisition of Intramolecular Adjuvant Fusion Gene AhpC-LTB(AL)

[0211] Primer design and synthesis

[0212] AhpC P1 5'-CG CATATG TTAGTTACAAAAACTTGCC-3'

[0213] Nde I

[0214] P2' 5'- GCTACCTCCGCCTCC AAGCTTAATGGAAT

[0215] Linker

[0216] LTB P5” 5’- GGAGGCGGAGGTAGC GCTCCCCAGTCTATT-3'

[0217] linker

[0218] P6 5'- CTCGAG GTTTTCCATGCTGATTGC-3'

[0219] wxya

[0220] Using the AhpC and LTB recovered in Example 1 as templates, use P1 and P2', P5" and P6 primers to amplify AhpC and LTB genes respectively, and the bacterial genome extraction is carried out on a daily basis with a spin-column type bacterial genomic DNA extraction kit The PCR amplification system is: template DNA 1μl; 10×PCR buffer 5μl; dNTPs (10mmol / L) 4μl; primers (0.025mmol / L) 1μl each; Taq DNA polymerase (5u / μl) 0.5μl; water to a final volume of 50 μl.

[0221] T...

Embodiment 3

[0225] Example 3 Acquisition of Intramolecular Adjuvant Fusion Gene AhpC-CTB(AC)

[0226] Primer design and synthesis

[0227] AhpC P1 5'-CG CATATG TTAGTTACAAAAACTTGCC-3'

[0228] Nde I

[0229] P2' 5'- GCTACCTCCGCCTCC AAGCTTAATGGAAT

[0230] Linker

[0231] CTB P7” 5’- GGAGGCGGAGGTAGC ATTAAATTAAAATTTGGT-3'

[0232] linker

[0233] P8 5'- CTCGAG ATTTGCCATACTAATTGCG-3'

[0234] wxya

[0235] Using the AhpC and CTB recovered in Example 1 as templates, using P1 and P2', P7" and P8 as primers, respectively amplify the AhpC and CTB genes, and extract the bacterial genome by days. The PCR amplification system is: template DNA 1μl; 10×PCR buffer (containing magnesium chloride) 5μl; dNTPs (10mmol / L) 4μl; primers (0.025mmol / L) each 1μl; Taq DNA polymerase (5u / μl ) 0.5 μl, add deionized water to a final volume of 50 μl.

[0236] The reaction system was mixe...

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Abstract

The disclosed vaccine employs alkyl hydrogen peroxide reductase (AhpC) or its fusion gene with neutral granular leukocyte activated protein NapA as active constituent, as well as intra-molecule external adjuvant such as bacillus coli heat-labile toxin B subunit LTB, cholera morbus toxin B subunit CTB.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a genetic engineering vaccine used for immunity and treatment of human Helicobacter pylori infection and a preparation method for a multivalent subunit vaccine thereof. Background technique [0002] Helicobacter pylori (H. pylori) is a gram-negative bacillus that colonizes the human gastric mucosa and causes stomach and duodenal diseases. More than 50% of the world's population is infected with Helicobacter pylori in the stomach, and most of them are in Acquired in childhood, if not treated, it will persist and cause chronic gastritis, peptic ulcer, and even gastric cancer. Since Warren and Marshall discovered Helicobacter pylori in 1983, people have been working on the research of this bacterium. After years of analysis and research by the World Health Organization and the National Institutes of Health, H. pylori was officially listed as the primary carcinogen of gastric cancer. ...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61P1/04C07K19/00
Inventor 邹全明张卫军杨珺毛旭虎郭刚童文德吴超郭鹰刘开云解庆华
Owner ARMY MEDICAL UNIV
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