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Urease epitope fusion peptide liposome bacterin for preventing the helicobacter pylori infecting

An anti-Helicobacter pylori and Helicobacter pylori technology, applied in the field of biomedicine, can solve the problem of not completely preventing Helicobacter pylori colonization and the like

Inactive Publication Date: 2007-10-31
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have shown that immunizing animals with intact urease does not inhibit the activity of urease well, so it cannot completely prevent the colonization of Helicobacter pylori in the stomach (Infect Immun 1992, 60: 4826-31)

Method used

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  • Urease epitope fusion peptide liposome bacterin for preventing the helicobacter pylori infecting
  • Urease epitope fusion peptide liposome bacterin for preventing the helicobacter pylori infecting
  • Urease epitope fusion peptide liposome bacterin for preventing the helicobacter pylori infecting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of Fusion Peptide CtUBE Expression Vector

[0040] (1) Design and synthesize PCR primers and introduce NcoI and EcoRI sites at the same time:

[0041] PCTB01: 5′-AAAAA CCATGG GCACACCTCAAAATATTACTGATTTG-3′,

[0042] PCTB02: 5′-AA GAATTC GCCGCCATTTGCCATACTAATTGCGGCAATCGCAT-3';

[0043] (2) Using the Vibrio cholerae genomic DNA as a template, the CTB coding sequence was amplified by PCR (94°C pre-denaturation for 3 minutes; 94°C for 45s, 56°C for 50s, 72°C for 50s, 40 cycles; 72°C extension for 10min) to amplify the CTB coding sequence 1);

[0044] (3) The PCR product was double digested with NcoI and EcoRI and cloned into the multiple cloning site of pET-28a to obtain the intermediate vector pET-CtUBE-Pre, which was transformed into Escherichia coli DH5α for amplification;

[0045] (4) Synthesize the UreB epitope coding sequence and introduce EciRI and XhoI sites simultaneously:

[0046] UBP1: 5′- AATTC TGCCACCACTTGGATAAAAGCATTAAAGAAGATGTTC...

Embodiment 2

[0051] Example 2 Establishment of fusion peptide CtUBE expression, separation, renaturation and purification system

[0052] (1) Transfer the positive transformants with correct sequencing into LB medium, induce expression with 1mM IPTG (accompanying drawing 4), analyze the expression form of the fusion protein through 12% SDS-PAGE, and analyze the expression amount with electronic scanning simultaneously;

[0053] (2) The fusion peptide is expressed in the form of inclusion bodies. After expanding the expression culture, the bacterial cells are lysed with a lysate containing lysozyme, and the inclusion bodies are collected by centrifugation;

[0054] (3) After the inclusion bodies were washed 5 times with Wash Buffer, they were dissolved with inclusion body dissolution buffer, and about 10 times the volume of Renaturation Buffer was slowly added, and transferred to 4°C for renaturation for more than 48 hours;

[0055] (4) After the refolded fusion protein is fully dialyzed wi...

Embodiment 3

[0058] Example 3 Preparation of fusion peptide CtUBE liposome vaccine

[0059] (1) Add 0.314g lecithin and 0.126g cholesterol respectively in eggplant-shaped bottle, and add 6ml chloroform to make lecithin and cholesterol dissolve and mix;

[0060] (2) Evaporate chloroform to dryness on the rotary evaporator, add 2.26ml chloroform and 3.74ml ether, dissolve lecithin and cholesterol film;

[0061] (3) Add 2ml of 1mg / ml CtUBE solution (dissolved in PBS with pH 8.04), and sonicate in ice bath for 4min;

[0062] (4) Reduce the pressure on the rotary evaporator to about 0.05MPa, and evaporate the organic solvent at 37°C;

[0063] (5) Add 10ml of PBS (pH8.04) to the eggplant-shaped bottle and rotate at high speed for 3-4h to obtain a light milky yellow liposome suspension;

[0064] (6) Regulate the liposome particle size with 1.0 μm, 0.8 μm and 0.4 μm microporous membrane successively;

[0065] (7) Take 1ml of liposome suspension and put it into a 1.5ml centrifuge tube, centrifug...

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Abstract

The invention discloses an urea enzyme epitope fuse peptiolipid plastid vaccine to against pylorus bolt bacteria infection, which is characterized by the following: choosing fuse peptide of pylorus bolt bacteria urea enzyme B subunit and stick film adjuvant cholera morbus toxin B subunit as immunogen; coating the immunogen with liposome; producing the liposome vaccine. This liposome vaccine can evoke organism to generate special immune response and inhibit planting of pylorus bolt bacteria in stomach.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the construction, transformation, expression, Purification and preparation of CtUBE liposome vaccine provide a preventive and therapeutic vaccine against Helicobacter pylori infection. Background technique [0002] Helicobacter pylori is the main cause of diseases such as gastritis, peptic ulcer and gastric cancer. All clinically isolated strains of Helicobacter pylori express urease and are highly conserved at the same time. Urease can decompose urea to produce ammonia, neutralize gastric acid, and provide a favorable environment for the colonization of Helicobacter pylori. Due to these characteristics of urease, it has become a hotspot of candidate antigens in the development of anti-Helicobacter pylori vaccines. However, studies have shown that immunizing animals with intact urease does not inhibit the activity of urease well, and thus cannot completely prevent the colonization of...

Claims

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Application Information

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IPC IPC(8): A61K9/127A61K39/116A61K39/106A61P31/04A61P1/04C12N15/62
Inventor 吴梧桐赵文锋徐旭东
Owner CHINA PHARM UNIV
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