Helicobacter pylori multiple-epitope fusion protein and multiple-epitope vaccine prepared by helicobacter pylori multiple-epitope fusion protein

A technology of Helicobacter pylori and fusion protein, which is applied in the field of fusion protein and multi-epitope vaccine, and can solve problems affecting the therapeutic effect of the vaccine

Inactive Publication Date: 2012-12-26
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above adjuvants can elicit a strong mucosal immune response, they cannot induce naive CD4 + T cells differentiate into Th1 cells, and the Th1 response is crucial for eradicating Helicobacter pylori, and the inability to induce Th1 cell differentiation affects the therapeutic effect of the vaccine to a certain extent

Method used

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  • Helicobacter pylori multiple-epitope fusion protein and multiple-epitope vaccine prepared by helicobacter pylori multiple-epitope fusion protein
  • Helicobacter pylori multiple-epitope fusion protein and multiple-epitope vaccine prepared by helicobacter pylori multiple-epitope fusion protein
  • Helicobacter pylori multiple-epitope fusion protein and multiple-epitope vaccine prepared by helicobacter pylori multiple-epitope fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of recombinant plasmid

[0039] E. coli preferred codons are used to synthesize the vaccine protein coding gene EpiVac, and its nucleotide sequence is shown in SEQ ID NO:1. Insert EpiVac into the plasmid vector pET 30a to obtain a recombinant plasmid named pET 30a-EpiVac. Transform the recombinant plasmid into E.coli DH5α competent cells, inoculate the transformed competent cells on LB plates containing a final concentration of 30μg / ml kanamycin for resistance selection, pick a single clone, and use whole bacteria PCR method and restriction enzyme digestion of recombinant plasmids identify recombinants. Recombinant plasmid was identified correctly by restriction enzyme digestion, and it was also correct by sequencing. Please refer to the restriction enzyme digestion electropherogram figure 1 .

Embodiment 2

[0040] Example 2 Induced expression of vaccine protein and identification of expression form

[0041] The recombinant plasmids prepared in Example 1 were respectively transformed into E.coli BL21(DE3) competent cells, and single clones were picked and inoculated in 10ml LB medium containing a final concentration of 50μg / ml kanamycin at 37°C , 200rpm / min shaking culture overnight, and then inoculating in fresh LB medium at a volume ratio of 1:100, 37℃, 200rpm / min shaking culture, until the cell density OD 600nm When it reached about 0.6, IPTG was added to a final concentration of 1 mmol / L, and the culture was induced for 5 hours. The cells were collected and sonicated and centrifuged at 12000rpm / min for 30min. The supernatant and precipitate obtained were analyzed by SDS-PAGE to determine the expression form of the target protein. The results show that the recombinant protein EpiVac is an inclusion body protein. The expression and expression form of the recombinant protein EpiVac...

Embodiment 3

[0042] Example 3 Purification of vaccine protein

[0043] Centrifuge the 1L of the induced recombinant bacteria and collect the cells, wash them once with PBS, then resuspend the bacteria in 100ml A solution (20mM Tris, 5mM EDTA), break it with a high pressure homogenizer, and centrifuge at 800×g for 20min at 4℃ , Discard the precipitate, centrifuge the supernatant at 8000 rpm / min for 30 min, discard the supernatant to obtain inclusion bodies. The inclusion bodies were dispersed with liquid B (20mM Tris, 5m MEDTA, 1% Triton) and stirred for 1h. Centrifuge at 8000 rpm / min for 30 min. Disperse the pellet with solution C (20mM Tris, 5mM EDTA, 2M urea) and stir for 10 min. Centrifuge again to dissolve the pellet in the lysate (20mM Tris, 8M urea, pH 8.5). Filter with a 0.22um membrane, and the filtrate is purified by affinity chromatography. Using Invitrogen's Ni-NTAAgarose column, the protein was purified by the affinity between the 6×His tag at the N-terminal of the recombinant p...

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Abstract

The invention relates to a helicobacter pylori multiple-epitope fusion protein and a multiple-epitope vaccine prepared by the helicobacter pylori multiple-epitope fusion protein. An amino acid sequence of the helicobacter pylori multiple-epitope fusion protein is shown as SEQ ID NO:1. Fusion proteins in the multiple-epitope vaccine can induce generation of specificity sIgA in stomach tissues and formation of phigh-potency specificity IgG in blood serum, can effectively reduce constant value quantity of helicobacter pylori in a stomach of a mouse and has obvious protection effects.

Description

Technical field [0001] The invention relates to a fusion protein and a multi-epitope vaccine, in particular to a Helicobacter pylori multi-epitope fusion protein and a multi-epitope vaccine prepared therefrom. Background technique [0002] Helicobacter pylori is a gram-negative bacterium that inhabits human gastric mucosa. It has been identified as an important pathogen of chronic gastritis, peptic ulcer and a type I pathogenic factor of gastric cancer. More than 50% of the world’s population has Helicobacter pylori infection. In China, there are more than 600 million Helicobacter pylori infections. Among them, about 30% of infected people develop chronic gastritis, 10%-20% of people can develop peptic ulcers (gastric ulcer and duodenal ulcer), and some infected people can develop gastric mucosal atrophy and intestinal epithelialization. Birth or even stomach cancer. At present, the clinical eradication of Helicobacter pylori is mainly through the combination of antibiotics and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/02A61P31/04C12R1/01
Inventor 吴超李海波邹全明章金勇刘开云杨武晨陈立李滨赵卓毛旭虎郭刚童文德鲁东水
Owner ARMY MEDICAL UNIV
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