Peptide carrier fusion proteins as allergy vaccines

A carrier and vaccine technology, applied in the direction of carrier-antigen complex structure, vaccine, carrier-bound antigen/hapten components, etc.

Active Publication Date: 2017-09-15
WORG PHARM (HANDZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no protocol has been described suitable for human vaccination with peptides containing allergen-specific T-cell epitopes

Method used

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  • Peptide carrier fusion proteins as allergy vaccines
  • Peptide carrier fusion proteins as allergy vaccines
  • Peptide carrier fusion proteins as allergy vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0448] Embodiment 1: Construction of HBV Phlpl_4xP5 (BM321) expression plasmid

[0449] Synthetic BM321 genes were assembled from synthetic oligonucleotides and / or PCR products and cloned into an appropriate standard vector (pMK-RQkanR). Plasmids were purified from transformed E. coli K12 strain (DH10B-T1R), and concentrations were determined by UV spectroscopy. The final synthesized and codon-optimized BM321 DNA sequence was further cloned into the expression vector pET28b(+) using appropriate restriction sites (NcoI site at the 5' end and EcoRI at the 3' end). Plasmid DNA was purified from transformed E. coli K12DH10B (dam+dcm+), and the concentration was determined by UV spectroscopy. The final construct was confirmed by sequencing of the insert. A summary of the plasmid data and plasmid map of the final expression vector "pBM-321" is shown below.

[0450] Summary of the BM321 sequence cloned into the final expression vector pET-28b(+).

[0451] sequence

Embodiment 2

[0452] Embodiment 2: the transformation of expression plasmid to the expression host that is used for HBV_Phlpl_4xP5 (BM321)

[0453] Chemically competent E. coli BL21(DE3) cells were transformed with expression plasmids by heat shock method. Transformed cells were plated on LB agar plates consisting of 0.5% sodium chloride, 1% soy peptone, 0.5% yeast extract, 1.5% agar and 50 μg / mL kanamycin for selection. Cells were grown on LB plates by overnight incubation at 37°C. Individual clones of transformed BL21(DE3) E. coli cells were isolated, cultured in LB medium, and screened for growth and expression of BM321. The best performing clones were selected for further master cell bank establishment.

Embodiment 3

[0454] Embodiment 3: Preparation of the master cell bank of HBV Phlpl_4xP5 (BM321)

[0455] Aliquots of selected clones were used for inoculation of 150 mL of medium (composition: 0.5% NaCl, 1% soy peptone, 0.5% yeast extract, 50 μg / mL kanamycin). Incubate the master cell bank (MCB) culture at 37 °C with continuous agitation at 200 rpm until the culture reaches the optical density OD 600 =1-2. Glycerol was added to obtain a final glycerol concentration of 15% v / v, the MCBs were aliquoted into 1 mL vials and stored in a -75 ± 10 °C ultra-low temperature freezer.

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Abstract

The present invention relates to polypeptides comprising at least 3 peptide fragments consisting of 10 to 50 contiguous amino acid residues of at least 1 wild-type allergen fused to the Hepadnaviridae family N- and C-termini of viral surface polypeptides or at least 1 fragment fused to said surface polypeptides.

Description

technical field [0001] The present invention relates to novel polypeptides and uses thereof. Background technique [0002] Type I allergy is an IgE-mediated hypersensitivity disorder that affects almost 25% of the population. It is based on the specific immunoglobulin E response to harmless airborne, insect, venom, food allergen and contact allergen antigens (derived from antigenic sources that are not themselves harmful, such as pollen, insects, molds and animal proteins ) identification. Cross-linking of effector cell-bound IgE antibodies causes release of inflammatory mediators (eg histamine, leucotrienes) and thus immediate symptoms of allergy (eg rhinoconjunctivitis, asthma, dermatitis, anaphylaxis). T cell activation through IgE-dependent and IgE-independent mechanisms contributes to chronic allergic inflammation. [0003] The only potentially causative form of allergy treatment is allergen-specific immunotherapy, which is based on repeated administration of increas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/36C07K14/02
CPCA61K39/36A61K2039/6075A61K2039/645C07K2319/00C07K14/005C12N2730/10122A61K2039/57C12N2730/10134A61K39/12A61K39/292A61P37/08A61K39/385C07K14/02C07K14/415A61K2039/53A61K2039/64
Inventor K·尼斯波德兹阿纳M·福克-泰克尔S·威塔拉S·班纳吉K-W·陈M·韦伯R·瓦伦察K·马特
Owner WORG PHARM (HANDZHOU) CO LTD
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