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Carrier protein of bacterial polysaccharide conjugate vaccine and application thereof

A fusion protein and polysaccharide technology, applied in the direction of anti-bacterial immunoglobulin, anti-bacterial drugs, medical preparations containing active ingredients, etc., can solve the problems of limited immunogenicity and low polysaccharide protein

Active Publication Date: 2017-03-22
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The natural glycoprotein prepared by biological cross-linking technology, the polysaccharide and the protein are combined with a single end of N-glycosidic bond or O-glycosidic bond, which can maximize the exposure of the antigenic epitope of the polysaccharide, but due to the small number of glycosylation sites on the carrier protein, Low polysaccharide protein ratio, limiting its immunogenicity

Method used

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  • Carrier protein of bacterial polysaccharide conjugate vaccine and application thereof
  • Carrier protein of bacterial polysaccharide conjugate vaccine and application thereof
  • Carrier protein of bacterial polysaccharide conjugate vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1. Preparation and Application of Shigella flexneri O Polysaccharide-Recombinant Cholera Toxin B Subunit Fusion Protein Conjugate

[0124] Shigella spp., commonly known as Shigella spp., is a highly contagious Gram-negative enteropathogenic bacterium, which mainly invades human colonic epithelial cells and finally locates in the large intestine, causing typical bacillary dysentery ( fever, abdominal pain, tenesmus, fecal pus and blood), and the large virulence plasmid is the most important factor of its pathogenicity. The large virulence plasmid contains about 32 genes related to virulence, mainly including System-related mxi-spa gene, virulence genes such as ipaBCD and ipgC closely related to bacterial invasion of epithelial cells. In order to develop it as a safe host bacterium, the large plasmid pCP encoding the virulence factor must be removed first, and on this basis, the O antigen ligase gene waaI is deficient, thereby developing a host bacterium suitable ...

Embodiment 2

[0273] Example 2. Preparation and Application of Salmonella Paratyphi A O Polysaccharide-Recombinant Cholera Toxin B Subunit Fusion Protein Conjugate

[0274] A. Exploring the construction of recombinant bacteria to extend the sugar chain of Salmonella paratyphi A O antigen

[0275] 1. Knockout of the O antigen chain length control enzyme gene cld of Salmonella paratyphi A CMCC50973 strain

[0276] 1. Preparation of linear targeting DNA fragment 1

[0277] 1) Design of PCR primers

[0278] According to the S. paratyphi ATCC9150 genome sequence (CP000026) published by GeneBack, a targeting fragment of the cld gene (position 887812 to 888789) was designed to knock out the cld gene of Salmonella paratyphi A CMCC50973. Cut off 500bp at the upstream and downstream of the cld gene as the homology arm, amplify the target fragment of the chloramphenicol resistance gene containing 500bp homologous sequence and FRT site at both ends by PCR, and identify it with 50973cldup 5' and 50973...

Embodiment 3

[0417] Example 3. Preparation and Application of Escherichia coli O157 O Polysaccharide-Recombinant Cholera Toxin B Subunit Fusion Protein Conjugate

[0418] When exogenous polysaccharides are used to modify recombinant fusion proteins, bacteria that are double-deficient in O-antigen ligase gene and host O antigen synthesis should be used as the host. None of the polysaccharides can be used by the host LPS synthesis pathway, and the host's O antigen synthesis defect ensures that the glycosylation modification system only uses exogenous polysaccharides to modify the recombinant fusion protein, and does not appear to be modified by the host's own O antigen polysaccharides. pollution phenomenon. The host bacteria co-express the recombinant fusion protein gene, Neisseria meningitidis O-oligosaccharide transferase PglL gene, and exogenous polysaccharide synthesis gene cluster. Under the catalysis of oligosaccharide transferase PglL, the exogenous polysaccharide is transferred to th...

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Abstract

The invention discloses a bacterial polysaccharide O-glycosylation modified recombinant cholera toxin B subunit fusion protein and an application thereof. The present invention provides a conjugate of polysaccharide and protein which is coupled by the bacterial polysaccharide and recombinant cholera toxin B subunit fusion protein. The bacterial polysaccharide is connected with the glycosylation sites of the recombinant cholera toxin B subunit fusion protein in the form of O-glucosidic bond. The experiment indicates that the preparation of the bacterial polysaccharide protein conjugate vaccine from O-glycosylation modified recombinant cholera toxin B subunit fusion protein can enhance the ability of inducing animals to generate anti-polysaccharide antibodies, avoid miscellaneous problems of pathogen culture, enhance the vaccine homogeneity and production efficiency, and reduce the vaccine preparation cost, therefore possessing wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bacterial polysaccharide O-glycosylated recombinant cholera toxin B subunit fusion protein and an application thereof, belonging to the field of biomedicine. Background technique [0002] O-polysaccharide (O-PS) and capsular polysaccharide (CPS) of pathogenic bacteria are often important protective antigens, such as type O157 of Escherichia coli, type O1 and O139 of Vibrio cholerae, type 4, 6B, 9V, 14, 18C, 19F, 23F, etc., CP5 and CP8 of Staphylococcus aureus, etc. Many antibodies against polysaccharides are neutralizing antibodies to pathogenic bacteria, so the development of polysaccharide vaccines has always been one of the focuses of bacterial vaccine research and development. However, the immune memory and immunogenicity of polysaccharide vaccines are weak, and now the research and development focus of such vaccines has shifted to polysaccharide-protein conjugate vaccines, fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/385A61K39/095A61K39/106C07K16/12A61P31/04A61P37/04
CPCY02A50/30
Inventor 吴军孙鹏王恒樑潘超刘波朱力唱韶红巩新曾明
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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