Fusion protein capable of being self-assembled into protein nanoparticle and application of fusion protein

A fusion protein and nanoparticle technology, applied in the field of fusion proteins, can solve problems such as weak immune response ability

Active Publication Date: 2020-06-26
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the polysaccharide conjugate vaccine prepared by the current technology still has weak immune response ability against certain bacteria, and the response level needs to be further improved

Method used

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  • Fusion protein capable of being self-assembled into protein nanoparticle and application of fusion protein
  • Fusion protein capable of being self-assembled into protein nanoparticle and application of fusion protein
  • Fusion protein capable of being self-assembled into protein nanoparticle and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1. Construction and expression of nanoparticle vectors

[0141] 1) Construction of B5Tri fusion protein expression vector

[0142] In this example, a vector expressing a fusion protein (B5Tri) of B5 protein and Tri sequence is constructed, wherein the B5 protein includes cholera toxin B subunit (CTB), Shiga toxin B subunit (StxB), Escherichia coli heat-labile enterotoxin (LTB), Bacillus subtilisin B subunit (SubB), the Tri sequence is SEQ ID NO.9. The build method is as follows:

[0143]Construction of CTBTri expression vector: According to the amino acid sequence of cholera toxin B subunit (X76390.1) published by GeneBack, its signal peptide (the first 21 amino acids) was replaced with DsbA signal peptide (SEQ ID NO.31), at the C-terminal of CTB The Tri sequence was fused to construct the CTB fusion protein CTBTri. In the CTBTri expression cassette, the expression of CTBTri is initiated by the tac promoter, and the expression cassette is named as tacCTBTri, ...

Embodiment 2

[0151] Embodiment 2. Purification and detection of nanoparticles

[0152] Inoculate BL21 / pET28-tacB5Tri in LB medium with a final concentration of 50 μg / mL kanamycin, culture at 37°C, 220 rpm for 10 hours, then transfer to 5L LB medium at 37°C, and culture at 220 rpm When the OD600 was about 0.6, IPTG with a final concentration of 1 mM was added, and the temperature was lowered to 30° C. for 10 h.

[0153] Sample treatment: Take the above-mentioned bacteria induced at 30°C for 10 hours, add 400mL loading buffer (20mM pH7.5 Tris-HCl, 0.2M NaCl, 10mM imidazole), and ultrasonically break the bacteria (sonication for 3s and pause for 5s, and the cumulative ultrasonication time is 30min) , centrifuged at 12000g, and collected the supernatant.

[0154] Sample purification: first wash the column bed with 0.5M NaOH aqueous solution for at least 3 column bed volumes, then equilibrate to neutral pH with deionized water, and then use 0.5M NiSO 4 Equilibrate at least 3 column bed volume...

Embodiment 3

[0156] Example 3. Preparation of Nanoscale Bacterial Polysaccharide Conjugated Vaccines by Biological Method

[0157] 3.1 Construction of B5Tri (B5 respectively CTB, StxB, LTB and SubB) glycosylation expression vector

[0158] 3.1.1 Construction of Neisseria meningitidis glycosyltransferase PglL expression vector: The amino acid sequence of Neisseria meningitidis glycosyltransferase PglL (GeneBank: JN200826.1) is shown in SEQ ID No.19, and its coding sequence As shown in the 180th-1994th nucleotides of SEQID No.20. The 1st-6th nucleotide of SEQ ID No.20 is the XbaI recognition site, and the 105th-2240th nucleotide is the sequence of the PglL expression cassette. In the PglL expression cassette, the expression of PglL is initiated by the tac promoter. This expression cassette was named tacpglL. Wherein, the 105th-133rd nucleotide of SEQ ID No.20 is the sequence of the tac promoter, the 180th-1994th nucleotide is the coding sequence of Neisseria meningitidis glycosyltransferas...

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Abstract

The invention relates to recombinant fusion proteins and polysaccharide conjugate vaccines, specifically to a fusion protein comprising a substrate protein of a pentamer and a peptide sequence capableof forming a trimer, an immunogenic composition comprising the fusion protein, the application of the fusion protein in immunization, a polynucleotide sequence encoding the fusion protein, an expression vector comprising the polynucleotide sequence, and a host cell comprising the expression vector.

Description

technical field [0001] The invention relates to a fusion protein that can be self-assembled into protein nanoparticles and its application, belonging to the field of biomedicine. Background technique [0002] Vaccines play an important role in the prevention of pathogenic bacterial infections. The new generation of vaccine products (subunit vaccines) have higher safety due to their single and clear ingredients, but these vaccines often have low immunogenicity, and will be rapidly diluted and degraded by body fluids after injection, so Often some adjuvant and suitable delivery system are required to promote a maximal immune response. In recent years, research has continuously found that the immune effect can be significantly improved by nano-scale vaccines, especially vaccines based on nano-carriers, which have been continuously applied. The most important feature is that they can replace adjuvants and simultaneously stimulate Cellular immunity and humoral immunity, especia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/385A61K39/095A61P31/04
CPCC07K14/28C07K14/25C07K14/245C07K14/32C07K14/22A61K39/385A61K39/095A61P31/04C07K2319/55C07K2319/91C07K2319/735A61K2039/6037A61K2039/552A61K39/00A61K2039/55555C12N15/62
Inventor 王恒樑潘超朱力吴军黄竞张璐璐孙鹏曾明王斌刘先凯王东澍冯尔玲刘波
Owner ACADEMY OF MILITARY MEDICAL SCI
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