A fusion protein that can self-assemble into protein nanoparticles and its application
A fusion protein and nanoparticle technology, applied in the field of fusion proteins, can solve problems such as weak immune response ability, and achieve the effect of treating bacterial infection and significant clinical value
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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 B5 protein and Tri sequence fusion protein (B5Tri) is constructed, wherein, 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, and its ...
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...
PUM
Property | Measurement | Unit |
---|---|---|
diameter | aaaaa | aaaaa |
particle size | aaaaa | aaaaa |
particle size | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com