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O-type FMDV capsid protein-ferritin fusion protein, protein cage nanoparticle and preparation method of O-type FMDV capsid protein-ferritin fusion protein

A technology for capsid protein and fusion protein, which is applied in the direction of expression enhancement/folding protein fusion, biochemical equipment and methods, nanotechnology, etc. No activity or poor activity, etc., to achieve the effect of promoting soluble expression, improving high immunogenicity and good immunogenicity

Inactive Publication Date: 2020-04-24
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when expressing recombinant proteins in the E. coli bacterial system, there are many shortcomings that are difficult to overcome: 1. The recombinant proteins often appear in the form of inactive inclusion bodies; 2. Lack of eukaryotic post-translational modifications (glycosylation, phosphorylation) and acetylation, etc.) mechanism, although the obtained recombinant protein is correct in the primary amino acid sequence, it is quite different from the natural protein in the advanced structure and conformation, and has no activity or very poor activity; 3. Host cell (Escherichia coli) own protein Becoming a pyrogen, difficult to remove, and safety, these problems limit the further application of prokaryotic expressed recombinant proteins in practice
However, there is currently no good solution that can improve both the solubility of the fusion protein and the immunogenicity of the antigen

Method used

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  • O-type FMDV capsid protein-ferritin fusion protein, protein cage nanoparticle and preparation method of O-type FMDV capsid protein-ferritin fusion protein
  • O-type FMDV capsid protein-ferritin fusion protein, protein cage nanoparticle and preparation method of O-type FMDV capsid protein-ferritin fusion protein
  • O-type FMDV capsid protein-ferritin fusion protein, protein cage nanoparticle and preparation method of O-type FMDV capsid protein-ferritin fusion protein

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Embodiment 1

[0039] 1. Materials and methods

[0040] The expression vector pET-21b / EW29 / AfFtn with EW29 gene (the insertion site of EW29 in pET-21b is Nde I and Nhe Between Ⅰ, the insertion site of AfFtn in pET-21b is Bam H I and xho Between Ⅰ) is preserved by the Key Open Laboratory of Animal Biochemistry and Nutrition of Henan Agricultural University.

[0041] Carrier PUC57-SeFnt16599 with O-type FMDV capsid protein-ferritin fusion protein (the insertion site of SeFnt16599 in PUC57 is Bam HI and xho Between I) was constructed and synthesized by Nanjing GenScript. SeFnt16599 represents an epitope-ferritin fragment of type O FMDV capsid protein. O-type FMDV capsid protein epitope is according to the O-type foot-and-mouth disease amino acid of FMDV O / GER / Wup / 82 strain, and its epitope is carried out the sequence that Escherichia coli expresses codon optimization, and this amino acid is as shown in SEQ ID NO.10, The nucleotide sequence expressing the epitope of the O-type FMDV ...

Embodiment 2

[0050] 1. Materials and methods

[0051] 8 recombinant plasmids pET21b-His with lytic tags and target genes (FMDV epitope sequence + ferritin) 6 -Grifin / GST / MBP / Sumo / Thioredoxin / γ-crystallin / ArsC / PpiB / -SeFnt16599 (abbreviated as pET21b1-pET21b8), and the expression vector pET-21b / EW29 / AfFtn with EW29 gene (EW29 in pET-21b The insertion site is Nde I and Nhe Between Ⅰ, the insertion site of AfFtn in pET-21b is Bam H I and xho Between Ⅰ) is preserved by the Key Open Laboratory of Animal Biochemistry and Nutrition of Henan Agricultural University.

[0052] References for the construction process of pET21b1-pET21b8 (Guo Yukun, Guo Wanying, Ming Shengli, et al. Soluble expression, purification and electron microscope detection of O-type foot-and-mouth disease virus capsid protein in Escherichia coli[J]. China Veterinary Medicine, 2018(7). ), wherein SeFnt16599 represents the O-type FMDV capsid protein epitope-ferritin fragment. O-type FMDV capsid protein epitope is based ...

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Abstract

The invention discloses O-type FMDV capsid protein-ferritin fusion protein, a protein cage nanoparticle and a preparation method of the O-type FMDV capsid protein-ferritin fusion protein. The preparation method comprises the following steps: connecting O-type FMDV dominant epitopes with the nucleotide sequence of a ferritin fragment in series to synthesize an O-type FMDV capsid protein epitope-ferritin fragment; and connecting the O-type FMDV capsid protein epitope-ferritin fragment with a solubilizing label and an affinity label EW29 to obtain an EW29 / solubilizing label / O-type FMDV capsid protein epitope-ferritin fragment, and carrying out induction and affinity purification to obtain the O-type FMDV capsid protein-ferritin fusion protein. An electron microscope result shows that the fusion protein forms the protein cage nanoparticle with a particle size of 20-25 nm. The invention lays a foundation for further development of a safe and effective O-type FMDV capsid protein vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an O-type FMDV capsid protein-ferritin fusion protein, protein cage nanoparticles and a preparation method thereof. Background technique [0002] Through DNA recombination technology, fusion protein has been easily expressed in prokaryotic expression system (Escherichia coli) and eukaryotic expression system (yeast and mammalian cells), and its products have been widely used in biology and medicine. Rapid development. Compared with eukaryotic expression systems, Escherichia coli is still the main host for recombinant protein production due to its advantages of easy operation, low cost and high yield. However, when expressing recombinant proteins in the E. coli bacterial system, there are many shortcomings that are difficult to overcome: 1. Recombinant proteins often appear in the form of inactive inclusion bodies; 2. Lack of eukaryotic post-translational modifications (g...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/385A61K39/135A61K47/64A61P31/14B82Y5/00B82Y30/00B82Y40/00
CPCC07K14/005C07K14/47C12N15/70A61K39/385A61K39/12A61K9/5169A61K47/64B82Y5/00B82Y30/00B82Y40/00C07K2319/20C07K2319/35C12N2770/32122C12N2770/32134A61K2039/6031A61K2039/552
Inventor 杨国宇郭玉堃王梦珂郭豫杰明胜利张爽曾磊
Owner HENAN AGRICULTURAL UNIVERSITY