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A-type FMDV 1D protein-ferritin fusion protein, protein cage nanoparticles and preparation method of protein cage nanoparticles

A fusion protein and nanoparticle technology, applied in the biological field, can solve the problems of not obtaining the soluble antigen immunogenicity of fusion protein, limiting the application of recombinant protein, inactivity or poor activity, avoiding denaturation and renaturation treatment, and improving expression. , the effect of easy separation and purification

Inactive Publication Date: 2020-05-19
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A-type FMDV 1D protein-ferritin fusion protein, protein cage nanoparticles and preparation method of protein cage nanoparticles
  • A-type FMDV 1D protein-ferritin fusion protein, protein cage nanoparticles and preparation method of protein cage nanoparticles
  • A-type FMDV 1D protein-ferritin fusion protein, protein cage nanoparticles and preparation method of protein cage nanoparticles

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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-CcFnt166AS with type A FMDV 1D protein epitope-ferritin fusion protein (the insertion site of CcFnt166AS in PUC57 is Bam HI and xho Between I) was constructed and synthesized by Nanjing GenScript. CcFnt166AS represents the type A FMDV 1D protein epitope-ferritin fragment. Type A FMDV 1D protein epitope is based on the amino acid of 1D protein of FMDV A / GDMM / CHA / 2013 strain, and its epitope is subjected to the sequence obtained by Escherichia coli expression codon optimization, the amino acid is shown in SEQ ID NO.10, The nucleotide sequence expressing the epitope of the type A FMDV 1D protein is shown in...

Embodiment 2

[0049] 1. Materials and methods

[0050] 8 recombinant plasmids pET21b-His with lytic tags and target genes (FMDV epitope + ferritin sequence) 6 -Grifin / GST / MBP / Sumo / Thioredoxin / γ-crystallin / ArsC / PpiB-CcFnt166AS (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.

[0051] References for the construction process of pET21b1-pET21b8 (Guo Yukun, Ming Shengli, Guo Wanying, et al. Soluble expression, purification and electron microscope detection of 1D protein of foot-and-mouth disease virus type A in Escherichia coli[J]. Acta Anatomy (06):97 -102.), wherein CcFnt166AS represents the type A FMDV 1D protein epitope-ferritin fragment. Type A FMDV 1D protein epitope is based on the amino acid of...

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Abstract

The invention discloses an A-type FMDV 1D protein-ferritin fusion protein, protein cage nanoparticles and a preparation method of the protein cage nanoparticles. According to the invention, nucleotidesequences containing an A-type FMDV dominant epitopes and ferritin fragments are connected in series, and an A-type FMDV 1D protein epitope-ferritin fragment is designed and synthesized; and then theA-type FMDV 1D protein epitope-ferritin fragment is connected with a solubilization label and an affinity label EW29 to obtain an EW29 / solubilization label / A-type FMDV 1D protein epitope-ferritin fragment, and induction and affinity purification are carried out to obtain the A-type FMDV 1D protein-ferritin fusion protein. Electron microscope results show that the fusion protein forms protein cagenanoparticles of 20-25nm. The invention lays a foundation for further developing safe and effective A-type FMDV 1D protein vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a type A FMDV 1D 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 (glycos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C07K1/22C12N1/21A61K39/135A61K47/64A61P31/14C12R1/19
CPCC07K14/005C07K14/79C12N15/70A61K39/12A61K47/64A61P31/14C12N2770/32122C07K2319/42A61K2039/552C12N2770/32134
Inventor 郭玉堃于朋伟郭豫杰曾磊杨国宇
Owner HENAN AGRICULTURAL UNIVERSITY