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O-type FMDV VP1 protein-ferritin fusion protein, protein cage nanoparticle and preparation method thereof

A fusion protein and nanoparticle technology, applied in the direction of expression enhancement stability/folding protein fusion, biochemical equipment and methods, nanotechnology, etc., can solve the problem of soluble antigen immunogenicity of fusion protein, inactivity of recombinant protein, Activity or poor activity, etc., to avoid denaturation and refolding treatment, improve 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 VP1 protein-ferritin fusion protein, protein cage nanoparticle and preparation method thereof
  • O-type FMDV VP1 protein-ferritin fusion protein, protein cage nanoparticle and preparation method thereof
  • O-type FMDV VP1 protein-ferritin fusion protein, protein cage nanoparticle and preparation method thereof

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Experimental program
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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-SeFnt16798 with O-type FMDV VP1 protein-ferritin fusion protein (the insertion site of SeFnt16798 in PUC57 is Bam HI and xho Between I) was constructed and synthesized by Nanjing GenScript. SeFnt16798 represents the O-type FMDV VP1 protein epitope-ferritin fragment. O-type FMDV VP1 protein epitope is based on the amino acid of the 1D protein of the FMDV O / MYA / 7 / 98 strain, and its epitope is subjected to the sequence obtained by codon optimization of Escherichia coli expression, the amino acid is shown in SEQ ID NO.10, expressed The nucleotide sequence of the O-type FMDV VP1 protein epitope is shown in SE...

Embodiment 2

[0049] 1. Materials and methods

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

[0051] SeFnt16798 (abbreviated as pET21b1-pET21b8), and 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.

[0052] References for the construction process of pET21b1-pET21b8 (Guo Yukun, Ming Shengli, Guo Wanying, et al. Soluble expression, purification and nano-like structure observation of O-type foot-and-mouth disease virus VP1 protein in Escherichia coli[J]. Acta Anatomy, 2018, v .49(01):121-126.), wherein SeFnt16798 represents the O-type FMDV VP1 protein epitope-ferritin fragment. The O-type FMDV VP1 protein epi...

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Abstract

The invention discloses an O-type FMDV VP1 protein-ferritin fusion protein, a protein cage nanoparticle and a preparation method of the O-type FMDV VP1 protein-ferritin fusion protein. According to the invention, nucleotide sequences containing O-type FMDV dominant epitope and ferritin fragments are connected in series, and the O-type FMDV VP1 protein epitope-ferritin fragment is designed and synthesized; and connecting with a solubility-enhancing tag and an affinity tag EW29 is carried out to obtain an EW29 / solubility-enhancing tag / O-type FMDV VP1 protein epitope-ferritin fragment, and induction and affinity purification are carried out to obtain the O-type FMDV VP1 protein-ferritin fusion protein. It is shown by electron microscope results that the fusion protein forms protein cage nano-particles with the particle size of 20-25 nm. The preparation method lays a foundation for further developing a safe and effective O-type FMDV VP1 protein vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an O-type FMDV VP1 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 (glyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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 郭玉堃马英先王梦珂明胜利郭豫杰杨国宇
Owner HENAN AGRICULTURAL UNIVERSITY