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A nanobody that neutralizes Ebola virus

An Ebola virus and nanobody technology, applied in nanotechnology, antibodies, antiviral agents, etc. for materials and surface science, can solve the problems of high production cost and low expression, and achieve low production cost and cycle time. Short, good tissue penetration effect

Active Publication Date: 2019-07-23
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, several candidate antibody drugs against Ebola are all full-length IgG proteins, which need to be expressed in eukaryotic cells, so their expression levels are low and production costs are high

Method used

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  • A nanobody that neutralizes Ebola virus
  • A nanobody that neutralizes Ebola virus
  • A nanobody that neutralizes Ebola virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] [Example 1] Expression and purification of Ebola virus envelope protein

[0042] According to the gene sequence of Ebola virus (GenBank No.AY354458), its amino acid sequence was analyzed, and the gene Gp499 of the extracellular domain (amino acid 33-311, 463-632) of its envelope protein was synthesized, and the gene was connected with the vector pSecTag2A, transformed, and constructed Vector pSecTag2A-Gp449. 1 day before transfection, 293F cells (control cell density was 5-×10 5 cells / ml) 40mL was inoculated in a 125mL suspension cell culture flask. Dilute 40 μg of the plasmid (pSecTag2A-Gp449) in 4 mL of medium and mix gently, then dilute 120 μL of PEI (polyethyleneimine) in the culture medium and mix gently. They were added dropwise to the cells after 20 min incubation at room temperature. The cells were placed in a suspension incubator at 250 rpm at 37°C for suspension culture.

[0043] After 144 hours, the culture supernatant was collected, and the envelope prot...

Embodiment 2

[0045] [Example 2] Construction of phage display library for screening

[0046] Using m01s as the backbone, a phage library was constructed according to existing literature (Gong R, et al., PLoS ONE, 2012), and screened with antigens expressed by mammalian cells. After the purified antigen was incubated overnight at 4°C in a 96-well plate, panned with a phage library, the specific phage was captured by the antigen, washed with PBS+0.05% Tween-20, and after 7 rounds of screening, a The enriched clone was named ebo7. Using ebo7 as the backbone, the gene sequence was randomly mutated, and a specific high-affinity clone was obtained from the mutant, which was named ebo7c2.

Embodiment 3

[0047] [Example 3] Expression and purification of ebo7c2

[0048] The ebo7c2 was expressed and purified according to the existing literature (Gong R, et al., Methods Mol Biol., 2012). The prokaryotic ebo7c2 expression vector was constructed and transformed into E.coli HB2151. Re-inoculate the strains in SB medium containing 100 μg / ml ampicillin (1L medium contains 30g tryptone, 20g yeast extract and 10g MOPS, the pH value is adjusted to 7.0 with NaOH), and the OD 600 When it reaches 0.7-1.0, add IPTG to a final concentration of 200 μg / ml, and induce expression at 37° C. and 220 rpm for 14-16 hours. Collect the bacteria by centrifugation at 4°C, 6000rpm, 15min, discard the medium, resuspend the pellet in Buffer A (50mM Tris-HCL, 450mM NaCL, pH 8.0), treat with polymyxin B (polymyxin B) for 1 hour and then centrifuge Collect the supernatant. Purified with Ni-NTA filler and verified its purity by SDS-PAGE. Then it was concentrated by ultrafiltration with an ultrafiltration ce...

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Abstract

The invention provides a nanometer antibody ebo7c2 for neutralizing Ebola viruses and application of the nanometer antibody to preparing medicines for preventing or treating Ebola virus infection. Amino acid sequences of the nanometer antibody are shown as SEQ ID NO:1, and nucleotide sequences for genes for encoding the nanometer antibody ebo7c2 are shown as SEQ ID NO:2. The nanometer antibody and the application have the advantages that CH2 structural domains m01s of human antibodies IgG1 are used as skeletons of the nanometer antibody, and amino acid of the nanometer antibody is different from amino acid sequences of the CH2 structural domains m01s in three loop zones; the nanometer antibody ebo7c2 can be bound with envelope proteins of the Ebola viruses; the nanometer antibody ebo7c2 has small molecular weights and is excellent in tissue permeability and ability of being bound with antigen epitopes with steric hindrance effects; the nanometer antibody is provided with the m01s skeletons and can be bound with FcRn, and the plasma half-life of the nanometer antibody can possibly reach 10 hours; the nanometer antibody ebo7c2 can be expressed in prokaryotic expression systems and is low in production cost and short in cycle.

Description

technical field [0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to an antiviral nanobody directed against the envelope protein of Ebola virus. Background technique [0002] Therapeutic monoclonal antibody (monoclonal antibody, monoclonal antibody) has become an ideal drug that people expect because it can accurately attack target molecules and has less toxic side effects. At present, monoclonal antibody drugs at home and abroad are mainly used in the treatment of tumors, immune diseases and viral infections. For example Palivizumab (Synagis) for the treatment of respiratory syncytial virus RSV. [0003] The traditional preparation method of monoclonal antibody is hybridoma technology. With the rapid development of genetic engineering technology, therapeutic monoclonal antibodies have evolved from mouse monoclonal antibodies, to chimeric antibodies, humanized antibodies, and to fully human antibodies in recent years, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/569A61K39/42A61P31/14B82Y30/00
CPCA61K39/00B82Y30/00C07K16/10G01N33/56983G01N2333/08
Inventor 龚睿石剑
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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