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Single-domain antibody for neutralizing Xinjiang hemorrhagic fever virus

A technology of hemorrhagic fever virus and single domain antibody, applied in the direction of antibodies, antiviral agents, antibody mimics/scaffolds, etc., can solve the problems that have not yet been achieved, and achieve the effects of low production cost, short cycle and good tissue permeability

Active Publication Date: 2016-12-07
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

Currently, there are no candidate antibody drugs for Xinjiang hemorrhagic fever

Method used

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  • Single-domain antibody for neutralizing Xinjiang hemorrhagic fever virus
  • Single-domain antibody for neutralizing Xinjiang hemorrhagic fever virus
  • Single-domain antibody for neutralizing Xinjiang hemorrhagic fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] [Example 1] Expression and purification of envelope protein of Xinjiang hemorrhagic fever virus

[0040] According to the gene sequence of Xinjiang hemorrhagic fever virus (GenBank No.gi|16271971), its amino acid sequence was analyzed, and the domain III (amino acid 406-521) of its envelope protein Gc was synthesized, and the gene was connected and transformed with the vector pET22b to construct the vector pET22b-Gc Domain III. 100ng of plasmid (pET22b-Gc DomainIII) ​​was transformed into BL21(DE3) competent cells. Re-inoculate the bacteria in LB medium containing 100 μg / ml ampicillin (1L medium contains 10 g tryptone, 5 g yeast extract and 10 g NaCl), and wait until OD 600 When it reached 0.6-1.0, IPTG was added to a final concentration of 0.1 mmol / ml, and expression was induced at 30°C and 220 rpm for 16-20 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), a...

Embodiment 2

[0043] [Example 2] Construction and screening of phage display library

[0044] Using m0 as the backbone, a phage library was constructed according to existing literature (W Chen, et al., Methods Mol Biol, 2009:81-99), and screened with antigens expressed in Escherichia coli. After the purified antigen was incubated overnight at 4°C in a 96-well plate, it was panned with a phage library, and the specific phage was captured by the antigen, washed with PBS+0.05% Tween-20, and after four rounds of screening, the obtained One enriched clone was named XHF4RC8.

Embodiment 3

[0045] [Example 3] Expression and purification of XHF4RC8

[0046] XHF4RC8 was expressed and purified according to the existing literature (Gong R, et al., Methods Mol Biol., 2012). The prokaryotic XHF4RC8 expression vector was constructed and transformed into E.coli HB2151 competent cells. 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), and treat it with polymyxin B (polymyxin B) for 1 hour Collect the supernatant by centrifugation. Purified with Ni-NTA filler and verified its purity by SDS-PAGE. It was then concentrated by ultrafiltration using ...

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Abstract

The invention provides a single-domain antibody XHF4RC8 for neutralizing the Xinjiang hemorrhagic fever virus and application thereof in preparing medicine for preventing or treating Xinjiang hemorrhagic fever virus infection. The amino acid sequence of the single-domain antibody XHF4RC8 is shown as SEQ ID NO: 1, and the nucleotide sequence of a gene for coding XHF4RC8 is shown as SEQ ID NO: 2. The VH structural domain m0 of a human antibody IgG1 serves as the framework of the single-domain antibody. XHF4RC8 can be bound with the structural domain III of Xinjiang hemorrhagic fever virus envelope protein Gc and has the virus neutralizing effect. XHF4RC8 is small in molecular weight and has higher tissue penetration and higher capability for being bound with epitope with the steric effect. XHF4RC8 can be expressed in a prokaryotic expression system and is low in production cost and short in production cycle. XHF4RC8 has the potential function of treating the Xinjiang hemorrhagic fever virus clinically or can be used in combination with other antibodies to achieve an ideal treatment effect.

Description

technical field [0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to an antiviral single-domain antibody directed at the envelope protein of Xinjiang hemorrhagic fever 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. An example is Palivizumab (Synagis) for the treatment of respiratory and packet 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 h...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C07K19/00G01N33/569A61K39/42A61P31/14
CPCA61K39/42C07K16/10C07K2317/569C07K2319/00G01N33/56983G01N2333/08A61K2300/00
Inventor 龚睿张怀东
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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