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Anti-filovirus monoclonal neutralizing antibody as well as preparation method and application thereof

A filovirus, monoclonal technology, applied in chemical instruments and methods, antiviral immunoglobulin, botanical equipment and methods, etc., to achieve the effect of high homology and good neutralizing activity

Active Publication Date: 2019-12-10
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the deficiencies of the prior art, the present invention provides anti-filovirus monoclonal neutralizing antibody and its preparation method and application. The monoclonal neutralizing antibody is prepared based on a rhesus monkey experimental technology platform, which solves the problem of filovirus antibody in practice. Diagnostic and therapeutic issues, providing new options for establishing filovirus detection, diagnosis, prevention and treatment

Method used

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  • Anti-filovirus monoclonal neutralizing antibody as well as preparation method and application thereof
  • Anti-filovirus monoclonal neutralizing antibody as well as preparation method and application thereof
  • Anti-filovirus monoclonal neutralizing antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0096] Example 2 Marking and sorting of antigen-specific B cells

[0097] The obtained rhesus monkey PBMC was labeled with antibody labeling technology, CD3 antibody, CD20 antibody, CD27 antibody, IgG antibody and anti-histidine antibody, and a single GP protein-specific B cell was sorted by flow cytometry. Proceed as follows:

[0098] (1) Centrifuge the prepared PBMCs to discard the incubation supernatant, add 1 mL of PBS buffer, centrifuge at 400×g for 5 min, and discard the supernatant;

[0099] (2) First add 1 μL Aqua, incubate at 4°C for 20 minutes, then add 1 mL PBS, centrifuge at 400×g for 5 minutes, and discard the supernatant;

[0100] (3) Add the fluorescently labeled antibodies shown in Table 1, stain at 4°C for 30 minutes, add 1 mL of PBS after staining, centrifuge at 400×g for 5 minutes, and finally add 300 μL of PBS to resuspend, incubate at 4°C in a refrigerator, and perform sorting on the machine.

[0101] Table 1 Antibody labeling system

[0102]

[0103...

Embodiment 3

[0104] Example 3 Isolation of Antibody Variable Region Genes from Single B Cells Using RT-PCR

[0105] The reverse transcription kit produced by Promega was used to prepare the cell lysate, which was divided into 96-well plates, and the cells were centrifuged. After adding the reverse transcription reagent, they were reversed into cDNA. After the reverse transcription was completed, they were stored at -80°C;

[0106] Using rhesus monkey-specific primers to amplify the heavy and light chain variable region genes of the antibody through nested PCR using B cell cDNA as a template, the 50 μL system contains 5 μL cDNA, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM specific primers, Carry out PCR amplification according to the following conditions: pre-denaturation at 94°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 50s, 35 cycles; 72°C for 7min; the obtained PCR products were identified by 1% agarose gel electrophoresis, recombinant antibodies The electrophoresis results of 7D10 he...

Embodiment 4

[0108] Embodiment 4 constructs the expression vector of monoclonal neutralizing antibody

[0109] Use homologous recombination primers to add homologous recombination arms at the two ends of the antibody heavy chain variable region gene and the two ends of the light variable region gene, and use double enzymes to linearize the expression plasmid containing the human antibody heavy and light chain IgG1 constant regions Homologous recombination arm is generated; the variable region gene fragment added to the homologous recombination arm and the linearized plasmid are connected by homologous recombination to form a complete expression vector, in which the recombinant antibody heavy chain humanized plasmid The map is shown in Figure 4 (A) or Figure 4 (C), and the light chain humanized plasmid map is shown in Figure 4 (B) or Figure 4 (D). The recombinant product was transformed into TOP10 Escherichia coli competent, Amplify the plasmid.

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Abstract

The invention provides an anti-filovirus monoclonal neutralizing antibody as well as a preparation method and application thereof. A heavy chain variable region of an antigen binding segment of the monoclonal neutralizing antibody comprises amino acid sequences of SEQ ID NO:13 or SEQ ID NO:15 shown in the specification, and a light chain variable region of the antigen binding segment of the monoclonal neutralizing antibody comprises amino acid sequences of SEQ ID NO:14 or SEQ ID NO:16 shown in the specification. The antigen binding segment of the monoclonal neutralizing antibody is obtained from adult Chinese rhesus monkeys, after humanization, the antibody has good neutralizing activity upon three pseudoviruses of a Zaire type, a Sultan type and a Marburg type of filovirus, and has good affinity upon GP (glycoprotein); and the monoclonal neutralizing antibody is potentially applied to research on anti-filovirus treatment medicines, research on anti-filovirus detection kits, molecularbiological reagents for anti-filovirus antigen detection, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an anti-filovirus monoclonal neutralizing antibody and a preparation method and application thereof. Background technique [0002] Filovirus is a single-stranded negative-sense RNA virus, which is a pathogen with a very high lethality that infects primates including humans. It can cause severe hemorrhagic fever to infected primates, mainly divided into Ebola Laviruses, Marburgviruses, and Cuevaviruses. Among them, the Ebola virus genus includes five species: Zaire type (ZEBOV), Sudan type (SUDV), Reston type (RESTV), Tai Forest type (TAFV) and Bundibugyo type (BDBV). The average fatality rate of Ebola virus is about 50%. The incubation period from infection to onset of Ebola virus is 2 to 21 days. After infection with Ebola virus, the initial symptoms are fatigue, fever, muscle pain, headache and sore throat. Pain, followed by vomiting, diarrhea, rash, impaired kidney and liver funct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10G01N33/569
CPCC07K16/10G01N33/56983C07K2317/56Y02A50/30
Inventor 陈凌冯玉鹏冯立强王龙雨
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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