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Antibody targeting influenza A virus N1 subtype NA protein

A type A influenza virus and antibody technology, applied in the field of cellular immunology and genetic engineering, can solve the problems that it is difficult to quickly develop vaccines and antiviral drugs are not completely reliable

Active Publication Date: 2022-03-01
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, when a pandemic infection occurs, it is difficult to quickly develop a vaccine against the pandemic influenza virus
In addition, antiviral drugs are not entirely reliable due to problems associated with the emergence of resistant mutant viruses

Method used

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  • Antibody targeting influenza A virus N1 subtype NA protein
  • Antibody targeting influenza A virus N1 subtype NA protein
  • Antibody targeting influenza A virus N1 subtype NA protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Screening Anti-NA Protein Antibody

[0062] 1. Construction of phage antibody library

[0063] An antibody library targeting the N1 subtype NA protein of influenza A virus was constructed by means of computer-aided molecular design, and candidate antibodies were screened using phage display technology.

[0064] (1) The displayed phage library adheres to the N1 subtype NA protein of H1N1;

[0065] (2) Repeated washing to remove non-specific binding, eluting and collecting phages with N1 subtype NA protein of H1N1;

[0066] (3) Re-infect Escherichia coli, select a single clone to display on the surface of the phage, and screen positive clones that recognize the N1 subtype NA protein of H1N1 by ELISA.

[0067] 2. Determination and analysis of antibody variable region gene sequence

[0068] Positive clones were sent to Shanghai Sangon Biotechnology Co., Ltd. for antibody variable region gene sequence determination, and the sequencing results were searched and a...

Embodiment 2

[0083] Example 2 Preparation of Monoclonal Antibody FNA1

[0084] 1. Use the PCR method to amplify the heavy chain variable region sequence of the antibody FNA1 (the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.17) and the light chain variable region sequence (the amino acid sequence is shown in SEQ ID NO.8, the nucleotide sequence is shown in SEQ ID NO.18), and the fragment is cloned into the expression vector by the method of molecular cloning.

[0085] 2. Transfect the vector containing both the light chain and heavy chain genes of the monoclonal antibody into mammalian cells for expression.

[0086] 3. Collect the expression supernatant, and use GE's Protein A FF protein column for purification.

[0087] 4. Elute with pH 3.0 citric acid buffer, collect the effluent, and immediately neutralize with 1mol / L pH 8.5 TRIS-HCL buffer, dialyze with pH 7.2, 0.01mol / L PBS for 72h, 0.22μm Sterilize by membrane filtration.

[0088] ...

Embodiment 3

[0090] Example 3 Specific detection of monoclonal antibody FNA1

[0091] 1. Detection of the specificity of the monoclonal antibody FNA1 by ELISA

[0092] 1. Coat 2 μg / ml of NA antigens of influenza A virus H1N1, H3N2, H5N1 and H7N9 on an ELISA plate, overnight at 4°C;

[0093] 2. Block unbound sites with skimmed milk powder, and then wash 5 times with PBS buffer containing 0.1% Tween;

[0094]3. Prepare different concentrations of antibody FNA1, add to the ELISA strip in step 2, incubate at 37°C for 1 hour, and wash 5 times with PBS buffer containing 0.1% Tween;

[0095] 4. Add HRP-labeled goat anti-human antibody, incubate at 37°C for 30 minutes, wash 5 times with PBS buffer containing 0.1% Tween; add TMB to develop color, and detect OD after 1mol / L sulfuric acid terminates 450 value.

[0096] 5. Results

[0097] The result is as figure 2 As shown, the antibody FNA1 can effectively bind the NA antigens of influenza A viruses H1N1 and H5N1 in a dose-dependent manner. ...

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Abstract

The invention discloses an antibody targeting an influenza A virus N1 subtype NA protein. The invention also discloses a coding nucleic acid, a vector and a host cell of the anti-influenza A virus antibody. In addition, the invention also discloses an immunoconjugate and a detection kit for detecting the influenza A virus. The antibody provided by the invention can identify NA antigens of H5N1 and H1N1 N1 subtypes, and has the effect of inhibiting H5N1 pseudovirus release so as to limit intercellular propagation.

Description

technical field [0001] The invention belongs to the fields of cellular immunology and genetic engineering, and relates to an antibody targeting the N1 subtype NA protein of influenza A virus. Background technique [0002] Influenza is an illness caused by influenza viruses that infect the respiratory tract and often occur in winter. Influenza is known to be very contagious and can affect people of all age groups, especially the elderly. Influenza viruses are negative-strand, enveloped RNA (ribonucleic acid) viruses belonging to the Orthomyxoviridae family. Influenza viruses have 8 segments of single-stranded RNA and are classified as A (A), B (B) and C (C). Influenza A viruses are further divided into subtypes based on their major surface proteins hemagglutinin (HA) and neuraminidase (NA). To date, 16 HAs and 9 NAs have been identified (Cheung TK and Poon LL 2007, AnnNY Acad Sci. 1102: 1-25). Influenza viruses have a wide range of infections according to their type (incl...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/16G01N33/569
CPCC07K16/1018A61P31/16G01N33/56983C07K2317/565C07K2317/567C07K2317/56C07K2317/24C07K2317/76G01N2469/10G01N2333/11
Inventor 肖鹤冯健男沈倍奋陈楠陈国江王晶乔春霞李新颖刘成华
Owner ACADEMY OF MILITARY MEDICAL SCI
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