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Influenza virus ha protein stem specific monoclonal antibody and its preparation method and application

A monoclonal antibody, influenza virus technology, applied in antiviral immunoglobulin, virus/phage, botanical equipment and methods, etc., can solve problems such as difficulty and few antigenic epitopes

Active Publication Date: 2022-04-19
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the HA stem contains fewer epitopes, and most of them are not immunodominant epitopes, so it is difficult to obtain HA stem-specific monoclonal antibodies

Method used

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  • Influenza virus ha protein stem specific monoclonal antibody and its preparation method and application
  • Influenza virus ha protein stem specific monoclonal antibody and its preparation method and application
  • Influenza virus ha protein stem specific monoclonal antibody and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Preparation of HA protein-specific monoclonal antibody

[0046] 1. Expression of recombinant H7N9 HA protein

[0047] 1.1 Construction of recombinant baculovirus expressing H7N9 HA gene

[0048] PCR amplified the extracellular region of the H7N9 (A / chicken / Guangdong / GD15 / 2016, GD15) HA gene, added 6×His tags at the C-terminus, added NotI and XbaI restriction sites at both ends, and ligated to the transfer vector In pVL1393, a transfer plasmid (pVL-GDHA) was constructed. After the intermediate transfer plasmid was digested and verified by sequencing, it was co-transfected with the linearized genomic DNA of Autographa californica polynuclear polyhedrosis virus into insect cells sf9 to rescue the recombinant baculovirus.

[0049] The brief procedure for virus rescue is:

[0050] (1) Preparation of transfection complex: In a centrifuge tube, add 0.5mL Sf900 III SFM serum-free medium and 5μL DNA shuttle transfection reagent, mix well; in another centrifuge tube, ...

Embodiment 2

[0066] Example 2. Characterization of Monoclonal Antibodies

[0067] 1. Affinity determination of monoclonal antibody and HA protein

[0068] Dilute the purified monoclonal antibody (500, 1000, 5000, 10000, 100000, 1000000 times), and use the ELISA method to detect the reactivity of antibodies with different dilutions and HA protein. The brief process is as follows:

[0069] (1) Coat the ELISA plate with purified HA protein (0.25 μg / mL) and incubate overnight at 4°C.

[0070] (2) Pour off the coating solution, add 200 μL of PBST containing 5% skim milk to each well, and block at 37° C. for 1 hour.

[0071] (3) Wash 5 times with PBST, add 100 μL of antibody diluted in each well, and incubate at 37°C for 1 hour.

[0072] (4) Wash 5 times with PBST, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:2000) to each well, and incubate at 37°C for 1 hour.

[0073] (5) Wash 5 times with PBST, add 100 μL TMB substrate to each well, and develop color for 10 min at ro...

Embodiment 3

[0081] Example 3. Epitope Identification of Monoclonal Antibodies

[0082] The epitope recognized by 5D3 1B5 was screened with a phage surface display random polypeptide library, and the library used was ph.D. TM -12Phage Display Peptide Library (NEB), according to the instructions of the kit. The DNA of the positive phage clone was extracted, sequenced with primer-96gIII (5'-CCCTCATAGTTAGCGTAACG-3'), and the sequence of 12 peptides inserted in the phage was deduced. By comparing with the HA gene sequence, the matching site was located, and the mimic epitope recognized by the antibody was determined.

[0083] Image 6 For the identification of the epitope recognized by the monoclonal antibody, the results showed that 10 positive phage clones were selected for sequencing, and the deduced analog polypeptide was AWQYSLTLPDVL, which was completely consistent with the 431st W, 433rd Y and 437th L of the HA protein, suggesting that These three amino acids are the core motif of th...

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Abstract

The invention belongs to the field of biotechnology, and relates to a monoclonal antibody for recognizing the HA stem of influenza virus, an antigenic epitope, a coding gene and applications thereof. The present invention provides an influenza virus HA stalk-specific monoclonal antibody, including a heavy chain variable region and a light chain variable region, and the nucleotide and amino acid sequences of the heavy chain variable region are respectively shown in SEQ ID NO.1‑2 shown; the nucleotide and amino acid sequences of the light chain variable region are shown in SEQ ID NO.6-7 respectively. The monoclonal antibody specifically recognizes the epitope of the stem of the HA protein with the 431st W, 433rd Y, and 437th L as core motifs. The monoclonal antibody is obtained by immunizing mice with H7N9 subtype avian influenza virus HA protein as an antigen. It has high affinity and strong sensitivity, and has good cross-reactivity with group 1 and group 2 influenza viruses. It has high application value in scientific detection.

Description

technical field [0001] The invention relates to an influenza virus HA stem-specific monoclonal antibody and a preparation method and application thereof, belonging to the field of biotechnology. Background technique [0002] Type A influenza virus (hereinafter referred to as influenza virus) is an important pathogen causing human and animal influenza. Influenza viruses can be divided into two groups (group 1 and group 2), which contain many subtypes. According to the antigenic differences of the glycoproteins hemagglutinin (HA) and neuraminidase (NA) on the surface of the viral envelope, it is divided into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11). Influenza virus is a single-stranded positive-sense RNA virus with a genome divided into 8 segments. Therefore, during the infection process of influenza virus, gene reassortment between different subtypes of viruses occurs frequently, and under the immune pressure of the body, the HA protein of the virus will undergo ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C07K14/11C12N5/20C12N15/13G01N33/577G01N33/569
CPCC07K16/1018C07K14/005G01N33/577G01N33/56983C07K2317/56C07K2317/565C07K2317/34C07K2317/33C07K2317/92C12N2760/16122G01N2333/11G01N2469/10
Inventor 胡增垒赵江艳石磊刘秀梵
Owner YANGZHOU UNIV
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