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Monoclonal antibody for identifying EB virus gH glycoprotein, and application of monoclonal antibody

A monoclonal antibody and antigen technology, applied in the direction of antiviral immunoglobulin, antibody, antiviral agent, etc., can solve the problems of monoclonal antibody marketing and other issues

Active Publication Date: 2021-09-10
SUN YAT SEN UNIV CANCER CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still no monoclonal antibody against the EBV envelope glycoprotein on the market

Method used

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  • Monoclonal antibody for identifying EB virus gH glycoprotein, and application of monoclonal antibody
  • Monoclonal antibody for identifying EB virus gH glycoprotein, and application of monoclonal antibody
  • Monoclonal antibody for identifying EB virus gH glycoprotein, and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] The preparation of embodiment 1 anti-Epstein-Barr virus gLgH protein monoclonal antibody (monoclonal antibody)

[0073] (1) Preparation of protein antigen: refer to the complete gene sequence of Epstein-Barr virus M81 strain (KF373730.1) and connect the C-terminus of the gL protein ectoregion sequence (corresponding to virus BKRF2 gene aa24-aa137) to the gH protein ectoregion through a flexible amino acid sequence Sequence (corresponding to the virus BXLF2 gene aa19-aa678), the N-terminal of the gL protein is connected to the signal peptide coding sequence, and the C-terminal of the gH protein is connected to the polyhistidine polypeptide (6×His) that is convenient for affinity chromatography purification. The above sequence is constructed into a suitable eukaryotic expression vector, and the successfully constructed recombinant plasmid is transfected into 293F cells for expression and purified to finally obtain gLgH protein (the amino acid sequence of gLgH protein is sh...

Embodiment 2

[0080] Example 2 Isolation and sequence analysis of the monoclonal antibody 6H2 light chain gene and heavy chain gene against Epstein-Barr virus gLgH protein

[0081] Semi-adherent culture about 10 7Blow up the adherent cells to make them suspend, transfer the suspension to a new 4mL centrifuge tube, centrifuge at 1500rpm for 3min, collect the cell pellet, resuspend in 100μL sterile PBS, and transfer to a new 1.5mL centrifuge tube Add 800uL Trizol (Roche, Germany), gently invert and mix, let stand for 10min; add 200μL chloroform, shake vigorously for 15s, let stand for 10min, centrifuge at 12000rpm at 4°C for 15min, transfer the upper layer to a new 1.5mL centrifuge tube , add an equal volume of isopropanol, mix well, let stand for 10 minutes; centrifuge at 12,000 rpm at 4°C for 10 minutes, discard the supernatant, add 600 μL of 75% ethanol to wash, centrifuge at 12,000 rpm at 4°C for 5 minutes, discard the supernatant, and dry the precipitate at 60°C for 5 minutes ; The tran...

Embodiment 3

[0083] Example 3 Comparison of EC50 of neutralizing antibody 6H2 and other antibodies AMMO1, M3, 1D8

[0084] The gLgH protein was washed with CB buffer (NaHCO 3 / Na 2 CO 3 buffer, the final concentration is 50mM, the pH value is 9.6) and the final concentration is 2 μg / mL; add 100 μL of coating solution to each well of a 96-well microplate plate, and coat at 37 ° C for 2 hours; wash with PBST ( 20mM PBS7.4, 150mM NaCl, 0.1% Tween20) to wash once; then add 200μL blocking solution (20mM NaCl with pH value of 7.4 containing 20% ​​calf serum and 1% casein) 2 HPO 4 / NaH 2 PO 4 buffer solution), placed at 37°C for 2 hours; discard the blocking solution; dry and store in an aluminum foil bag at 2-8°C for later use. Monoclonal antibodies 6H2, AMMO1 (references for the preparation method of AMMO1 Snijder et al., 2018, Immunity 48,799–811; PDB of AMMO1 light chain: 6C5V_L; PDB of AMMO1 light chain: 6C5V_H), M3 (already published in patent document CN111548411A Disclosed in), 1D8...

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Abstract

The invention belongs to the technical field of antibodies, and discloses a monoclonal antibody for recognizing EB (Epstein-Barr) virus gH glycoprotein, and application of the monoclonal antibody. The monoclonal antibody or the antigen binding fragment thereof is subjected to specific binding with the 573th, 625th, 627th and 655th amino acids of the gH protein, wherein the amino acid sequence of the gH protein is as shown in SEQ ID NO. 11. The binding activity of the monoclonal antibody or the antigen binding fragment thereof is superior to the binding activity of other monoclonal antibodies (AMMO1, M3 and 1D8); affinity to the gLgH protein is high; strong neutralizing activity is realized in all epithelial cell infection models; an obvious inhibition effect for cell membrane fusion is achieved; and a way that the monoclonal antibody or the antigen binding fragment thereof is subjected to the specific binding with the 573th, 625th, 627th and 655th amino acids of the gH protein is different from other reported gLgH neutralizing antibody recognition and binding epitopes.

Description

technical field [0001] The invention belongs to the technical field of antibodies, in particular to a monoclonal antibody for recognizing gH glycoprotein of Epstein-Barr virus and its application. Background technique [0002] Epstein-Barr virus (EBV) was first successfully cultured and established from Burkitt lymphoma cells by Epstein and Barr in 1964. EBV belongs to the gamma subtype herpes virus and is the first human cancer-causing virus discovered. EBV infection is very common in the population, it is reported that more than 95% of adults in the world carry EBV. In children and adolescents, EBV infection frequently causes infectious mononucleosis. EBV latent infection is related to the occurrence of various human lymphoid tumors and epithelial tumors, such as Hodgkin's lymphoma, Burkitt's lymphoma and NK / T cell lymphoma, etc. Epithelial tumors include nasopharyngeal carcinoma and about 10% of gastric cancer, etc. . For organ transplant patients and immunosuppressed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08A61K39/42A61P31/22G01N33/577G01N33/569
CPCC07K16/085A61P31/22G01N33/577G01N33/56994C07K2317/565C07K2317/56C07K2317/92C07K2317/76C07K2317/24G01N2333/05G01N2469/10
Inventor 张晓陈毅歆洪俊平徐淼吴倩钟玲曾益新夏宁邵
Owner SUN YAT SEN UNIV CANCER CENT
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