Monoclonal antibody 10G12 and application thereof

A monoclonal antibody, antibody technology, applied in the direction of application, antibody, antiviral agent, etc.

Active Publication Date: 2019-10-01
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no specific therapeutic drug against adenovirus for clinical use in the world, and the research and development of drugs is mostly limited to chemical drugs—nucleoside analogs against DNA viruses, while the research on specific biological drugs for adenovirus is still blank

Method used

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  • Monoclonal antibody 10G12 and application thereof
  • Monoclonal antibody 10G12 and application thereof
  • Monoclonal antibody 10G12 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, the discovery of antibody

[0074] Mice were immunized with inactivated and purified human adenovirus type 7 and a mouse single-chain antibody phage library was constructed. After extensive screening, analysis, and verification, an antibody sequence was obtained, which was named 10G12 antibody.

[0075] The amino acid sequence of the heavy chain variable region of the 10G12 antibody is shown in Sequence 2 of the sequence listing (the 26th-33rd amino acid residues from the N-terminal form CDR1, the 51-66th amino acid residues form CDR2, and the 97th-109th amino acid residues Amino acid residues constitute CDR3), and its coding gene is shown in sequence 1 of the sequence listing.

[0076] The amino acid sequence of the light chain variable region of the 10G12 antibody is shown in Sequence 4 of the Sequence Listing (among them, amino acid residues 27-37 from the N-terminal form CDR1, amino acid residues 55-57 form CDR2, and amino acid residues 94- The 102nd ...

Embodiment 2、10G12

[0077] Example 2, Preparation of 10G12 Antibody

[0078] 1. Construction of recombinant plasmids

[0079] 1. Replace the small fragment between the SalI and AgeI sites of the pMABG1 vector (Beijing Baite Meibo Biological Co., Ltd.) with the DNA molecule shown in Sequence 1 of the sequence listing to obtain a recombinant expression vector containing the heavy chain variable region ( verified by sequencing).

[0080] 2. Replace the small fragment between the SalI and AgeI sites of the pMABKa vector (Beijing Best Meibo Biological Co., Ltd.) with the DNA molecule shown in sequence 3 of the sequence listing to obtain a recombinant expression vector containing the light chain variable region ( verified by sequencing).

[0081] 2. Preparation of 10G12 antibody

[0082] 1. The day before transfection, FreeStyle TM HEK 293-F cells (Invitrogen, product number: R79007) were adjusted to a concentration of 1.0×10 6 / ml, inoculated into culture flasks, 37°C, 5% CO 2 , 125rpm cell sha...

Embodiment 3、10G12

[0087] Example 3, Detection of Binding Ability of 10G12 Antibody

[0088] 1. Preparation of human adenovirus type 7 stock solution, concentrated solution and inactivated virus

[0089] 1. Preparation of adenovirus liquid

[0090] Adenovirus culture: conventional DMEM+10% (volume percentage) FBS medium to culture A549 cells (Beijing Concorde Cell Resource Center, article number: 25), and pass A549 cells to 75cm one day before virus inoculation. 2 In the cell flask, make the cell density reach 75% to 90% when the virus is inoculated the next day; on the day of inoculation, slowly suck out the cell culture medium in the culture flask, add 5ml DMEM to gently wash the cells and discard, and then add 3ml DMEM+2 % (volume percentage content) FBS; use a micropipette to draw an appropriate amount of HAdV7 virus into the cell bottle, and infect according to MOI ≈ 0.001, shake the bottle evenly several times to make the virus evenly dispersed, and place it at 37 ° C, 5% CO 2 Adsorb in ...

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Abstract

The invention discloses a monoclonal antibody 10G12 and application thereof. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chainvariable region comprises three complementary determining regions HCDR1, HCDR2 and HCDR3; the light chain variable region comprises three complementary determining regions LCDR1, LCDR2 and LCDR3; theHCDR1, HCDR2 and HCDR3 are sequentially shown as the positions 26-33, 51-66 and 97-109 from the N end of a sequence 2 in a sequence table; the LCDR1, LCDR2 and LCDR3 are sequentially shown as the positions 27-37, 55-57 and 94-102 of the N end of a sequence 4 in the sequence table. Based on clinical requirements, an inventor of the invention finds that the anti-human adenovirus type 7 antibody 10G12 is used for preventing and treating adenovirus infection and has important biological and medical significance.

Description

technical field [0001] The present invention relates to a monoclonal antibody 10G12 and its application. Background technique [0002] Human adenovirus belongs to the Adenoviridae mammalian adenovirus genus. Human adenovirus was first discovered in 1953. It was isolated and cultured from the atrophic tonsil adenoid tissue of healthy people; the genome of the virus is double-stranded DNA with a total length of about 36kb. These double-stranded DNA and viral structural proteins combine to form the core of the virus. The virus has no envelope, and the core is covered with a capsid. The capsid is composed of 252 capsomers, of which 240 are hexon proteins, 12 penton proteins, and the shell It is a regular 20-hedron structure with a diameter of about 70-90nm. [0003] There are 7 subgroups of adenoviruses found so far, A-G, and 67 different serotypes, of which 55 subtypes can infect humans and cause disease. The most common adenovirus infection is to infect the respiratory tract...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13A61K39/42A61P31/20
CPCA61P31/20C07K16/081C07K2317/24C07K2317/56C07K2317/565C07K2317/76C07K2317/92
Inventor 杨志新陆健昇王荣余云舟黄莺周权周晓巍
Owner ACADEMY OF MILITARY MEDICAL SCI
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