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Monoclonal antibody 3-3E for human adenovirus type 7 and application of monoclonal antibody 3-3E

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

Active Publication Date: 2019-06-18
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 3-3E for human adenovirus type 7 and application of monoclonal antibody 3-3E
  • Monoclonal antibody 3-3E for human adenovirus type 7 and application of monoclonal antibody 3-3E
  • Monoclonal antibody 3-3E for human adenovirus type 7 and application of monoclonal antibody 3-3E

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, the discovery of antibody

[0071] The peripheral blood of convalescent DENV-1 infected patients was collected, and peripheral blood mononuclear lymphocytes were separated and collected. Collected peripheral blood mononuclear lymphocytes were added with anti-cell surface marker antibodies (BD, #555332, #555415, #555441, #560677, #555622) or control antibodies (BD, #555748, #555742, #555751, # 555749, #557872) were labeled and flow sorted, and the sorted cells were used for antibody gene amplification. Using the isolated single B cell as a template, the antibody gene was amplified, and the amplified positive clone was sequenced and expressed, and the purified sample of adenovirus type 7 was used as the antigen. After a large number of screening, analysis, and verification, an antibody sequence was obtained, named as 3-3E antibody.

[0072] The amino acid sequence of the heavy chain variable region of the 3-3E antibody is shown in Sequence 2 of the Sequenc...

Embodiment 2、3-3

[0074] The preparation of embodiment 2, 3-3E antibody

[0075] 1. Construction of recombinant plasmids

[0076] 1. Replace the small fragment between the SalI and PmlI sites of the pTSE-G1n 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 (Already sequenced and verified).

[0077] 2. Replace the small fragment between the SalI and PmlI sites of the pTSE-K vector (Beijing Baite 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 (Already sequenced and verified).

[0078] 2. Preparation of 3-3E antibody

[0079] 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% C...

Embodiment 3、3-3

[0084] Example 3, the binding ability detection of 3-3E antibody

[0085] 1. Preparation of human adenovirus type 7 stock solution, virus concentrate and inactivated virus

[0086] 1. Preparation of adenovirus liquid

[0087] 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 an i...

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Abstract

The invention discloses a monoclonal antibody 3-3E for a human adenovirus type 7 and an application of the monoclonal antibody 3-3E. The monoclonal antibody comprises a heavy chain variable region anda light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions HCDR1, HCDR2 and HCDR3; the light chain variable region comprises three complementary determining regions LCDR1, LCDR2 and LCDR3; HCDR1, HCDR2 and HCDR3 are sequentially represented as 26-33-posiition, 51-58-poisiton and 97-110-position from the N end of a sequence 2 in a sequence table; LCDR1, LCDR2 and LCDR3 are sequentially represented as 27-32-posiition, 50-52-poisiton and 89-96-position from the N end of a sequence 4 in a sequence table. On the basis of the clinical demand, the antibody 3-3E resistant to the human adenovirus type 7 is discovered, can be used for preventing and treating adenovirus infection and has great biological and medical significance.

Description

technical field [0001] The invention relates to a human type 7 adenovirus monoclonal antibody 3-3E 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 80-110nm. [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 r...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13A61K39/42A61P31/20
CPCA61K39/42A61P31/20C07K16/08
Inventor 杨志新王荣陆健昇余云舟周权周晓巍
Owner ACADEMY OF MILITARY MEDICAL SCI
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