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

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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