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Humanized avian influenza virus H7N9 resisting neutralizing antibody M5 as well as preparation method and application thereof

An avian influenza virus and antibody technology, applied in the direction of antiviral agents, antiviral immunoglobulins, botanical equipment and methods, etc., can solve the problems of restriction, blood-borne disease infection, and time-consuming

Active Publication Date: 2014-09-10
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Immunoglobulin (Ig), as an antibody component, mainly comes from the immune serum of donors (patients in convalescence), it takes a long time from obtaining positive serum to passing safety testing, and requires a lot of manpower and financial resources, which makes it Large-scale production is limited, and because the antibody is mainly derived from serum, it is prone to infection of blood-borne diseases

Method used

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  • Humanized avian influenza virus H7N9 resisting neutralizing antibody M5 as well as preparation method and application thereof
  • Humanized avian influenza virus H7N9 resisting neutralizing antibody M5 as well as preparation method and application thereof
  • Humanized avian influenza virus H7N9 resisting neutralizing antibody M5 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1 Materials and methods

[0033]1.1 Source of virus, cell and vector: H7N9 virus A / Anhui / 1 / 2013 strain has been published in the literature (Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Li X, Yang L, Guo J, Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Zhang Y, Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y.2013.Human infection with a novel avian-origin influenza A(H7N9)virus.N Engl J.Med.368:1888–1897), provided by the Institute of Pathogenic Biology, Chinese Academy of Medical Sciences. The cells used for the in vitro neutralization experiment of H7N9 avian influenza virus were MDCK cells, which were purchased from ATCC. The bacterial strain used to construct the phage antibody library was XL1-Blue (Stratagene, USA), and the vector pComb3H (provided by Scripps Research Institute, USA).

[0034] 1.2 Antigen prepa...

Embodiment 2

[0104] Example 2 Application of Human Anti-H7N9 Avian Influenza Virus Neutralizing Antibody M5

[0105] At present, there are no specific vaccines and drugs for the prevention and treatment of acute respiratory infectious diseases caused by the H7N9 virus. The immunoglobulins used clinically are often derived from the convalescent serum of patients infected with the virus, which limits their mass production. At the same time, because it comes from serum, it is prone to infection of blood-borne diseases. Using the human source anti-H7N9 avian influenza virus genetically engineered antibody M5 obtained by the invention to replace the blood-derived immunoglobulin provides a new approach for the treatment of acute respiratory infectious diseases caused by the H7N9 virus.

Embodiment 3

[0106] Example 3 Method for preparing full antibody immunoglobulin IgG by using neutralizing antibody M5

[0107] 1. Expression and purification of whole antibody IgG

[0108] ① Construction of whole antibody recombinant expression plasmid: first use primers (upstream primer 5'-CCCAAGCTTGTTGCTCTGGATCTCTGGTGCCTACGGGGAAATTGTGTTGACCCAGTCTCC-3', downstream primer 5'-CTAGTCTAGAATTAACACTCTCCCCTG-3') to amplify the light chain segment of FAB antibody, digest with XBAI / HINDIII, Cloned into the PIGG vector (provided by the SCRIPPS Institute of the United States) (CHRISTOPH RADER, MIKHAIL POPKOV, JOHN A.NEVES, AND CARLOS F.BARBAS III. INTEGRINA VΒ3-TARGETED THERAPY FOR KAPOSI.S SARCOMA WITHHAN IN VITRO-EVOLVED ANTIBODY. THE FASEB JOURNAL. (OCTOBER18, 2002) 10.1096 / FJ.02-0281FJE.), to obtain the vector PIGG-L. Then use primers (upstream primer 5'-GAGGAGCTCACTCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTAC-3', downstream primer 5'-GAGGGGCCCTTGGTGGAGGCTGAGGAGACGGT-3') to amplify the heavy chain s...

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Abstract

The invention provides a humanized avian influenza virus H7N9 resisting neutralizing antibody M5 which is obtained through screening by using a phage surface display technology. Amino acid sequences of light-chain and heavy-chain variable regions of the antibody are respectively represented by SEQ ID No. 1 and SEQ ID No. 2. The antibody can specifically identify a particulate antigen of a virus H7N9, can be subjected to remarkable enzyme-linked immunoreaction with the virus H7N9 and has the neutralizing active function of resisting virus H7N9 infection; in addition, the antibody provided by the invention can be prepared into specific antibody drugs for preventing and treating the avian influenza virus H7N9 and sequentially is clinically used for preventing and treating acute respiratory tract infections caused by the virus H7N9.

Description

technical field [0001] The invention relates to genetic engineering and phage surface display technology, in particular to human neutralizing antibody M5 against H7N9 avian influenza virus, its preparation method and application. Background technique [0002] H7N9 avian influenza is an acute respiratory infectious disease caused by H7N9 avian influenza virus. Since February 2013, cases of severe pneumonia of unknown cause have occurred successively in Shanghai, Anhui Province, and Jiangsu Province. Patients generally present with flu-like symptoms, such as fever, cough, and less sputum, which may be accompanied by headache, muscle aches, and general malaise. The condition of severe patients develops rapidly, manifested as severe pneumonia, with body temperature mostly above 39°C, dyspnea, and may be accompanied by hemoptysis and sputum; acute respiratory distress syndrome, mediastinal emphysema, sepsis, shock, consciousness may develop rapidly Obstacles and acute kidney in...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/63A61K39/42A61P31/16G01N33/569
Inventor 陈哲金奇
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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