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Humanized neutralizing antibody D5 against avian influenza virus (AIV) H7N9 as well as preparation method and application thereof

A bird flu virus and antibody technology, applied in antiviral agents, antiviral immunoglobulins, botanical equipment and methods, etc., can solve the problems of infection, time-consuming, and limitations of blood-borne diseases

Inactive Publication Date: 2014-09-17
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 neutralizing antibody D5 against avian influenza virus (AIV) H7N9 as well as preparation method and application thereof
  • Humanized neutralizing antibody D5 against avian influenza virus (AIV) H7N9 as well as preparation method and application thereof
  • Humanized neutralizing antibody D5 against avian influenza virus (AIV) H7N9 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Human anti-H7N9 avian influenza virus neutralizing antibody D5 and its preparation

[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 Pathogen 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, U...

Embodiment 2

[0104] Example 2 Application of Human Anti-H7N9 Avian Influenza Virus Neutralizing Antibody D5

[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. The invention provides a new approach for the treatment of acute respiratory infectious diseases caused by the H7N9 virus by using the human source anti-H7N9 avian influenza virus genetically engineered antibody D5 to replace the blood-derived immunoglobulin.

Embodiment 3

[0106] Example 3 Method for preparing full antibody immunoglobulin IgG by using neutralizing antibody D5

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

[0108] ① Construction of whole antibody recombinant expression plasmid: first use primers (upstream primer 5'-CCCAAGCTTGTTGCTCTGGATCTCTGGTGCCTACGGGGACATCCAGATGACCCAGTCTCCATCCTCC-3', downstream primer 5'-CTAGTCTAGAATTAACACTCTCCCCTG-3') to amplify the light chain segment of the Fab antibody, digest with XbaI / HindIII, Cloned into PIgG vector (provided by Scripps Research Institute, USA) (Christoph Rader, Mikhail Popkov, John A.Neves, and Carlos F.Barbas III. Integrinαvβ3-targeted therapy for Kaposi.s sarcoma with an in vitro-evolved antibody. The FASEB Journal .(October18, 2002) 10.1096 / fj.02-0281fje.), the vector PIgG-L was obtained. Then use primers (upstream primer 5'-GAGGAGCTCACTCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGAC-3', downstream primer 5'-GAGGGGCCCTTGGTGGAGGCTGAGGAGACGGT-3') to amplify the heavy chain segment of...

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Abstract

The invention provides a humanized neutralizing antibody D5 against the avian influenza virus (AIV) H7N9. The neutralizing antibody D5 is screened by utilizing the phage surface display technique. The amino acid sequences of light and heavy chain variable regions of the neutralizing antibody D5 are respectively shown in SEQ ID No.1 and SEQ ID No.2. The antibody can specifically recognize the antigens of H7N9 virions, can carry out obvious enzyme-linked immunosorbent assay with the virus H7N9, and has a neutralizing activity function of resisting infection with the virus H7N9. Besides, the antibody can be used for preparing specific antibody drugs for preventing and treating the AIV H7N9, and then the drugs are clinically used for preventing and treating acute respiratory infectious diseases caused by the virus H7N9.

Description

technical field [0001] The present invention relates to genetic engineering and phage surface display technology, in particular to human-source anti-H7N9 avian influenza virus neutralizing antibody D5, 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 acu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21A61K39/42A61P31/16G01N33/569
Inventor 陈哲金奇
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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