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Human derived anti-EV71 virus neutralizing antibody E1 and application thereof

An antibody and virus technology, used in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of blood-borne disease infection, limitation, and time-consuming

Active Publication Date: 2017-10-20
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] As an antibody component, immunoglobulin (Ig) mainly comes from the immune serum of donors (patients in convalescence), and 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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  • Human derived anti-EV71 virus neutralizing antibody E1 and application thereof
  • Human derived anti-EV71 virus neutralizing antibody E1 and application thereof
  • Human derived anti-EV71 virus neutralizing antibody E1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of Human Anti-EV71 Virus Antibody Library and Screening of Fab Antibody

[0033] 1.1 Materials and methods

[0034]1.1.1 Source of virus, cell and vector: EV71 virus strain FUYANG-0805 has been published in the literature (Yang F, Jin Q, He Y, Li L, Hou Y.2001. The complete genome of Enterovirus 71 China strain. SciChina C Life Sci and Wu Z, Yang F, Zhao R, Zhao L, Guo D, Jin Q.2009.Identification of small interfering RNAs which inhibit the replication of severalEnterovirus 71strains in China.J Virol Methods), by Institute of Pathogen Biology, Chinese Academy of Medical Sciences supply. The cells used for EV71 virus neutralization experiment in vitro were RD cells, which were purchased from ATCC. The strain is XL1-Blue (Stratagene, USA), and the vector pComb3H (provided by Scripps Research Institute, USA).

[0035] 1.1.2 Antigen preparation

[0036] Purification of EV71 virus: Harvest the culture supernatant of RD cells infected with EV71 viru...

Embodiment 2

[0109] Example 2 Application of Human Anti-EV71 Virus Neutralizing Antibody E1

[0110] At present, there are no specific vaccines and drugs for the prevention and treatment of hand, foot and mouth disease. The immunoglobulins used clinically are all derived from the positive serum of human anti-EV71, which limits their large-scale preparation. Susceptible to infection with blood-borne diseases. The human source anti-EV71 virus genetically engineered antibody obtained by the invention is used to replace the blood source immunoglobulin, which provides a new approach for the treatment of hand, foot and mouth disease.

Embodiment 3

[0111] Example 3 The method for preparing full antibody immunoglobulin IgG by using neutralizing antibody E1

[0112] 1.1 Expression and purification of whole antibody IgG

[0113] 1.1.1 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, and use XbaI / HindIII enzyme cut and cloned into the PIgG vector (provided by the Scripps Institute of the United States) (Christoph Rader, Mikhail Popkov, JohnA.Neves, and Carlos F.Barbas III. Integrinαvβ3-targeted therapy for Kaposi.ssarcoma with an in vitro-evolved antibody.The FASEB Journal. (October 18, 2002) 10.1096 / fj.02-0281fje.), the vector PIgG-L was obtained. Then use primers (upstream primer 5'-gagGAGCTCACTCCgaggtgcagctgttggagtctgggggaggcttggtac-3', downstream primer 5'-gagGGGCCCTTGGTGGAGGCTGAGGAGACGGT-3') to amplif...

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Abstract

The invention provides a human derived anti-EV71 virus neutralizing antibody E1 screened by utilizing the phage display technology, which has amino acid sequences of variable regions of light and heavy chains respectively as shown in SEQ ID No.1 and SEQ ID No.2. The antibody can be used for specifically identifying antigen of EV71 virus particles, can generate a remarkable enzyme-linked immunosorbent assay with EV71 virus, and has a neutralizing activity for resisting EV71 virus infection. Furthermore, the antibody can be prepared to form a specific antibody for preventing and treating the hand-foot-and-mouth disease, and can be used for preventing and treating the hand-foot-and-mouth disease caused by EV71 virus in clinical application.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and phage display, in particular to a human neutralizing antibody E1 against EV71 virus and its application. Background technique [0002] Hand foot mouth disease (Hand foot mouth disease, HFMD) is an infectious disease caused by enterovirus, mostly occurs in infants under 5 years old, can cause fever, rashes and ulcers on hands, feet, mouth and other parts, individual patients may Cause myocarditis, pulmonary edema, aseptic meningoencephalitis and other complications. There are more than 20 kinds of enteroviruses that cause HFMD, among which Coxsackievirus A16 (Cox A16) and enterovirus 71 (Enterovirus71.EV71) are the most common. In 1974, Schmidt et al. reported for the first time that EV71 was isolated from patients with neurological symptoms in the California outbreak (1969-1973). Subsequently, many countries in the world reported the prevalence of EV71 virus in different regions....

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/569
CPCA61K2039/505C07K16/1009C07K2317/24C07K2317/55C07K2317/565C07K2317/622C07K2317/76
Inventor 金奇陈哲
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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