Anti-Ebola virus vp40 protein monoclonal antibody a2g7 and its application

A monoclonal antibody, Ebola virus technology, applied in the direction of anti-viral immunoglobulin, instruments, biochemical equipment and methods, etc., to achieve a significant anti-viral effect

Active Publication Date: 2020-05-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, VP40 monoclonal antibodies with anti-Ebola virus effects have not been reported

Method used

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  • Anti-Ebola virus vp40 protein monoclonal antibody a2g7 and its application
  • Anti-Ebola virus vp40 protein monoclonal antibody a2g7 and its application
  • Anti-Ebola virus vp40 protein monoclonal antibody a2g7 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Preparation of monoclonal antibody A2G7 against Ebola VP40 protein

[0030] (1) Immunization of mice: For the first immunization, the 15-peptide (ARSNSTIARGGNSNC) of cross-linked KLH Ebola VP40 protein and QuickAntibody-mouse5W were mixed uniformly in a volume ratio of 1:1, with a total volume of 100ul. 0.1ml per BALB / C mouse (Ebola VP40 protein antigen peptide 100ug), intramuscularly injected into the inner thigh. On the 21st day, a dose of booster immunization was given in the same way. On the 35th day, a small amount of tail blood was collected for ELISA determination, and the antibody titer reached 1:32000, and then the tail vein was injected for a booster immunization, and cell fusion was carried out 3 days later.

[0031] (2) Culture of mouse myeloma cell SP2 / 0: The SP2 / 0 myeloma cell line from BALB / C mice was cultured and passaged in 10% FBS-DMEM medium, and in 5% CO 2 cultured in a humidified 37°C incubator. Subculture the day before fusion to ensu...

Embodiment 2

[0041] Example 2. Antiviral effect of monoclonal antibody A2G7 against Zaire-type Ebola VP40 protein, combined application of A2G7 with anti-GP monoclonal antibody (ZJEB8-01) and other anti-VP40 monoclonal antibodies (G7A6, F1B4) Research on Viral Effects

[0042] This experiment is realized through the following ways:

[0043] (1) Construction of Zaire-type Ebola virus-like particles (ZEBOV-trVLPs) in vitro replication model ( image 3 ). ZEBOV-trVLPs can simulate ZEBOV to synthesize mini-filament virus-like particles, which have basic functions such as invasion and replication, but are not biohazardous; the trVLPs system is widely used in the research of simulating ZEBOV, and is useful for screening virus receptors, antiviral drugs, etc. Research matters.

[0044] (2) Collect trVLPs particles by ultracentrifugation, give A2G7 monoclonal antibody dose gradients (3, 5, 10, 15 μg / ml) and incubate with trVLPs (MIO=3) for 1 hour in vitro, then add to 293T cell culture plate (2...

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PUM

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Abstract

The invention discloses an anti-Ebola virus VP40 protein monoclonal antibody A2G7 and an application thereof. The monoclonal antibody subtypes comprise IgG1 and kappa types and can specifically bind to the N-terminal antigen peptide of the ZEBOV-VP40 protein to exert an antiviral effect. The hybridoma cell producing the monoclonal antibody is the hybridoma cell derived from the immunized BALB / C mouse splenic lymphocyte and mouse myeloma cell SP2 / 0 through fusion, screening, cloning and passage after screening and can stably secrete A2G7. The heavy chain amino acid sequence of the antibody is shown in the formula of SEQ ID No. 1, and the light chain amino acid sequence is shown in the formula of SEQ ID No. 2. A2G7 has the antiviral effect of antagonizing Zaire Ebola virus-like particles andcan be combined with other monoclonal antibodies.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation and application of anti-Zaire Ebola virus VP40 protein monoclonal antibody, and is a hybridoma cell line that secretes anti-VP40 protein monoclonal antibody by using cell engineering and antibody engineering technology , through the induction of ascites in mice of the same strain, the monoclonal antibody A2G7 against VP40 protein was prepared, identified as IgG1, κ type, the sequence of the antibody was clarified by gene sequencing, and then realized by affinity purification, electrophoresis, immunization, genetic engineering and other technologies Application of the antibody. Background technique [0002] Ebola virus disease is a fatal hemorrhagic fever caused by Ebola virus infection. In 1976, the Ebola virus was first discovered in southern Sudan and Congo (Kinshasa) (formerly known as Zaire), and then broke out on a small scale in Central Africa. As of 2013, a tota...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N5/20G01N33/577G01N33/68
CPCC07K16/10G01N33/56983G01N33/577G01N2333/08
Inventor 姚航平余东山李兰娟
Owner ZHEJIANG UNIV
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