Anti-Ebola virus vp40 protein monoclonal antibody g7a6 and its application

A monoclonal antibody, Ebola virus technology, applied in antiviral immunoglobulin, instrument, biological material analysis, etc., to achieve significant antiviral effect

Active Publication Date: 2020-06-23
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 g7a6 and its application
  • Anti-Ebola virus vp40 protein monoclonal antibody g7a6 and its application
  • Anti-Ebola virus vp40 protein monoclonal antibody g7a6 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation of monoclonal antibody G7A6 against Ebola VP40 protein

[0029] (1) Immunization of mice: For the first immunization, 14 peptides of Ebola VP40 protein cross-linked to KLH (CTGKKVTSKNGQPI) and QuickAntibody-mouse5W were mixed 1:1 by volume, and the total volume was 100ul. Each BALB / C mouse 0.1ml (Ebola VP40 protein antigen peptide 100ug), intramuscular injection of the inner thigh. On the 21st day, boost the immunization in the same way. On the 35th day, a small amount of tail blood was collected for ELISA assay, and the antibody titer reached 1:32000. Immediately, the tail vein was injected to boost the immunization once, and cell fusion was carried out after 3 days.

[0030] (2) Culture of mouse myeloma cell SP2 / 0: The SP2 / 0 myeloma cell line from BALB / C mice was cultured and passaged in a medium containing 10% FBS-DMEM, and was cultured in a medium containing 5% CO. 2 Culture in a humidified 37°C incubator. Passage the day before fusion to e...

Embodiment 2

[0040] Example 2. Antiviral effect of monoclonal antibody G7A6 against Zaire Ebola VP40 protein, combined application of G7A6 with anti-GP monoclonal antibody (ZJEB8-01) and other anti-VP40 monoclonal antibodies (A2G7, F1B4) Research on viral effects

[0041] This experiment is realized by the following ways:

[0042] (1) Construction of Zaire Ebola virus-like particles (ZEBOV-trVLPs) in vitro replication model ( image 3 ). ZEBOV-trVLPs can simulate ZEBOV to synthesize miniature filovirus-like particles, which have basic functions such as invasion and replication, but are not biohazardous. Research is important.

[0043] (2) The trVLPs particles were collected by ultracentrifugation, and incubated with trVLPs (MIO=3) in a dose gradient of G7A6 monoclonal antibody (3, 5, 10, 15 μg / ml) for 1 h in vitro, and then added to a 293T cell culture plate (24-well). Plate); set the trVLPs without antibody as the control group. After 48 hours of incubation, the supernatant was disca...

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PUM

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Abstract

The invention discloses an ebola virus resisting VP40 protein monoclonal antibody G7A6 and application thereof. The subtype of the monoclonal antibody is an IgG1 and kappa type and the monoclonal antibody can be specifically combined with an N-terminal antigen peptide of ZEBOV-VP40 protein to express a virus resisting effect; a hybridoma cell for producing the monoclonal antibody is a hybridoma cell which is obtained by carrying out fusing, screening, cloning and passage flow screening on an immunized BALB / C mouse spleen lymphocyte and a mouse myeloma cell SP2 / 0, and can stably secrete G7A6; aheavy chain amino acid sequence of the antibody is shown as SEQ ID No. 1 and a light chain amino acid sequence of the antibody is shown as SEQ ID No. 2. The G7A6 has the virus resisting effect of antagonizing Zaire type ebola virus-like particles and can be combined with other single antibodies to use.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the preparation and application of an anti-Zaire Ebola virus VP40 protein monoclonal antibody. , through the induction of ascites in mice of the same strain, the monoclonal antibody G7A6 against VP40 protein was prepared, identified as IgG1, κ type, the sequence of the antibody was clarified by gene sequencing, and then achieved by affinity purification, electrophoresis, immunization, genetic engineering and other technologies application of this antibody. Background technique [0002] Ebola virus disease is a fatal hemorrhagic fever caused by Ebola virus infection. In 1976, Ebola virus was first discovered in southern Sudan and Congo (Kinshasa) (formerly known as Zaire), and then small-scale outbreaks occurred in Central Africa. As of 2013, a total of 2,387 people were sickened, 1,310 died, and the case fatality rate 54.9%. In 2014, the Ebola virus broke out again, with the Zaire Eb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10G01N33/569
CPCC07K16/10G01N33/56983G01N2333/08
Inventor 姚航平余东山李兰娟
Owner ZHEJIANG UNIV
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