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Neutralizing antibody against Junin virus, preparation method thereof and application thereof

A virus antigen and antibody technology, applied in the field of biomedicine

Active Publication Date: 2019-09-06
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although a live virus vaccine (Candid 1) has been approved in Argentina to prevent JUNV infection, there is currently no targeted drug for the clinical treatment of Argentine hemorrhagic fever

Method used

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  • Neutralizing antibody against Junin virus, preparation method thereof and application thereof
  • Neutralizing antibody against Junin virus, preparation method thereof and application thereof
  • Neutralizing antibody against Junin virus, preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This example provides a method for preparing a neutralizing antibody against Junin virus.

[0036] 1. Construction of expression vector

[0037] In this example, the full-length gene (GPC) of the envelope glycoprotein of Junin virus was obtained by gene synthesis, and the sequence of GPC is shown in SEQ ID No.1. The GPC was constructed into the multiple cloning region of the eukaryotic expression vector pCAGGS, and the expression plasmid pCAGGS-GPC containing GPC was obtained.

[0038] 2. Junin virus antigen preparation

[0039] 2.1. Fake virus packaging:

[0040] Transfect the expression plasmid pCAGGS-GPC constructed in step 1 into HEK293T cells (host cells), express for 24 hours, infect the cells with vesicular stomatitis virus pseudovirus VSVΔG-GFP / G, continue to culture for 36 hours, and collect the cells for culture supernatant to obtain virus fluid, which contains a large number of virus particles.

[0041] 2.2. Antigen purification:

[0042] Add 20% sucrose s...

Embodiment 2

[0052] The activity of the neutralizing antibody obtained in Example 1 was verified.

[0053] Dilute the GP1-specific antibody or GP1-specific antibody fragment obtained in Example 1 with serum-free medium, dilute 9-15 gradients starting from 200 μg / ml, and then mix it with Junin pseudovirus at 37°C Incubate for 1h.

[0054] Then the mixture of antibody and virus was added to the surface of Vero-E6 cells, infected at 37°C for 1 hour, and blank and virus were used as negative and positive controls, respectively. Subsequently, after the infection was fully removed, the cells were continued to be cultured for 24 hours, and the fluorescence value of each cell well was quantitatively detected by a luciferase activity detection kit.

[0055] The activity of the antibody was evaluated by calculating the inhibition rate of the antibody against virus infection. Through this scheme, the EC50 of polyclonal antibody, polyclonal antibody fragment, GP1-specific antibody and GP1-specific an...

Embodiment 3

[0057] It was verified that the antibody provided in Example 1 has a broad-spectrum neutralizing effect on the highly pathogenic New World arenavirus.

[0058] The neutralizing antibodies provided in Example 1 were used against Junin virus (JUNV), Tsushima Qiubo virus (Machupo virus, MACV), Guanarito virus (Guanarito virus, GTOV), Sabia virus ( Sabiavirus, SABV) and Chapare virus (Chapare virus, CHPV) antiserum inhibition experiment. Dilute the polyantiserum obtained in Example 1 with serum-free medium, dilute 9 gradients from 1: (20-5120), and then mix with JUNV, MACV, GTOV, SABV or CHPV pseudovirus respectively, 37 Incubate at ℃ for 1h.

[0059] Then the mixture of antiserum and virus was added to the surface of Vero-E6 cells, infected at 37°C for 1 hour, and blank and virus were used as negative and positive controls, respectively. Subsequently, after the infection was fully removed, the cells were continued to be cultured for 24 hours, and the fluorescence value of each ...

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Abstract

The invention provides a neutralizing antibody against Junin virus, a preparation method thereof and an application thereof, which relate to the technical field of bio-medicine. The method comprises the following steps: immunizing an animal with a prepared Junin virus antigen to obtain a polyclonal antibody, then performing specific purification of the Junin virus envelope glycoprotein subunit 1 on the polyclonal antibody, preparing the antibodies with high neutralizing activity, and obtaining a large amount of horse antiserum products for clinical treatment of Argentine hemorrhagic fever. Thepreparation method and the product prepared therefrom contribute to the emergency treatment of Argentine hemorrhagic fever.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a neutralizing antibody against Junin virus, its preparation method and its application. Background technique [0002] Junin virus (JUNV) is an important member of the Arenaviridae mammalian Arenavirus genus, and is a biosafety level 4 pathogen. It is mainly prevalent in South America and can cause Argentine hemorrhagic fever with a fatality rate of about 30%, second only to severe viruses such as Ebola and Marburg. In recent years, with the rapid circulation of international personnel and materials, JUNV has a tendency to spread to North America, Europe and Asia, posing a threat to the global biosecurity situation. [0003] Similar to Lassa virus of the same family, the JUNV genome contains a large segment L with a length of about 7.2k and a small segment S with a length of about 3.5k. The L segment encodes matrix protein Z and RNA polymerase L, while the S segment encodes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10A61K39/42A61K47/68A61P31/14
CPCA61K2039/505A61K47/6841A61P31/14C07K16/10C07K2317/20C07K2317/76C12N2760/10034
Inventor 潘晓彦吴妍肖庚富
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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