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Preparation method and application of GII. 4 type norovirus broad-spectrum monoclonal antibody

A monoclonal antibody, GII.4 technology, applied in virus immunology detection, biomedical field, can solve the problem of no small animal model, norovirus lack of cell culture model, vaccine and antiviral drug research obstacles, etc., to achieve sensitive Highly specific and highly specific effects

Inactive Publication Date: 2019-05-10
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Norovirus lacks a cell culture model, and there is no small animal model, which has brought great obstacles to the research of vaccines and antiviral drugs

Method used

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  • Preparation method and application of GII. 4 type norovirus broad-spectrum monoclonal antibody
  • Preparation method and application of GII. 4 type norovirus broad-spectrum monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0033] A preparation method for GII.4 type norovirus broad-spectrum monoclonal antibody, comprising the steps of:

[0034] 1), the preparation of the immunogen, specifically amplifying or designing and synthesizing the P sequence, constructing the PGEX-4T-1-P recombinant expression vector, using the Escherichia coli expression system to express the NoVGⅡ.4 type P protein, passing through the GST affinity chromatography column Purify and prepare P particles.

[0035] The immunogens prepared in the present invention are GII.4 type NoV P particles, which are respectively GII.4 type NoV96 cluster, 2004 cluster, 2006b cluster and 2010 cluster.

[0036] Specifically, four NoV96 clusters, 2004 clusters, 2006b clusters, and 2010 clusters were used to prepare four GII.4-type NoVP sequences according to the preparation method of the immunogen in step 1) to obtain four NoV96 clusters, 2004 clusters, 2006b clusters, and 2010 clusters. Type 4 P particles.

[0037]The preparation process ...

Embodiment 2

[0072] A kit for detecting NoV antigen or NoV IgG or IgM, the kit contains the anti-Norovirus type GII.4 murine monoclonal antibody prepared by the method in Example 1. The anti-Norovirus GII.4 murine monoclonal antibody can select at least one of the GII.4, GII.3 or GII.17 genotypes.

[0073] Since the monoclonal antibody prepared by the present invention has a high level of binding effect on the three genotypes of NoV GII.4, GII.3, and GII.17, the monoclonal antibody of the present invention or its active fragment or conservative variant , or any combination thereof can be used as a reagent, medicament or kit for preparing and detecting antigens of NoV GII.4, GII.3, and GII.17 genotypes, and can be further used for diagnosing NoV infection.

[0074] The anti-NoV monoclonal antibody of the present invention has strong specificity against NoV, high sensitivity and stability, and is suitable for preparing NoV detection kits.

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Abstract

The invention relates to a preparation method of a GII. 4 type norovirus broad-spectrum monoclonal antibody and application of the GII. 4 type norovirus broad-spectrum monoclonal antibody as an IgG orIgM kit for detecting NoV antigen or NoV, wherein the preparation comprises the following steps of: 1) preparing an immunogen, amplifying or designing and synthesizing a P sequence, constructing a PGEX-4T-1-P recombinant expression vector, expressing NoVGII. 4 type P protein by using an escherichia coli expression system, and purifying by using a GST affinity chromatography column to prepare P particles; 2) animal immunity; 3) preparing and screening hybridoma cell strains, wherein fusing mouse spleen cells and myeloma cells by PEG1500 to prepare hybridoma cells; detecting supernatant of cells by an enzyme-linked immunosorbent assay to screen the GII. 4 type norovirus antibody; 4) cloning of hybridoma cells. The anti-NoV monoclonal antibody prepared by the method has strong specificity toNoV, high sensitivity and stability, and is suitable for preparing a NoV detection kit.

Description

technical field [0001] The invention relates to the technical field of virus immunological detection in the field of biomedicine, in particular to a preparation method and application of a GII.4 norovirus broad-spectrum monoclonal antibody. Background technique [0002] Norovirus (NoV) is the most important pathogen causing viral gastroenteritis. Nearly 90% of viral gastroenteritis epidemics in the world are caused by NoV. In developing countries, there are at least 200,000 five-year-olds every year. The following children died from diarrhea caused by NoV. NoV is a single-stranded positive-sense RNA virus belonging to the family Caliciviridae, which has no envelope and no folding, and the capsid has an icosahedral symmetrical structure. Its whole genome encodes three open reading frames (ORFs), among which ORF2 encodes the main structural protein VP1, which consists of two parts, the S region and the protruding P region. NoV has genetic diversity and is divided into five g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10G01N33/577G01N33/569G01N33/68
Inventor 戴迎春侯玉珍张婷胡贵方张绪富
Owner SOUTHERN MEDICAL UNIVERSITY
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