Preparation and application of mouse monoclonal antibody against noroviruses GII.4

A technology of antibodies and vectors, applied in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of norovirus lack of cell culture models, no small animal models, vaccines and antiviral drug research obstacles, etc.

Active Publication Date: 2016-12-07
中国科学院上海免疫与感染研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 and application of mouse monoclonal antibody against noroviruses GII.4
  • Preparation and application of mouse monoclonal antibody against noroviruses GII.4
  • Preparation and application of mouse monoclonal antibody against noroviruses GII.4

Examples

Experimental program
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preparation example Construction

[0128] Preparation of monoclonal antibodies

[0129] Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, an antigen of the invention may be administered to an animal to induce the production of monoclonal antibodies. For monoclonal antibodies, hybridoma technology can be used to prepare (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol.6:511, 1976; Kohler et al., Eur.J.Immunol. 6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981) or can be prepared by recombinant DNA methods (US Patent No. 4,816,567).

[0130] Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to culture medium (HAT medium matrix), including myeloma cell lines, such as murine Myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (...

Embodiment 1

[0181] Example 1 Screening of hybridoma cells secreting GII.4 specific antibody

[0182] Spleen cells from mice immunized with GII.4 virus-like particles were used to prepare hybridoma cells. The hybridoma cell supernatant was screened by Elisa test, so as to obtain a hybridoma cell line capable of secreting virus-binding ability. Finally, five monoclonal antibodies with excellent binding ability were screened out, all of them could bind GII.4 VLP. Subtype identification showed that G9, D11, 7D8 and 8E1 belonged to IgG1, and 2D8 belonged to IgG3.

[0183] Table 1. Identification of hybridoma cell lines secreting monoclonal antibody

[0184] hybridoma cell line

heavy chain

light chain

Binding ability to GII.4VLP*

G9

IgG1

kappa

+++

D11

IgG1

kappa

+++

2D8

IgG3

kappa

+++

7D8

IgG1

kappa

+++

8E1

IgG1

kappa

+++

[0185] All samples used for analysis were 50ul hybridoma ...

Embodiment 2

[0187] Example 2 Specific Analysis of Anti-GII.4 Monoclonal Antibody

[0188] The purity and integrity of GII.4 mAb purified from ascitic fluid were first identified by SDS-PAGE. figure 1 It shows that the heavy chain and light chain of the five monoclonal antibodies are about 50KD and 25KD respectively. Next, the reactivity of the monoclonal antibody with different antigens, including GI.1 virus-like particles and GII.4 virus-like particles, was detected by Elisa method. figure 2 It shows that G9, D11, 2D8 and 8E1 can specifically recognize GII.4 virus-like particles, and there is no cross-reaction with GI.1 virus-like particles, while some monoclonal antibodies (such as 7D8) ​​can also bind to GI.1 virus-like particles Combined with GII.4 virus-like particles, it cannot specifically recognize GII.4 virus.

[0189] Finally, the binding of monoclonal antibody to GII.4 was analyzed by Western blot, image 3 It shows that among the five antibodies: D11, G9 and 8E1 can recogn...

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Abstract

The invention discloses preparation and application of a mouse monoclonal antibody against noroviruses GII.4. Experimental results indicate that the monoclonal antibody has high neutralizing activity for the noroviruses GII.4, does not generate a cross reaction with GI.1 virus-like particles, and can specifically recognize the GII.4 virus-like particles.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to the preparation and application of an anti-Norovirus GII.4 mouse monoclonal antibody. Background technique [0002] Noroviruses (NoVs) are one of the major pathogens causing sporadic cases and large outbreaks of acute gastroenteritis, and Noroviruses can infect people of all ages. Although the symptoms caused by norovirus infection are generally mild and self-limiting, and the course of the disease lasts for about 1-3 days, it can still cause more serious symptoms and even death in children, the elderly, and people with immunocompromised . According to the amino acid sequence of VP1 capsid protein, Norovirus can be divided into 6 genomes (G1-GVI), but only GI, GII and GIV can infect humans. Human norovirus infection is mainly caused by norovirus GII, and large outbreaks are mostly caused by norovirus GII.4. Norovirus infection caused by variants of the GII.4 v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12P21/08A61K39/42A61P31/14
Inventor 黄忠王晓黎
Owner 中国科学院上海免疫与感染研究所
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