Monoclonal antibody which binds to pseudorabies virus gD protein and application thereof

A pseudorabies virus and monoclonal antibody technology, applied in antiviral immunoglobulins, antibodies, antiviral agents, etc., can solve the problems that the expected expression effect cannot be verified and cannot be accurately quantified, so as to reduce morbidity and mortality Effect

Active Publication Date: 2019-01-15
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA detection kit containing the above-mentioned antibody overcomes the technical difficulties that the PRVgD protein cannot be accurately quantified and the expected expression effect cannot be verified after the tandem expression or fusion expression of the PRVgD protein and other proteins, and solves the key problem of vaccine preparation

Method used

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  • Monoclonal antibody which binds to pseudorabies virus gD protein and application thereof
  • Monoclonal antibody which binds to pseudorabies virus gD protein and application thereof
  • Monoclonal antibody which binds to pseudorabies virus gD protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1: Preparation and identification of gD protein containing porcine pseudorabies virus.

[0096] 1.1 Preparation of porcine pseudorabies virus gD protein

[0097] Inoculate the PRV HN1201 virus or its cultures of different generations on well-growing PK15 cells (PRV HN1201 strain preservation number is CCTCC NO.V 201311, see patent CN104004774A), and the cultures of different generations are within 5-35 generations Culture, extract PRV genomic DNA and prepare PRVgD protein according to patent CN104004774A, purify through His affinity chromatography and molecular sieve, refer to the BCA protein concentration assay kit method of Beyond Company to measure the content of PRVgD protein (abbreviated as PRVgD1) to 200 μg / ml .

[0098] 1.2 Preparation of tandem expression protein containing PRVgD protein

[0099] According to the patent CN105693827A, the tandem expression protein of PRVgD protein and PRVgB protein fragment (abbreviated as PRVgD2) was prepared respectiv...

Embodiment 2

[0103] Example 2: Preparation, purification and identification of porcine pseudorabies virus gD protein monoclonal antibody.

[0104] 2.1 Preparation and purification of PRVgD protein monoclonal antibody

[0105] First, the PRV HN1201 strain virus fluid (2×10 8 TCID 50 / ml) was inactivated by formaldehyde, and 10 mice were immunized after being emulsified with Freund's adjuvant in an equal volume, immunized once every 2 weeks, and treated with the ELISA method coated with PRVgD1 prepared in Example 1 for 3-7 times of immunization Continuous ELISA testing was performed on the serum of mice from all over the world. The results: the ELISA titers of the serum of mice in each group were all ≤1:320, and one mouse was selected for cell fusion. The positive rate after fusion was 0, so it was discarded.

[0106] Next, 10 mice were immunized with PRVgD1 prepared in Example 1 and Freund's adjuvant in equal volume after emulsification, and immunized once every two weeks. The serum ELIS...

Embodiment 3

[0130] Example 3: Preparation and application of pharmaceutical composition containing mouse monoclonal antibody 5G7

[0131] 3.1 Application of mouse monoclonal antibody 5G7 in the prevention and treatment of porcine pseudorabies

[0132] 3.1.1 Evaluation of the effect of mouse monoclonal antibody 5G7 on the prevention of porcine pseudorabies

[0133] Screen 30 1-3-day-old piglets with double-negative PRV antigen and antibody, and randomly divide them into ①-⑥6 groups, 5 piglets / group; screen 30 pre-delivery sows with PRV antigen and antibody double-negative, and randomly divide them into 6 groups. Among them ①, ③, ⑤, ⑦, ⑨, Six groups of odd groups were intramuscularly injected with 1ml of the mouse monoclonal antibody 5G7 prepared in Example 2, ②, ④, ⑥, ⑧, ⑩, 6 groups of even groups were intramuscularly injected with PBS buffer solution 1ml as a control group. After 24 hours of injection, ①-②, ⑦-⑧ 4 groups of animals were simultaneously inoculated with 1ml of PRVHN1201...

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Abstract

The invention relates to a variable region sequence of a mouse monoclonal antibody which specifically binds to a pseudorabies virus gD protein. The sequence is the variable region sequence of mouse monoclonal antibody 3B6 or the variable region sequence of mouse monoclonal antibody 5G7. The invention also relates to an antibody consisting of a heavy chain variable region sequence or its conservative variant and / or a light chain variable region sequence or its conservative variant of mouse monoclonal antibody 3B6, and an antibody consisting of a heavy chain variable region sequence or its conservative variant and / or a light chain variable region sequence or its conservative variant of mouse monoclonal antibody 5G7. According to the invention, an ELISA detection kit containing the antibody overcomes the technical problem that the PRVgD protein cannot be accurately quantified after tandem expression or fusion expression with other proteins and the expected expression effect cannot be verified, and the key problem of formulating a vaccine is solved. Meanwhile, a pharmaceutical composition containing the antibody has a wide spectrum.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody combined with pseudorabies virus gD protein and an application thereof. Background technique [0002] Pseudorabies, also known as Aujeszky's disease, is caused by porcine herpesvirus type Ⅰ (Suid herpesvirus 1strain, also known as pseudorabies virus Pseudorabies virus, PRV) in the herpesviridae (Herpesviridae) α subfamily. It is an acute infectious disease of a variety of domestic and wild animals such as dogs, cats, rabbits, rats, wild boars, mink, bears, and foxes, with fever, itching (except pigs) and encephalomyelitis as the main symptoms. [0003] Pseudorabies of pigs exists widely in our country and is seriously harmful. It is one of the main diseases restricting the production of large-scale pig farms. It can cause abortion, stillbirth or mummified fetuses in pregnant sows, and neurological symptoms and paralysis in piglets, especially within ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08G01N33/535G01N33/569G01N33/577G01N33/68A61K39/42A61P31/22
CPCA61K2039/505C07K16/085C07K2317/56G01N33/535G01N33/56994G01N33/577G01N33/68
Inventor 田克恭王莹
Owner LUOYANG PULIKE WANTAI BIOTECH
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