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

A pseudorabies virus and monoclonal antibody technology, applied in antiviral immunoglobulin, immunoglobulin, immunoassay, etc., can solve the problems of inaccurate quantification, inability to clearly locate by PCR, inability to track the dynamic distribution of pathogens, etc.

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 monoclonal antibody overcomes the technical problems that the PRVgB protein and other proteins cannot be accurately quantified after tandem expression or fusion expression, and the expected expression effect cannot be verified, and solves the key problem of vaccine preparation
At the same time, the monoclonal antibody can also be used for immunohistochemical detection, which solves the problem that PCR cannot be clearly positioned in tissues and cells and cannot track the dynamic distribution of pathogens in the body

Method used

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

Examples

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Effect test

preparation example Construction

[0078] Step 2) Preparation of paraffin slices: fix with fixative, rinse with running water, then dehydrate, make transparent, soak in wax with an automatic dehydrator, slice, spread, and mount on treated glass slides after embedding with an embedding machine. Obtain paraffin sections after baking;

[0079] Step 3) Inhibit endogenous enzymes, dewax the slides to distilled water, add 3% H 2 o 2 Stand to inhibit endogenous enzymes;

[0080] Step 4) Antigen retrieval Use antigen heat restoration such as high pressure heat restoration, boiling heat restoration, microwave heat restoration, or enzymatic digestion to treat the baked slides to restore antigens.

[0081] Step 5) Blocking is washed 3 times with phosphate buffered saline, and a blocking solution such as horse serum or bovine serum albumin BSA is added dropwise to block;

[0082] Step 6) After staining, remove the blocking solution and add diluted primary antibody, incubate at room temperature for 1 hour or at 37°C for ...

Embodiment 1

[0089] Example 1: Preparation and identification of porcine pseudorabies virus gB protein.

[0090] 1.1 Preparation of porcine pseudorabies virus gB protein

[0091] Inoculate PRV HN1201 virus or its cultures of different generations on well-growing PK15 cells (PRVHN1201 strain preservation number is CCTCC NO.V 201311, see patent CN104004774A), prepare PRVgB protein (abbreviated as PRVgB1) according to patent CN105693827A, and pass His After affinity chromatography and molecular sieve purification, the content of PRVgB1 was determined to be 5 μg / mL according to the instructions in the BCA protein concentration assay kit (purchased from Shanghai Beyontian Biotechnology Co., Ltd.).

[0092] 1.2 Preparation of fusion protein containing porcine pseudorabies virus gB protein

[0093] 1.2.1 According to the patent CN105693827A, the fusion protein of PRVgB protein fragment and PRVgD protein (abbreviated as PRVgB2) was prepared. After His affinity chromatography and molecular sieve p...

Embodiment 2

[0109] Example 2: Preparation, purification and identification of porcine pseudorabies virus gB protein monoclonal antibody.

[0110] 2.1 Preparation of PRVgB protein monoclonal antibody

[0111] The PRVgB1 prepared in Example 1.1 was concentrated and then emulsified with Freund's adjuvant at a final content of 100 μg / mouse to immunize 4 mice as group ①, immunized at intervals of 14 days (short-range), accumulatively immunized 3-5 times, And 7 days after each immunization, the blood was collected and the ELISA titer of the mouse serum was continuously detected (PRVgB1 prepared in Example 1 was used as the coating source). The results are shown in Table 1. It can be seen from Table 1 that the ELISA titers of the mouse sera continuously tested after 3-5 times of immunization were all ≤1:8000 and the upward trend was not obvious. If there is no positive cell in any one fusion, discard it.

[0112] The interval between each immunization was extended to 21 days (long-term immuniza...

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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 gB protein. The sequence is the variable region sequence of mouse monoclonal antibody 3C12 or the variable region sequence of mouse monoclonal antibody 5G12. 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 3C12, 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 5G12. According to the invention, an ELISA detection kit containing the antibody overcomes the technical problem that the PRVgB protein cannot be accurately quantified after tandem expression or fusion expression with other proteins and the expected expression effect cannot beverified, and the key problem of formulating a vaccine is solved. Meanwhile, the antibody can be used for immunohistochemical detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody combined with a pseudorabies virus gB 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 withi...

Claims

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Application Information

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IPC IPC(8): C07K16/08G01N33/577G01N33/569
CPCC07K16/085C07K2317/56G01N33/56994G01N2333/032G01N2469/10
Inventor 田克恭郝丽影
Owner LUOYANG PULIKE WANTAI BIOTECH
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