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Prion isomer monoclonal antibody gene and application thereof

A technology of monoclonal antibodies and isomers, applied in applications, antibodies, genetic engineering, etc., can solve the problems of inability to specifically recognize prion protein isomers and unsatisfactory immune effects.

Active Publication Date: 2012-06-20
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of the prior art can not specifically recognize the prion protein isomer at present, so the immune effect is not ideal

Method used

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  • Prion isomer monoclonal antibody gene and application thereof
  • Prion isomer monoclonal antibody gene and application thereof
  • Prion isomer monoclonal antibody gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 Preparation of anti-prion protein isomer monoclonal antibody APR3

[0052] 1. Preparation of Immunogen

[0053] The immunogenic PrP polypeptide fragment was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., and was coupled to keyhole limpet hemocyanin at the N-terminus.

[0054] 2. Immunization of Mice

[0055]Take 5 female BALB / c mice aged 6-8 weeks. A total of 4 immunizations were carried out with PrP polypeptide fragments: the first time the Aβ oligomerization mixture was mixed with an equal volume of Freund's complete adjuvant (Sigma) at a dose of 100 μg per mouse, and injected intraperitoneally. The injection was repeated after 2 weeks interval, using Freund's incomplete adjuvant (Sigma), and the dose of the conjugated polypeptide was 50 μg / mouse. Repeat once after an interval of 2 weeks; at another two-week interval, intrasplenic booster immunization was carried out with 50 μg of pure antigen, and the spleen was taken for ...

Embodiment 2

[0069] Embodiment 2 Immunohistochemical experiment

[0070] 1. Method

[0071] (1) Dewaxing and hydration.

[0072] (2) Wash twice with PBS for 5 minutes each

[0073] (3) Prepare fresh 3% H with distilled water or PBS 2 o 2 , sealed at room temperature for 5-10 minutes, and washed 3 times with distilled water.

[0074] (4) Antigen retrieval.

[0075] (5) Wash with PBS for 5 minutes.

[0076] (6) Add normal goat serum blocking solution dropwise, room temperature for 20 minutes. Shake off excess liquid.

[0077] (7) Add monoclonal antibody APR3 dropwise for 1 hour at room temperature or overnight at 4°C or 1 hour at 37°C (rewarm at 37°C for 45 minutes after overnight at 4°C).

[0078] (8) Wash with PBS three times for 2 minutes each time.

[0079] (9) Add biotinylated secondary antibody (goat anti-mouse IgG) dropwise, at 20°C-37°C for 20 minutes.

[0080] (10) Wash with PBS 3 times for 2 minutes each time.

[0081] (11) Add the reagent SABC dropwise, at 20°C to 37°C ...

Embodiment 3

[0088] Example 3 Western blot (Western blot) experiment

[0089] 1. Method

[0090] Take 50-100μg sample, 5×sample buffer, mix well and load the sample, first make the protein pass through the stacking gel with a voltage of 100V. When the sample enters the separating gel, adjust the voltage to keep it constant at 120V. When the bromophenol blue swims to the bottom of the gel, end the electrophoresis, remove the gel, and stain it with Coomassie Brilliant Blue R-250 routinely; put the gel and nitrocellulose membrane into containers containing blotting buffer Equilibrate in the chamber for 10 minutes, put filter paper, gel, NC membrane, and filter paper in order to form a "sandwich" shape, pour the transfer buffer, with the gel side facing the negative electrode and the NC membrane facing the positive electrode, carefully avoiding and driving away air bubbles. Turn on the power, make the constant current 80mA transfer continuously for 2h, cut off the power.

[0091] After the ...

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Abstract

The invention relates to a prion isomer monoclonal antibody gene, an application thereof and a monoclonal antibody prepared by using the gene. Antibody light chain and heavy chain variable region genes are cloned in a specific monoclonal antibody APR3 hybridoma cell of a cultured prion isomer. The obtained gene can be used for correctly coding a mouse antibody variable region. The invention also relates to the qualitative and quantitative detection of the monoclonal antibody in the prion isomer and an application in the aspects of preparing diagnostic reagents and therapeutic drugs for prion diseases. The monoclonal antibody provided by the invention can be used for specifically recognizing the prion isomer in a brain tissue and has stronger targeting activity when being applied to the prevention and the early treatment of the prion diseases.

Description

Technical field: [0001] The invention relates to a monoclonal antibody gene for specifically recognizing and binding prion protein isomers, and the monoclonal antibody prepared by the gene. [0002] The invention also relates to the use of the monoclonal antibody in the qualitative and quantitative detection of prion protein isomers, and in the preparation of diagnostic reagents and therapeutic drugs for prion disease. Background technique: [0003] Prion protein isoform (PrPSc) is the main cause of pathological changes and transmissibility of prion disease. The prion protein isoform PrPSc is also a marker of prion disease, and is the most important infectious factor of prion disease, with the strongest toxicity to neurons. [0004] Immunotherapy targeting the prion protein isoform PrPSc molecule to treat or prevent prion diseases is an important way of treatment. The methods in the prior art cannot specifically recognize prion protein isomers at present, so the immune eff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C07K16/08G01N33/577G01N33/569A61K39/42A61P31/20
Inventor 洪涛赵丽张莹
Owner BEIJING JIAOTONG UNIV
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