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Mouse anti-human PRRT2 monoclonal antibody, and preparation method and application thereof

A monoclonal antibody and antibody technology, applied in the biological field, can solve the problems of human PRRT2 expression profile research and functional research limitations, poor specificity, etc.

Inactive Publication Date: 2015-01-21
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, several commercialized PRRT2 antibodies are polyclonal antibodies with poor specificity. Western blot and immunofluorescence methods were used to detect the antibody. The results showed obvious non-specific heterosexuality, thereby limiting expression profiling and functional studies of human PRRT2

Method used

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  • Mouse anti-human PRRT2 monoclonal antibody, and preparation method and application thereof
  • Mouse anti-human PRRT2 monoclonal antibody, and preparation method and application thereof
  • Mouse anti-human PRRT2 monoclonal antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Design method of human PRRT2 immune antigen tandem polypeptide

[0070] The antigen epitope design of the present invention adopts the Antigen Auto Designer antigen design program of Abmart Company. Surface peptides are determined by calculating parameters such as solvent accessibility, disorder index, domain prediction of protein-protein interactions, or any combination of the above. Six peptides with a length of 10 amino acids, high hydrophilicity, high antigenicity, non-signal peptide, non-transmembrane region and disordered region were selected as antigenic epitopes (attached figure 1 ). In order to avoid the generation of new antigenic epitopes between different peptide fragments, a weak immunogenic linker GGGGS was inserted between different peptide fragments to connect the peptide fragments in series. Finally, the tandem polypeptide sequence of PRRT2 protein was determined as: PPQVLAGVPD-GGGGS-PKPALQPELP-GGGGS-QETVSKPE VS-GGG GS-PSSQLAGPGV-EGPAPEPHS...

Embodiment 2

[0073] Example 2: Antigen expression and purification

[0074] The antigen sequence synthesized by the whole gene was digested with BamHI / EcoRI, then digested with BamHI / EcoRI and the recovered PET32a vector was ligated by T4 ligase at room temperature for 2 hours. The ligation product was incubated with Rosetta competent cells (Novagen, Merck, Germany) prepared by the CaCl2 method on ice (4°C) for 0.5h, and then heat-activated at 42°C for 90s, and 500ul of non-resistant LB was added to the bacterial solution after the heat shock After the liquid medium, slowly shake at 250rpm on a shaker at 37°C for 45min, and finally plate on LB plates containing corresponding antibiotics. Bacterial plates were grown overnight (16h) in a 37°C incubator.

[0075] The next day, the transformed colony was picked and placed in the self-inducing system medium, and expressed overnight at 37°C and 250rpm. The next day, the bacterial solution was centrifuged (6000g, 5min), the supernatant was disc...

Embodiment 3

[0077] Example 3: Preparation of Monoclonal Antibody

[0078] A. Animal immunity

[0079] Oligonucleotide adjuvant: 50ul aluminum adjuvant (Thermo Fisher, USA) + 1 μg DNA adjuvant, the sequence is tccatgacgttcctgacgtT, the bases in lowercase are the sites that need sulfo-modification, commissioned by Cyparkson (Shanghai, China) to synthesize .

[0080] Immunization method: 3 Balb / c mice, 8-10 weeks old, weighing 18-20g. 20 μg of antigen was fully emulsified with Fluorine’s complete adjuvant (Sigma), and the immunization sites were the hind paws, the root of the tail, the underarms of the forelimbs and the groin, about 50 μL per point. 20μg antigen was completely mixed with oligonucleotide adjuvant, and immunized mouse hind leg muscle, 50-100μL per spot.

[0081] On the 8th day, 10 μg of antigen was emulsified completely with Fluorine’s complete adjuvant (Sigma), and immunized the forelimb armpit and groin of mice, 50-100 μL per point. Mix 20μg of antigen with oligonucleoti...

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Abstract

The invention discloses a mouse anti-human PRRT2 monoclonal antibody, and a preparation method and an application thereof. The invention also provides a hybridoma cell strain producing the monoclonal antibody. PRRT2 is a newly discovered gene. The mutation of the gene can lead to paroxysmal kinesigenic dyskinesia (PKD). The invention provides an anti-human PRRT2 monoclonal antibody. A sequence of the antigenic epitope corresponding to the antibody is represented by any one selected from SEQ ID NO3-SEQ ID NO8. The antibody provided by the invention has high specificity and sensitivity, such that a tool is provided for further research of the effect of PRRT2 in PKD development. The antibody has an important application prospect. The invention also relates to the preparation method and the application of the anti-human PRRT2 monoclonal antibody.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the preparation and application of PRRT2 antibodies. Specifically, the present invention provides a mouse monoclonal antibody against human PRRT2 protein, its preparation method and application. Background technique [0002] Paroxysmal kinesigenic dyskinesia (PKD) is a recurrent, exercise-induced, short-duration movement disorder with autosomal dominant inheritance, manifested as chorea, athetosis, throwing disorder, Dystonia and other involuntary movements. In 2011, the inventors of the present application discovered for the first time in the world that PRRT2 is the causative gene of familial PKD. [0003] Human PRRT2 is located at 16p11.2, has 4 exons, and encodes a proline-rich transmembrane protein 2 (proline-rich transmembrane protein 2, PRRT2) containing 340 amino acids. The PRRT2 protein belongs to the CD225 family, and it has two transmembrane domains near the C-terminus. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N5/20G01N33/68C12R1/91
Inventor 吴志英刘功禄李宏福
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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