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Method and system for identifying protein

A protein and target protein technology, applied in the biological field, can solve problems such as identification of polyclonal antibodies, and achieve the effects of easy promotion and application, reliable specificity, and improved screening efficiency

Inactive Publication Date: 2019-03-05
SHENZHEN HUADA GENE INST
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Problems solved by technology

[0007] Aiming at the deficiencies of the existing technology and the actual needs, the present invention provides a method and system for identifying proteins, which can make up for the defects in the prior art that polyclonal antibodies cannot be identified, and can establish a protein that corresponds to the polyclonal antibody. Immune Repertoire Reference Database Based on Next Generation Sequencing Technology

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  • Method and system for identifying protein

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Embodiment 1

[0052] Example 1 Identification of Rabbit GPC1 Polyclonal Antibody

[0053] (1) Sample processing: the polyclonal antibody is treated with multi-enzyme serial enzymatic hydrolysis;

[0054] (a) Preliminary enzymatic hydrolysis: pepsin enzymatic hydrolysis

[0055] In 25mM phosphate buffer (pH 2.5), hydrolyze the GPC1 polyclonal antibody with pepsin, cut SDS-PAGE gel to obtain F(ab') 2 Fragment, for F(ab') 2 The fragments were subjected to in-gel enzymatic hydrolysis, and the resulting peptides were eluted with gradients by high-performance liquid chromatography, divided into 6 components, freeze-dried in vacuum, dissolved in mobile phase A, and the sample volume was 1 μg, and analyzed by mass spectrometry;

[0056] Among them: pepsin hydrolysis conditions are: polyclonal antibody 50 μg, pepsin: polyclonal antibody (w / w) ratio is 1:100, polyclonal antibody concentration 1 μg / μl, 37 ℃ water bath for 16 hours, add Tris buffer (pH 9.0 ) adjust the reaction system to neutrality;...

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Abstract

The invention relates to a method and system for identifying a protein, especially to a proteomic method and system for efficiently identifying polyclonal antibodies. The method includes the followingsteps: (1) a step of sample treatment, namely a step of performing enzymatic hydrolysis treatment on a to-be-tested protein sample by using multiple enzymes in series; (2) a step of liquid chromatography-mass spectrometry detection, namely a step of performing liquid chromatography separation and mass spectrometric detection on peptide fragments after the enzymatic hydrolysis treatment; and (3) astep of data analysis, namely a step of filtering mass spectrometry original down data, performing database searching identification of the mass spectrometry data according to a constructed databaseto obtain identified peptide fragment information, and performing merging according to the identified peptide fragment information to obtain the reliable to-be-tested protein. The method and system for identifying a protein provides a standard bioinformation analysis process for proteomics analysis of the polyclonal antibodies, and can effectively, simply and reliably obtain specific antibodies with high specificity and good immunoreactivity from the polyclonal antibodies.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method and system for identifying proteins, in particular to a proteomics method and system for efficiently identifying polyclonal antibodies. Background technique [0002] Antigens stimulate the body to produce an immunological response, and the plasma cells of the body synthesize and secrete immunoglobulins that have the ability to specifically bind to antigens, that is, antibodies. Specific antibodies produced by one clone are called monoclonal antibodies. The mixture of various monoclonal antibodies formed in the body is called polyclonal antibody. The identification, screening and preparation of monoclonal antibodies have been developed from mouse hybridoma cell method and phage display technology to proteomics method, but none of them can be directly used for polyclonal antibodies. The preparation of polyclonal antibodies is mainly obtained by immunizing animals and separating...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 任艳何燕斌任哲邹婉婷李新洋丁权刘斯奇杨乃波
Owner SHENZHEN HUADA GENE INST
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