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Brat-protein-resistant monoclonal antibody and application

A monoclonal antibody and protein technology, applied in the direction of immunoglobulin, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of multiple bands, high specificity of monoclonal antibodies, and unstable production

Inactive Publication Date: 2015-06-24
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many heterogeneous bands detected by Western blot of the polyclonal antibody, and the polyclonal antibody is generally poor in uniformity and cannot be produced stably, while the monoclonal antibody has high specificity, and once it is successfully prepared, it can permanently and continuously produce completely identical antibodies , so it is more suitable for systematic and comprehensive inspection of the specific system, so the preparation of monoclonal antibodies to Brat protein is of great significance for the current study of Brat protein function

Method used

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  • Brat-protein-resistant monoclonal antibody and application
  • Brat-protein-resistant monoclonal antibody and application
  • Brat-protein-resistant monoclonal antibody and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1, the preparation of the hybridoma that secretes anti-Brat protein monoclonal antibody

[0019] 1. Immunization of Mice

[0020] Selection of mice: select healthy Balb / c female mice homologous to the used myeloma cells, and the age of the mice is 8-12 weeks.

[0021] Selection of immunogen: select amino acids 367-766 and amino acids 723-1037 of Brat protein as immunogens, wherein, the amino acid sequence of Brat protein 367-766 is shown in SEQ ID No. 1 in the sequence table, and the amino acid sequence of 723-1037 is shown in SEQ ID No. 2 in the sequence listing.

[0022] The 367-766th amino acid immunogen of Brat protein with histidine tag is His-Brat 367-766 Preparation: Using the cDNA of the full-length brat gene as a template, design primers, PCR amplify the gene sequence shown in SEQ ID No.3, and insert the sequenced correct gene fragment into the EcoRI and XhoI enzymes of the expression vector pET30a with His tag Between the cutting sites, a recombi...

Embodiment 2

[0041] Embodiment 2, preparation and performance test of monoclonal antibody

[0042] 1. Preparation of monoclonal antibody

[0043] After intraperitoneally injecting 3A9 hybridoma cells into Balb / c mice, the ascites was collected and purified by protein-G immunoaffinity chromatography to obtain the monoclonal antibody, which was named Brat-3A9.

[0044] Second, the sensitivity detection of monoclonal antibody

[0045] The sensitivity of the monoclonal antibody was detected by enzyme-linked immunosorbent assay (ELISA).

[0046] 1. Antigen coating

[0047] Recombinant protein His-Brat 367-766 and GST-Brat 723-1037 A gradient diluted to 100 ng / well with the coating solution was added to the wells of the polystyrene enzyme-linked detection plate. After coating overnight at 4°C, the washing solution was washed 3 times.

[0048] 2. closed

[0049] Add 200 μl of blocking solution to each well, wash 3 times at 37°C for two hours (or overnight at 4°C), and pat dry.

[0050] 3. ...

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Abstract

The invention discloses a Brat-protein-resistant monoclonal antibody of a rat source and hybridoma secreting the antibody. The preserving number of the hybridoma provided by the invention is CGMCC No.7410. Western detection result shows that the monoclonal antibody 3A9 of the rat source can specifically identify Brat protein in brain of Drosophila melanogaster, and a foundation is established for intensive study on the function of the Brat protein.

Description

technical field [0001] The invention relates to a monoclonal antibody against Brat protein, a hybridoma cell line producing the antibody and its preparation and application. Background technique [0002] The Brat protein is a protein encoded by the brain tumor gene, with a total length of 1037 amino acids, and two zinc finger domains B-box1 and B-box2 are arranged in sequence from the N-terminus to the C-terminus, and a Coiled-coil domain and a β-propeller domain (Arama et al., 2000). Loss of Brat protein causes brain tumors in Drosophila model animals (Arama et al., 2000). Brat protein is highly conserved in evolution, and its homologous gene trim family in mammals encodes proteins involved in biological processes such as apoptosis, cell cycle regulation, and tumorigenesis (Schwamborn et al., 2009). Brat in Drosophila plays a role in many biological processes such as embryonic somite development, asymmetric division of neural stem cells, and differentiation of reproductiv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/00G01N33/68C12R1/91
Inventor 石文文张永清汪奇馥
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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