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Preparation method of cotton GraiRGA transcription factor specific recognition antibody

A transcription factor and specific recognition technology, applied in the fields of bioinformatics and molecular genetics, can solve the problems of few successful application of transcription factor antibodies, difficulty of antibodies, slow work progress, etc., and achieve the effect of authenticity and reliability of antibodies

Active Publication Date: 2021-07-16
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation of antibodies is often affected by many factors such as antigen sequence design and immune effect. It is difficult to prepare antibodies that can specifically and efficiently recognize the target antigen protein. This is relatively slow in the related work in plants, especially the important crop cotton. Successful application of transcription factor antibody reports

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Obtaining of protein sequence of cotton GraiRGA transcription factor

[0027] Different transcription factors have certain conserved structures. According to the conservation of RGA transcription factors in plants, the RGA gene (XM_018628495.1) from radish was used to search the cotton genome by BLAST homology, and a highly homologous gene from Raymond cotton was obtained. The source gene sequence obtained the full-length nucleotide sequence of the gene, thereby obtaining the gene sequence and protein sequence of the cotton GraiRGA transcription factor.

[0028] The present invention uses the BLAST sequence alignment method to obtain the corresponding sequence information of the cotton GraiRGA protein according to the certain conservation of RGA transcription factors in plants.

[0029] It should be noted that the BLAST sequence alignment method used here is a prior art, and it is not a technical solution protected by the present invention, so the specific...

Embodiment 2

[0030] Example 2: Design and preparation of antigens

[0031] According to the obtained amino acid sequence of cotton GraiRGA protein, its protein structure was analyzed by bioinformatics software. Firstly, the transmembrane region was analyzed by using TMHMM software to exclude the sequence of the transmembrane region; Select the segment with a higher score as the antigen candidate region; combined with protein property analysis, analyze the parameters of the candidate antigen such as hydrophilicity, antigenicity, flexibility, and surface exposure, and select the antigen with good solubility and antigenicity, flexibility, and exposure. It is more ideal to combine the helical folding structure to finally determine the 1-318 amino acid sequence of the high segment, as the final antigen sequence.

[0032] The prokaryotic expression vector is constructed according to the determined antigen sequence, and the prokaryotic expression is carried out, and the corresponding antigenic pr...

Embodiment 3

[0033] Example 3: Preparation and evaluation of antibodies

[0034] (1) Immunize animals with the antigenic protein obtained above to prepare antiserum.

[0035] To immunize 2 New Zealand white rabbits, blood should be pre-collected first, and then complete Freund’s adjuvant + antigen on day 0, 600-800ug / rabbit for initial immunization, and incomplete Freund’s adjuvant + antigen on day 21, 400-500ug / monkey, for the first booster immunization, on the 35th day with incomplete Freund's adjuvant + antigen, 400-500ug / bird, for the second booster immunization, and serum testing on the 42nd day, the required The ELISA titer of the antiserum is 1:20000. If the titer meets the requirements, collect whole blood on the 49th day and separate the antiserum. If it does not meet the requirements, use incomplete Freund’s adjuvant + antigen on the 49th day, 200ug / bird, the third booster immunization was carried out, and the serum test was carried out on the 56th day. If the titer reached th...

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Abstract

The invention relates to the technical field of bioinformatics and molecular genetics, in particular to a preparation method of a cotton GraiRGA transcription factor specific recognition antibody. The preparation method comprises the following steps: analyzing an amino acid sequence of the cotton GraiRGA transcription factor, selecting a sequence with the length of 318 amino acids, performing prokaryotic expression and New Zealand white rabbit immunization to prepare antiserum, and performing antigen affinity purification to obtain the specific binding antibody of the GraiRGA transcription factor. The GraiRGA antibody prepared by the invention can be used for subcellular localization research of the GraiRGA transcription factor, and can also be used for performing chromatin action site and interaction protein analysis of the transcription factor in cells through a chromatin immunoprecipitation technology (ChIP), so that an important tool is provided for research of a transcription factor mediated cotton stress resistance mechanism, and the GraiRGA antibody has relatively high application value and potential.

Description

technical field [0001] The invention relates to the technical fields of bioinformatics and molecular genetics, in particular to a method for preparing a cotton GraiRGA transcription factor-specific recognition antibody. Background technique [0002] Transcription factors are proteins that can bind to specific chromatin sequence sites (ie, cis-regulatory elements), and are also called trans-acting factors. By interacting with cis-acting elements or interacting with other regulatory factor proteins, it regulates gene transcription, thereby exercising its biological functions such as regulating the growth and development of organisms, signal transfer, and stress response. RGA (repressor of GA1-3 mutant) belongs to the plant-specific GRAS transcription factor family. Members of this protein family have their own unique GRAS domain, and some members also have a DELLA protein structure, which is closely related to the stress resistance mechanism of plants. It is an important targ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/16C07K16/06
CPCC07K16/16C07K16/065
Inventor 王凯韩金磊余光润张会
Owner NANTONG UNIVERSITY
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