Preparation method of cotton grairga transcription factor specific recognition antibody

Abstract

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. After analyzing the amino acid sequence of the cotton GraiRGA transcription factor, a sequence with a length of 318 amino acids is selected, expressed by prokaryotic, and New Zealand white Rabbit immunization was carried out to prepare antiserum, and after antigen affinity purification, the specific binding antibody of GraiRGA transcription factor could be obtained. The GraiRGA antibody prepared by the present invention can be used for the subcellular localization research of the GraiRGA transcription factor, and the chromatin interaction site and interaction protein analysis of the transcription factor in the cell can also be carried out by chromatin immunoprecipitation (ChIP). The research on the stress resistance mechanism of cotton mediated by transcription factors provides an important tool with 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...

Examples

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...

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...

Examples

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...