Brachypodium distachybes functional centromere antigen polypeptide and its application

A technology of Brachypodium bismuth and functional centromere, which is applied in the fields of bioinformatics and molecular cytogenetics, can solve the problems such as the inability of universal antibodies and the difficulty of antibodies, and achieve the real and reliable effect of antibodies

Inactive Publication Date: 2019-05-07
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the amino acid sequence of CENH3 histone and H3 histone has a high degree of homology, and only the amino-terminal sequence changes, so it is relatively difficult to prepare antibodies that specifically recognize CENH3 histone but do not recognize normal H3; at the same time, CENH3 in Evolution is species-specific, and different species have large differences, and antibodies cannot be used universally among different species

Method used

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  • Brachypodium distachybes functional centromere antigen polypeptide and its application
  • Brachypodium distachybes functional centromere antigen polypeptide and its application

Examples

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Embodiment 1

[0023] Example 1: Obtaining the functional centromeric histone CENH3 sequence of Brachypodium bismuth

[0024] According to the known sequence information of H3 proteins and the relationship between H3 proteins and CENH3 proteins, they have a high degree of homology in the histone fold domain, but there are obvious differences in the N-terminus, using BLAST homology search, to identify The H3 protein sequence of Brachypodium erinacea was used as the query sequence, and the homology search and alignment was performed in the EST database, and the full-length cDNA of the CENH3 protein of Brachypodium erinacea was found. The gene sequence of CENH3 protein and the protein sequence of CENH3.

Embodiment 2

[0025] Example 2: Selection of Antigens

[0026] According to the obtained protein sequence of the functional centromere CENH3 of Brachypodium bismuth, the CENH3 protein sequence and H3 protein sequence were compared by bioinformatics software, and the specific sequence of the amino acid N-terminal of Brachypodium bismuth CENH3 protein (MARTKRPAIRKSKPQPKKQL) was obtained. This sequence of artificially synthesized polypeptides is identified by HPLC and mass spectrometry, and the purity of the polypeptides is more than 85%, and the content is 10-14 mg.

Embodiment 3

[0027] Example 3: Preparation and Evaluation of Antibodies

[0028] 1. The polypeptide obtained above is used as an antigen.

[0029] 2. Carrier coupling, the synthetic polypeptide is coupled to the protein carrier.

[0030] 3. Animal immunization, antiserum preparation. To immunize 2 New Zealand white rabbits, firstly, pre-collect blood, then use complete Freund's adjuvant + antigen on the 0th day, 600~800ug / rabbit for the primary immunization, and use incomplete Freund's adjuvant + antigen on the 21st day, 400~500ug / pc, for the first booster immunization, use incomplete Freund's adjuvant + antigen on the 35th day, 400~500ug / pc, for the second booster immunization, on the 42nd day for serum testing, required The ELISA titer of the antiserum is 1:20000. If the titer meets the requirements, whole blood is collected on the 49th day, and the antiserum is separated; / Only, the third booster immunization was carried out, and the serum test was carried out on the 56th day. If th...

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Abstract

The invention provides a brachypodium distachyon functional centromere histone CENH3 and application. Firstly, the nucleotide sequence of a brachypodium distachyon functional centromere histone CENH3 gene is predicted through the bioinformatics sequence comparison method, a polypeptide is synthesized according to a segment of specific sequence of the N end of an amino acid sequence encoded by the nucleotide sequence, New Zealand white rabbits are conventionally immuned through the polypeptide, antiserums are prepared, and a CENH3 protein antibody is obtained through antigen affinity purification. The CENH3 antibody can be used for immunostaining experiments, physical positioning of centromere and chromatin immunoprecipitation (ChIP) experiments, the functional DNA sequence of a brachypodium distachyon functional centromere is obtained, positioning of centromere genome genetic maps and physical maps is carried out, and important tools and information are provided for research of centromere formation and function exercise mechanisms and building of artificial chromosomes.

Description

technical field [0001] The invention relates to the fields of bioinformatics and molecular cytogenetics, in particular to a functional centromeric antigen polypeptide of Brachypodium biscuits and application thereof. Background technique [0002] The centromere is an important functional element on the chromosomes of higher eukaryotes, responsible for the equal separation of chromosomes in the process of cell mitosis and meiosis, thus playing an important role in ensuring the stable transmission of genetic material. However, because the centromeric region is rich in repetitive sequences and difficult to sequence and assemble, it is relatively difficult to study. [0003] With the deepening of research, scientists pointed out that a variant of histone H3, CENH3, which is a DNA packaging protein, is commonly found in the region where eukaryotic centromeres perform functions. Localization, function and inheritance play a decisive role, so the true centromere is defined as the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C07K16/16C07K16/06
CPCC07K14/415C07K16/06C07K16/16
Inventor 王凯官清娜单文波韩金磊
Owner FUJIAN AGRI & FORESTRY UNIV
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