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Brachypodium distachyon functional centromere antigen polypeptide and application

A technology of Brachypodium distachyon and functional centromere, which is applied in the fields of bioinformatics and molecular cytogenetics, and can solve the problems of antibody difficulties and antibodies that cannot be used universally

Inactive Publication Date: 2016-04-06
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 distachyon functional centromere antigen polypeptide and application
  • Brachypodium distachyon functional centromere antigen polypeptide and application

Examples

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Embodiment 1

[0023] Example 1: Acquisition of Brachypodium distachyon functional centromere histone CENH3 sequence

[0024] According to the known sequence information of H3 protein and the relationship between H3 protein and CENH3 protein, they have a high degree of homology in the histone fold domain, but have obvious differences in the N-terminus, use BLAST homology search to find The H3 protein sequence of Brachypodium distachyon was used as the query sequence, and the homology search and comparison were carried out in the EST database, and the full-length CDNA of the CENH3 protein of Brachypodium distachyon was found, thereby obtaining the Gene sequence of CENH3 protein and protein sequence of CENH3.

Embodiment 2

[0025] Example 2: Selection of antigens

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

Embodiment 3

[0027] Example 3: Preparation and evaluation of antibodies

[0028] 1. Using the polypeptide obtained above as an antigen.

[0029] 2. Carrier coupling, coupling the synthesized polypeptide to the protein carrier.

[0030] 3. Animal immunization and preparation of antiserum. 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 thi...

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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 the functional centromere antigen polypeptide of Brachypodium distachyon and its application. Background technique [0002] The centromere is an important functional element on the chromosomes of higher eukaryotes. It is responsible for the equal separation of chromosomes during cell mitosis and meiosis, thus playing an important role in ensuring the stable transmission of genetic material. However, because the centromere region is rich in repetitive sequences, it is difficult to sequence and assemble, so it is relatively difficult to study. [0003] With the deepening of research, scientists have pointed out that there is a variant of histone H3 commonly found in the functional region of eukaryotic centromeres, namely CENH3, which is a DNA packaging protein that regulates the centromere of chromosomes. Positioning, function, and genetics play a decisive...

Claims

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Application Information

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Patent Type & Authority Applications(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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