Pineapple function centromere antigenic polypeptide and application thereof

A technology of functional centromere and antigenic polypeptide, which is applied in the fields of bioinformatics and molecular cytogenetics, and can solve the problem that the specific boundary of the centromere is vaguely defined.

Inactive Publication Date: 2016-03-23
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Due to the importance of the centromere function, people have carried out a lot of research on the centromere. In cytology, we define the centromere as the main constriction area on the chromosome. When observing the shape

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  • Pineapple function centromere antigenic polypeptide and application thereof

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Embodiment 1

[0020] Example 1 Acquisition of pineapple functional centromere histone CENH3 sequence

[0021] 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 pineapple H3 protein sequence was used as the query sequence, and the homology search and comparison was carried out in the EST database. The full-length CDNA of the pineapple CENH3 protein was found, and the gene sequence and the CENH3 protein sequence of the pineapple CENH3 protein were obtained.

[0022] The present invention uses the BLAST sequence alignment method to obtain the corresponding sequence information of the CENH3 protein according to the relationship between the CENH3 protein and the H3 protein.

Embodiment 2

[0023] Example 2: Selection of antigens

[0024] According to the obtained protein sequence of pineapple functional centromere CENH3 ， By comparing the CENH3 protein sequence with the H3 protein sequence through bioinformatics software, the specific sequence of the amino acid N-terminal of the pineapple CENH3 protein (MARTKHPAVRKSKAEPKKKL) was obtained. This sequence was used to artificially synthesize the polypeptide. After identification by HPLC and mass spectrometry, the purity of the polypeptide was above 85%. , and the content is 10-14mg.

Embodiment 3

[0025] Example 3: Preparation and evaluation of antibodies

[0026] 1. Using the polypeptide obtained above as an antigen.

[0027] 2. Carrier coupling, coupling the synthesized polypeptide to the protein carrier.

[0028] 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 t...

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Abstract

The invention provides pineapple function centromere antigenic polypeptide and application thereof, an amino acid sequence is as shown in SEQ ID NO.2, one polypeptide is synthesized according to a specific sequence at an N terminal of the amino acid sequence, a New Zealand white rabbit is conventionally immunized by using the polypeptide, antiserum is prepared, an antibody of CENH3 protein is obtained by affinity purification of an antigen, an immunofluorescence test of the antibody is performed, the antibody of the pineapple CENH3 protein can be used for cytological localization of position of pineapple function centromere, also can be used for ChIP-seq combined with a chromatin immunoprecipitation technology of the pineapple, the position, size and sequence information of function centromere on chromosome of the pineapple are determined, the location of a centromere genome genetic map and a physical map is performed, and important tools and information are provided for formation of the centromere and study of a function performing mechanism and the construction of artificial chromosome.

Description

technical field [0001] The invention relates to the fields of bioinformatics and molecular cytogenetics, in particular to pineapple functional centromere antigen polypeptide and application thereof. Background technique [0002] Centromeres are important functional elements on 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. Therefore, the study of centromere has important theoretical and practical significance for revealing the genetic law of organisms. Due to the importance of the centromere function, people have carried out a lot of research on the centromere. In cytology, we define the centromere as the main constriction area on the chromosome. When observing the shape of the chromosome, this area can It is clearly seen by us, but the specific boundary of the centromere is vaguely defined. In order to carry ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K16/16C07K16/06
CPCC07K14/415C07K16/06C07K16/16
Inventor 王凯单文波韩金磊官清娜
Owner FUJIAN AGRI & FORESTRY UNIV
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