Pawpaw functional centromere antigen polypeptide, and applications thereof

A technology of functional centromere and antigenic polypeptide, applied in the fields of bioinformatics and molecular cytogenetics, can solve the problem of vague definition of the specific boundary of the centromere, and achieve the effect of being conducive to the construction and improvement, and the real and reliable antibody

Inactive Publication Date: 2016-03-16
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 sha...

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  • Pawpaw functional centromere antigen polypeptide, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Acquisition of papaya functional centromere histone CENH3 sequence

[0019] 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 papaya 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 CENH3 protein of papaya was found, and the gene sequence and protein sequence of CENH3 protein of papaya were obtained.

[0020] 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

[0021] Example 2: Selection of antigens

[0022] According to our purpose, to obtain the specific antibody of papaya CENH3 protein, we need to select a specific sequence of this protein as the antigen for antibody preparation. Based on the comparison between the CENH3 protein sequence and the H3 protein sequence, we selected a sequence (MARTKKSAHKKRKPQSAAGV) that only specifically exists on the CENH3 protein and is located at the N-terminus. This sequence was used to artificially synthesize the polypeptide, and it was identified by HPLC and mass spectrometry. The purity of the polypeptide is above 85%, and the content is 10-14mg.

Embodiment 3

[0023] Example 3: Preparation and evaluation of antibodies

[0024] (1) Using the polypeptide obtained above as an antigen.

[0025] (2) Carrier coupling, coupling the synthesized polypeptide to the protein carrier.

[0026](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 / only, the...

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Abstract

The invention provides a pawpaw functional centromere antigen polypeptide, and applications thereof. The amino acid sequence of pawpaw functional centromere histone CENH3 is represented by SEQ ID No.2; a specific sequence of the N terminal of the amino acid sequence coded based on the pawpaw functional centromere histone CENH3 is used for synthesis of a polypeptide; routine immune-inoculation of New Zealand white rabbits with the polypeptide is carried out, antiserum is prepared, the antibody of CENH3 protein is obtained via antigen affinity purification, and immunofluorescence assay of the antibody is carried out. The antibody of CENH3 protein can be used for cellular localization of pawpaw functional centromere, and can be applied to pawpaw chromatin immunoprecipitation assay sequencing (ChIP-seq), so that the position, size, and sequence information of functional centromeres on pawpaw chromosomes can be determined, obtained physical map information is more complete, and centromere-related information can be provided for pawpaw genome assembly.

Description

technical field [0001] The invention relates to the fields of bioinformatics and molecular cytogenetics, in particular to the papaya functional centromere antigen polypeptide and its application. 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 out...

Claims

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Application Information

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