Papaya functional centromeric antigen polypeptide and its application

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: 2019-04-02
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 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 further research, we need to know the structure of the centromere more clearly

Method used

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  • Papaya functional centromeric antigen polypeptide and its application

Examples

Experimental program
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Effect test

Embodiment 1

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

[0019] According to the known sequence information of the H3 protein and the relationship between the H3 protein and the CENH3 protein, they have a high degree of homology in the histone folding domain, but there are obvious differences at the N-terminus. Use BLAST homology search to The H3 protein sequence of papaya was used as the query sequence. A homology search was performed in the EST database to find the full-length cDNA of the CENH3 protein of papaya, and the gene sequence of the CENH3 protein of papaya and the protein sequence of CENH3 were obtained.

[0020] The 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 antigen

[0022] According to our purpose, to obtain a specific antibody against the CENH3 protein of papaya, we need to select a specific sequence of the protein as an antigen for antibody preparation. Based on the comparison between the CENH3 protein sequence and the H3 protein sequence, we selected a sequence (MARTKKSAHKKRKPQSAAGV) that specifically exists on the CENH3 protein and is located at the N-terminus. This sequence was used to artificially synthesize a polypeptide. After HPLC and mass spectrometry, the 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) Use 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, first pre-collect blood, and then use complete Freund’s adjuvant + antigen, 600~800ug / head on day 0, for primary immunization, and use incomplete Freund’s adjuvant + antigen on day 21. 400~500ug / head, for the first booster immunization, use incomplete Freund’s adjuvant + antigen on the 35th day, 400~500ug / head, for the second booster immunization, and perform a serum test on the 42nd day. The ELISA titer of the antiserum is 1:20000. If the titer meets the requirement, collect whole blood on the 49th day and separate the antiserum. If it does not meet the requirement, use incomplete Freund's adjuvant + antigen on the 49th day, 200ug / Only, carry out the third booster i...

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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 papaya functional centromere antigen polypeptides and applications thereof. 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. Therefore, research on centromeres has important theoretical and practical significance for revealing the laws of the organism. Due to the importance of centromere function, people have carried out a lot of research on centromere. In cytology, we define centromere as the main constriction area on chromosome. When observing the chromosome shape, this area can be It is clearly seen by us, but the definition of the specific boundary of the centromere is rather vague. In order to carry out fur...

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 王凯官清娜韩金磊单文波
Owner FUJIAN AGRI & FORESTRY UNIV
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