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Spindle centromere associated protein Z (CENP-Z) and encoded gene as well as application thereof

A technology of CENP-Z and CENP-E, applied in application, gene therapy, genetic engineering, etc., can solve problems such as spindle checkpoint inactivation and chromosome instability

Inactive Publication Date: 2012-07-11
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Loss of CENP-E inactivates the spindle checkpoint, producing chromosomal instability (Weaver et al., 2007)

Method used

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  • Spindle centromere associated protein Z (CENP-Z) and encoded gene as well as application thereof
  • Spindle centromere associated protein Z (CENP-Z) and encoded gene as well as application thereof
  • Spindle centromere associated protein Z (CENP-Z) and encoded gene as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, the acquisition of CENP-E binding protein CENP-Z gene

[0055] Using the gene library of human testis tissue as a template, using primers CENP-Zf (upstream primer) and CENP-Zr (downstream primer), the coding region of the CENP-Z gene was amplified by polymerase chain reaction (PCR). PCR was catalyzed by LA Taq polymerase (Takara Company), hot start at 94°C, annealing temperature at 55°C, and extension at 72°C for 1 minute. After the reaction, the PCR amplified product was ligated into pMD 18-T vector (Takara Company) using T4 DNA ligase. Then, transform the ligation product into Escherichia coli (E.coli) DH5α competent cells (see the above reference book for specific preparation), apply it on an LB plate containing ampicillin to screen positive clones and extract the plasmid (conventional method in the above reference book) , to obtain a recombinant plasmid containing the target fragment, named pMD18-CENP-Z. It is sequenced, and the sequencing results sh...

Embodiment 2

[0058] Example 2, the subcellular localization of CENP-Z in the cell cycle

[0059] 1. Construction of GFP-eukaryotic cell expression plasmid

[0060] The CENP-Z gene was excised from the pMD18-CENP-Z plasmid using EcoR I and BamH I restriction endonucleases (Takara Company), and the CENP-Z gene was separated from the pMD18-T vector by agarose electrophoresis, and analyzed with UNIQ- 10-column DNA gel recovery kit (Sangon company) recovery gene fragments. The gene fragment obtained was inserted into the vector pEGFP-C2 (Clontech Company) with T4 ligase (Takara Company) (double digestion with EcoR I and BamH I in advance), and the ligation product was transformed into Escherichia coli TOP10 strain competent (purchased from Invitrogen Company), spread on the LB plate containing kanamycin to screen positive clones (the conventional method in the above-mentioned reference book). After the plasmid was extracted, it was confirmed by DNA sequencing that the CENP-Z gene was correctl...

Embodiment 3

[0066] Embodiment 3, identification of CENP-Z and CENP-E interaction region

[0067] 1. CENP-E 2132-2701 Prokaryotic expression of genes and purification of expression products

[0068] Artificially synthesized wild-type CENP-E gene cDNA containing amino acid sequence 2132-2610 (abbreviated as CENP-E 2132-2610 gene), CENP-E was first treated with restriction endonuclease BamHI 2132-2610 The gene was digested with a single enzyme, and then ligated into the vector pMAL-c2 (NEB Company) that had undergone the same enzyme digestion. The ligation product was then transformed into Escherichia coli Rosetta (DE3) pLysS competent cells (purchased from Invitrogen), and the transformed cells were inoculated on LB resistance plates containing 100 mg / L ampicillin and 34 mg / L chloramphenicol (LB+Amp+ Cm) for screening. Pick the grown single clone, inoculate it into 5mL LB+Amp+Cm liquid medium, and shake the bacteria at 37°C for 12-16 hours. Then inoculate it in 500mL LB+Amp+Cm liquid c...

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Abstract

The invention discloses a centromere associated protein and encoded gene as well as application thereof, and aims to provide the centromere associated protein and the encoded gene as well as the application in preparing drug for inhibiting tumor proliferation. The protein is one of the following amino acid sequences: 1) the amino acid sequence indicated in SEQ ID NO.1; or 2) the amino acid residue sequence of the SEQ ID NO.1 is replaced by one or multiple amino acid residues, lost and added so as to be the amino acid sequence that is derived from the SEQ ID NO.1 and has the regulating and controlling functions to the process of the cell mitosis as combined with the CENP-E protein. The protein participates in the spindle checkpoint signal transduction route of the cell mitosis through the mutual function with the CENP-E, and the active ingredient of the protein can be taken as the drug target for developing the drug for inhibiting tumor proliferation. The protein and the encoded gene thereof play a significant role in the field of medicine and pharmacy, and have a wide application range.

Description

technical field [0001] The present invention relates to a protein and its coding gene and application, in particular to a CENP-E binding protein and its coding gene and its application in the preparation of drugs for inhibiting tumor growth. Background technique [0002] The main task of cell mitosis is to equally distribute the duplicated genome (chromosomes) to the two daughter cells. The fidelity of chromosome segregation is crucial to the survival and reproduction of species. Defects in chromosome segregation lead to aneuploidy, which is closely related to tumorigenesis. During mitosis, the connection between chromosomes and spindle microtubules is mediated by kinetochores. The kinetochore is a superprotein complex located on the centromere. It not only directly maintains the connection between the spindle filament and the chromosome, but also regulates the time-space sequence and fidelity of chromosome movement and chromosome segregation. This signaling pathway locat...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/63C12N5/10C12N15/113A61K48/00A61P35/02
Inventor 姚雪彪窦震庄筱璇都建王茜玮王冬梅丁霞
Owner UNIV OF SCI & TECH OF CHINA
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