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Cyclin Y and use thereof

A cell cycle and factor technology, which can be used in gene therapy, medical preparations containing active ingredients, peptide sources, etc., and can solve problems such as cell carcinogenesis

Inactive Publication Date: 2010-12-22
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many evidences have proved that only when the cell cycle is tightly and precisely regulated, can cells maintain normal operation, and the uncontrolled ultra-fast operation of the cell cycle will lead to cancerous cells

Method used

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  • Cyclin Y and use thereof
  • Cyclin Y and use thereof
  • Cyclin Y and use thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0154] Preparation of CCNY polyclonal antibody

[0155] Primers: 5'-GATGGATCCAAGATGGGGAACACTACCTC-3'(SEQ ID NO: 14), 5'-ATACTCGAGCTCCGTAGTTAAGAGATGATG-3'(SEQ ID NO: 15) were used to screen the CCNY full-length cDNA fragment obtained from the human brain cDNA library by double hybridization As a template, PCR amplifies the full-length open reading frame of the CCNY gene, introduces BamH1 / Xho1 restriction sites in its upstream and downstream respectively, and inserts the full-length open reading frame of the CCNY gene into the pET28a(+) (purchased from MERCK) vector The pET28a-CCNY plasmid was constructed between the BamH1 / Xho1 restriction sites of the multiple cloning site. Introduce Escherichia coli expression strain BL21, induce 3h with 0.1mMIPTG, most of the expressed His-CCNY fusion protein is concentrated in the inclusion body, so take the precipitate. The precipitate was washed once with 1M, 2M, and 4M urea lysis buffer, and then centrifuged at 14,000 rpm at 4°C for 20 m...

Embodiment 1

[0242] Transcription of embodiment 1, CCNY gene

[0243] CCNY is a protein that can interact with PFTK1 and is screened by the present inventors using the yeast two-hybrid method, and it belongs to a new member of the cell cycle factor family.

[0244] The present inventors used the CCNY gene as a template to label the probe, and conducted a hybridization experiment, and Northern hybridization detected two CCNY transcripts with sizes of 4.0 kb and 2.0 kb, respectively. The 16 human tissues tested (the codes of each organization are as follows: He is the heart; Br is the brain; PL is the placenta; Lu is the lung; Li is the liver; SM is the skeletal muscle; Ki is the kidney; Pa is the pancreas; Sp is the spleen; Th In Thymus; Pr, Prostate; Te, Testis; Ov, Ovary; SI, Small Intestine; Co, Colon; Different, its expression in different tissues is very different, it is highly expressed in testis, it also has high expression in heart and skeletal muscle, but it is very low in other t...

Embodiment 2

[0246] Example 2, CCNY affects the progress of the cell cycle

[0247] From structural analysis, CCNY protein is a member of Cyclin family, from functional analysis, CCNY participates in the regulation of cell cycle. The eukaryotic expression vector pEGFPN3-CCNY of CCNY was constructed, and it and the empty vector pEGFPN3 (purchased from Clontech) were transfected into 293T cells, and flow cytometry analysis was performed after 48 hours of culture to analyze the effect of overexpression of CCNY on the cell cycle. The results showed that the number of cells in S phase and G2 / M phase increased in cells exogenously overexpressing CCNY, the number of cells in S phase increased from 32% to 41%, the number of G2 / M cells increased from 8% to 16%, and the number of cells in G1 The number of cells decreased from 58% to 41% ( figure 2 A).

[0248] The inventors also detected similar trend changes in other cell lines. Taking Hep3B cells (obtained from the Cell Bank of Chinese Academy...

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Abstract

The invention relates to a cyclin Y and use thereof. In the invention, a protein which is the cyclin Y (CCNY) correlative with the adjustment of a cell cycle is determined for the first time. The CCNY can interact with a protein kinase family member PFTAIRE protein kinase 1 (PFTK1) so as to function in control over the kinase activity of the PFTK1 or cellular localization.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to cell cycle factor Y and its application. Background technique [0002] PFTK1 protein is one of the members of CDK protein kinase family. Existing studies have proved that it plays an important functional role in the occurrence of liver cancer and cell cycle regulation. [0003] In recent years, studies on PFTK1 have shown that PFTK1 is a member of the CDK family (Shu, F. et al. (2007). Functional characterization of human PFTK1 as a cyclin-dependent kinase. ProcNatl Acad Sci USA 104, 9248-53). PFTK1 can interact with cell cycle factor Cyclin D3 (CCND3) and protein inhibitor p21 Cip1 Combine to form a ternary complex and participate in the regulation of cell cycle G1 / S. Many evidences have proved that only when the cell cycle is strictly and precisely regulated, can the cells maintain normal operation, and the uncontrolled ultra-rapid operation of the cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/18A61K45/00A61K39/395A61K48/00C07K14/435C12Q1/68C12N15/113G01N33/50G01N33/539G01N33/574A61P35/00
Inventor 陈江野姜梅高衍昆李珊
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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