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Applications of gene and protein coded thereby to promotion of cell cytoplasm cleavage and cell proliferation as well as research and development of drugs

A drug and coding technology, applied in application, drug combination, gene therapy, etc., can solve problems such as unclear substrates and biological functions

Active Publication Date: 2013-03-06
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Human ALKBH4 has 302 amino acids. Although it has been shown to have dioxygenase activity, its substrate and biological function are still unclear

Method used

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  • Applications of gene and protein coded thereby to promotion of cell cytoplasm cleavage and cell proliferation as well as research and development of drugs
  • Applications of gene and protein coded thereby to promotion of cell cytoplasm cleavage and cell proliferation as well as research and development of drugs
  • Applications of gene and protein coded thereby to promotion of cell cytoplasm cleavage and cell proliferation as well as research and development of drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Experimental methods and materials applied in the embodiments of the present invention

[0044] Experimental Materials:

[0045] 1. Information on the cell lines used

[0046]

[0047] 2. Antibody information used

[0048]

[0049]

[0050] 3. Plasmid information used

[0051]

[0052] 4. Chemical reagents used

[0053] Nocodazole (Sigma, M1404), Cytochalasin D (Sigma, C8273) and Blebbistatin (Sigma, B0560), taxol (Invitrogen P3456) and phalloidin (Invitrogen P3457), Lipofectamine2000 (Invitrogen), Lipofectamine RNAiMAX (Invitrogen), PI-stained kit Becton-Dickinson), Annexin V-FITC & PI Detection Kit (Jiamay Biotech, Beijing, China), DAPI (Vector Laboratories, Burlingame, CA), anti-flag M2 affinity gel (sigma, A2220), anti-HA affinity gel ( sigma), GFP-Trap-A beads (ChromoTek, gta-20), anti-Rabbit Ig IP Beads (eBioscience), anti-Mouse Ig IP Beads (eBioscience), ECL Western Blotting Detection Kit (GE)

[0054] experimental method:

[005...

Embodiment 2

[0198] Example 2: Purification of ALKBH4 fusion protein to prepare rabbit-derived antibody against ALKBH4 protein

[0199] 1. ALKBH4 protein domain and possible activity mechanism

[0200] Escherichia coli AlkB protein as mononuclear non-heme Fe 2+ And α-ketoglutarate-dependent dioxygenase superfamily members, which use metal ions as prosthetic groups and oxygen molecules as co-substrates, activate dioxygen molecules to catalyze the oxidation of methyl groups that are in contact with nitrogen atoms on stable heterocycles group, causing a conformational change that removes the methyl group by releasing formaldehyde. ALKBH protein is a member of the human dioxygenase AlkB protein family. The commonality of this family protein is that it has the functional domain of Escherichia coli AlkB protein: the ferrous ion binding domain is HxDx n The H domain, α-ketoglutarate and the substrate binding domain, the RxxxxxR domain. The ALKBH4 protein also has such domains: iron ion binding...

Embodiment 3

[0214] Example 3: Verification of the localization and expression of the ALKBH4 gene in cells

[0215] 1. The ALKBH4 protein is localized on the centrosome.

[0216] In this experiment, immunofluorescence technique was used to verify the localization of ALKBH4 in cells. MRC5 cells were inoculated into six-well plates covered with coverslips at a seeding density of 30-40%, and treated with Nocodazole at a final concentration of 0.04 μg / ml the next night. 16 hours, the purpose of adding Nocodazole is to arrest the cells in mitosis. After 16 hours, wash once with PBS, replace with fresh complete medium to continue culturing for 1 hour, wash twice with PBS, fix and osmotically drill holes, the specific method is the same as in Example 1. The centrosome marker γ-tubulin and the spindle marker α-tubulin were hybridized with ALKBH4 antibody as the primary antibody, and the secondary antibody was hybridized with FITC-conjugated anti-mouse secondary antibody and Cy3-conjugated anti-ra...

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Abstract

The invention discloses functions of an ALKBH4 gene and an ALKBH4 protein coded thereby in cytoplasm cleavage and cell proliferation and a potential application value in research and development of anti-cancer drugs for ALKBH4. The ALKBH4 protein is interacted with actin and non-muscle myosin (NM) II to demethylate actin K84mel, and the demethylation modification reaction is critical to combination of NM II on actin and slide of NM II along an actin fiber and has an effect on the cytoplasm cleavage efficiency. ALKBH4 gene silencing can cause excessive copying of a midbody, cell cleavage stage arrest and cytoplasm cleavage failure to finally cause cell apoptosis and multinucleate cell generation, the loss or inhibition of the ALKBH4 protein can cause a cell proliferation and migration defect, and finally, a cell is dead. Therefore, ALKBH4 can be used for the research and the development of the anti-cancer drugs.

Description

technical field [0001] The invention relates to the functions of the ALKBH4 gene and its encoded protein ALKBH4 in cytokinesis and cell proliferation and the potential application value in the research and development of ALKBH4 anticancer drugs. Background technique [0002] Cytokinesis usually goes through four main stages: cleavage plane specification, cleavage furrow ingression, midbody formation, and abscission. Failure of cytokinesis often leads to centrosome overduplication and genomic instability, which can lead to malignant proliferation and migration. To ensure that cytokinesis occurs correctly in space and time, many components of cytokinesis and other cellular pathway processes are involved in this tightly regulated process. Cytokinesis is generally considered to be accomplished by the contractile ring (contractile ring) formed by actin fibers (F-actin) and type II non-muscle myosin (non-muscle myosin II or NM II). Actin Fibers play a key role in the formation a...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61P35/00A61K39/00C12N15/53C12N9/02
Inventor 杨运桂李明明史悦
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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