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Bombina maxima non-crystalline beta gamma-crystallin and trefoil factor protein compound ,gene as well as preparation and use thereof

A technology of crystallin and trefoil factor, which is applied in the field of biomedicine, can solve the problems that there are no literature reports on the combined synergistic effect of trefoil factor family proteins and amorphous βγ-crystallin proteins, and the signal transduction pathway is unclear, so as to promote wound healing. Effects of repairing, inhibiting growth and promoting cell migration

Inactive Publication Date: 2010-06-09
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the discovery of trefoil factor protein, it has existed as an orphan ligand, and the receptors and detailed signal transduction pathways that interact with trefoil factor are unclear, and there are no literature reports on trefoil factor family proteins and non-crystalline βγ-crystal proteins capable of joint synergies

Method used

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  • Bombina maxima non-crystalline beta gamma-crystallin and trefoil factor protein compound ,gene as well as preparation and use thereof
  • Bombina maxima non-crystalline beta gamma-crystallin and trefoil factor protein compound ,gene as well as preparation and use thereof
  • Bombina maxima non-crystalline beta gamma-crystallin and trefoil factor protein compound ,gene as well as preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Separation and purification of the complex of amorphous βγ-crystallin and trefoil factor protein, structural determination and molecular cloning of α-subunit and β-subunit genes of Bombina maxima

[0038] (1) Separation and purification of the amorphous βγ-crystallin and trefoil factor protein complex of Bombina grandis

[0039] Step 1: Breed the live Bombina grandis collected in the field in the laboratory, rinse it with clean water, put it in a glass container with a cover, add an appropriate amount of anhydrous ether to stimulate it, and seal the container for 3 to 5 minutes. A large amount of foamy secretions are produced on the skin surface of Bombina grandis. Open the container, gently massage the skin on the back of the animal, and rinse Bombina grandis with a physiological saline solution containing 5mM EDTA, collect the skin secretions, and centrifuge (5000rpm, 4°C , 20 minutes), and the supernatant was freeze-dried to obtain the skin secretion.

[...

Embodiment 2

[0062] Example 2: Determination of ATPase and GTPase in Vitro and Nucleotide Binding Activity of Bombina grandis amorphous βγ-crystallin and trefoil factor protein complex βγ-CAT

[0063] Nucleotide binding activity test

[0064] The specific experimental process of βγ-CAT and nucleotide binding activity test is as follows: βγ-CAT and 10 μM TNP-GTP / ATP (dissolved in solution 50mM Tris-HCl (pH7.8)+20% (v / v) glycerol) in Incubate at room temperature for 5 minutes, and then perform a fluorescence excitation test (410nm for excitation, 565-600nm for emission), and the ability to bind to nucleotide analogues is judged by the change in the fluorescence emission intensity of TNP-GTP / ATP at 575nm.

[0065] GTPase / ATPase activity test

[0066] The GTPase / ATPase activity of βγ-CAT was tested using the GTPase / ATPase colorimetric kit (Innova Biosciences, UK, high sensitivity), according to the KIT guidelines, and the enzyme activity unit was nmol / min.

[0067] The results show that βγ-C...

Embodiment 3

[0069] Example 3: Cytotoxicity of Bombina maxima amorphous βγ-crystallin and trefoil factor protein complex βγ-CAT on various tumor cell lines

[0070] Tumor cell lines HCT116, AGS, K562 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences; tumor cell lines Hela, HT29, MCF-7, THP-1, A375, Hacat were purchased from Cell Bank of Kunming Institute of Zoology, Chinese Academy of Sciences.

[0071] Inverted microscope (Olympus, USA); laser confocal microscope (Zeiss, Germany); 35mm cell culture dish (Corning, USA), cell culture flasks of various specifications (Corning, USA). Collagenase (Sigma, USA); Pancreatin (Sigma, USA); Penicillin, Streptomycin, Tetracycline (Sigma, USA); Trypan Blue (Sigma, USA); Fetal Bovine Serum (Hyclone, Blood Source USA); Seed medium (M199, McCoy's 5a, MEM, Ham'sF12K, RP1640) (Gibco, USA), thrombomodulin antibody (Invitrogen, USA), fluorescein FITC-labeled goat anti-rabbit secondary antibody (Invitrogen, USA); Fluorescein-labeled LDL (...

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Abstract

The invention relates to a protein compound of the bombina maxima noncrystal beta gamma-crystallin and the trefoil factor, the gene, the manufacturing method and use for the same. The protein compound, with an apparent molecular weight of 72kDa, is linked by an alpha subunit and a beta subunit with non covalent bond in the molecular form of alpha-beta2, which is a novel ATPase and GTPase, and hasa plurality of cell bio-activities, such as killing a plurality of tumor cells in nano-mol level in vitro, inhibiting the growth of tumor cells and inducing the exfoliation and apoptosis of tumor cells. And the protein compound has an activity of promoting the restoration of wound in pico-mol level. The protein compound undergoes the extra-cellular mono-ubiquitination decoration, and can be directly transferred to the nucleus for controlling the gene expression and regulating the cell states, including cell migration, exfoliation and growth. The protein compound of the invention provides a brand-new biological function of the noncrystal beta gamma-crystallin and a mechanism of the synergistic effect with the trefoil factor protein, which can be applied to manufacturing biomedical reagent,resisting tumor and promoting the restoration of wound.

Description

Technical field: [0001] The invention relates to a complex of amorphous βγ-crystal protein and trefoil factor protein, a gene, a preparation method and an application of Bombina grandis, belonging to the field of biomedicine. Background technique [0002] Tissue wound repair is a dynamic process in which blood cells, extracellular matrix and various cellular regulatory factors participate and interact. Tissue wound repair consists of three phases: inflammatory response, tissue formation and tissue remodeling. During the inflammatory response phase, various growth factors, cytokines and small molecular compounds of low molecular weight are released from the serum through the surrounding damaged blood vessels, causing an inflammatory response, and coagulating with the extracellular matrix around the damaged blood vessels. , forming a barrier against external microorganisms, and also serving as a scaffold for the action of growth factors in the early wound repair process. At ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61P35/00C12N15/55A61K38/46A61P17/02C12N9/16
Inventor 张云刘树柏何英英李文辉
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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