Amolops Hainan ensis anticancer peptides Hainanenin-1 as well as gene and application thereof

A technology of hainanenin-1 and Hainan turbulent frog, which is applied in the field of biomedicine to achieve the effect of preventing multidrug resistance of tumor cells, strong effect and simple structure

Active Publication Date: 2014-04-30
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, some anticancer peptides also have a certain inhibitory effect on angiogenesis in tumor sites.

Method used

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  • Amolops Hainan ensis anticancer peptides Hainanenin-1 as well as gene and application thereof
  • Amolops Hainan ensis anticancer peptides Hainanenin-1 as well as gene and application thereof
  • Amolops Hainan ensis anticancer peptides Hainanenin-1 as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Cloning of the Anti-cancer Peptide Hainanenin-1 Gene of Hainan Frog

[0037] 1) Extraction of total RNA from the skin of the Hainan turbulent frog:

[0038] ①Take 300mg of Hainan Turbulent frog skin tissue, put it into a mortar and add liquid nitrogen to grind it into powder, transfer it to an EP tube, add 1m1 of total RNA extraction buffer (Trizol, product of Invitrogen, USA), mix well, and then in 4 Centrifuge at 12000rpm for 10min.

[0039] ② Centrifuge to take the supernatant, add 0.2ml chloroform solution, mix vigorously, let stand at room temperature for 10 minutes, then centrifuge at 4°C, 12000rpm for 10 minutes, and discard the precipitate.

[0040] ③ Add an equal volume of isopropanol to the supernatant, let it stand at room temperature for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, collect the precipitate, wash it once with 75% (V / V) ethanol, and dry it. The precipitate at the bottom of the tube is Hainan Turbulence Total RNA from frog skin....

Embodiment 2

[0053] Inhibition of cell proliferation by the anti-cancer peptide Hainanenin-1 from Hainan turbulent frog,

[0054] Cytotoxicity of Hainanenin-1 on different tumor cell lines detected by MTT assay

[0055] The experimental cell lines MCF-7, SGC-7901, MDA-MB-453, A-549, PC-3, and HUVEC were kindly provided by the Kunming Institute of Zoology, Chinese Academy of Sciences, and they were all adherent growth cells. Cell pellets were suspended in DMEM (Gibico, Life Tech, USA) medium containing 20% ​​fetal bovine serum (Gibico, Life Tech, USA) and 100 U / ml green chain double antibody (Gibico, Life Tech, USA), and transferred to In a 25ml flask, at 37°C, 5% CO 2 Cultured in the incubator to logarithmic phase. Digest the logarithmic phase cells with trypsin, collect by centrifugation after termination, and make a cell suspension, adjust its concentration to 5-10×10 by counting on a hemocytometer 4 pieces / ml. In a 96-well plate, add 100ul to each well so that the cell density to be...

Embodiment 3

[0063] Study on the Mechanism of Hainanenin-1 Killing Tumor Cells

[0064] 1) Inverted microscope to observe the morphology of MCF-7 cells

[0065] During the process of apoptosis, some morphological changes will occur in the cells, and the optical microscope can observe the phenomenon of cell shape rounding, volume reduction, cell shrinkage and nuclear agglutination. MCF-7 cells that are now growing in logarithmic phase were treated with 2×10 5 Inoculated into 6-well plates at a density of 2 ml per well. After the cells adhered to the wall, the old culture medium was discarded, and culture medium containing different concentrations of Hainanenin-1 was added, and the same volume of 0.1% DMSO was added to the control group. The concentration of Hainanenin-1 is 2.15 μM / L, 4.3 μMm / L, 8.6 μMm / L, 13 μMm / L. At 37°C, 5% CO 2 Cells were cultured for 24 hours under the conditions of inverted microscope.

[0066] Depend on image 3 It can be seen that the cells in the control grou...

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Abstract

The invention belongs to the field of biomedicine and in particular relates to an Amolops Hainan ensis anticancer peptides Hainanenin-1 as well as a gene and an application thereof. A sequence of the anticancer peptides Hainanenin-1 is shown by amino acid in SEQ ID No. 1. The Hainanenin-1 is small in molecular weight, has a simple structure, only contains one disulfide bonds, is convenient to carry out chemical synthesis and genetic engineering preparation, is applied in preparing an anticancer drug, has broad-spectrum antineoplastic activity, has obvious killing action to MCF (Human Breast Cancer)-7 cells, has obvious growth inhibition action to other various cancer cells, and also has the beneficial effects of low hemolytic activity, nearly no toxicity to normal cells and the like. Most important of all, by adopting Hainanenin-1, due to the special cancer cell killing mechanism of Hainanenin-1, the tumor multi-drug resistance generated when traditional chemical drugs are applied is difficult to generate.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a Hainan turbulent frog (Amolo p s hainanensis) anticancer peptide Hainanenin-1 and its gene and application. Background technique [0002] Tumor is one of the major malignant diseases with the highest mortality rate in the world. With the development of industry, environmental pollution and changes in people's life rhythm, the incidence and mortality of tumors are increasing year by year. In recent years, with the improvement of treatment methods, the high sensitivity of early diagnosis of tumors, and the continuous emergence of new anticancer drugs and monoclonal antibody drugs, many cancer patients have brought good news, but human beings want to conquer cancer. There are still several major difficulties: 1. Cancer treatment is often accompanied by multidrug resistance (MDR) of the tumor, and the resistance of cancer cells to anticancer drugs is becoming more and more intense, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P35/00C12Q1/02
CPCA61K38/00C07K14/463
Inventor 于海宁王义鹏张嵩岩
Owner DALIAN UNIV OF TECH
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