Natural anti-infection anti-tumor double-function polypeptide ECCT and gene and application thereof

An anti-infection and anti-tumor technology, applied in the field of biomedicine, can solve the problems of large side effects, tumors prone to drug resistance, and side effects of radiotherapy, etc., and achieve the effect of strong anti-inflammatory activity, small molecular weight, and high antibacterial effect

Active Publication Date: 2015-10-21
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment methods for malignant tumors currently mainly include surgery, radiotherapy and drug chemotherapy. However, surgical treatment is only suitable for early solid t

Method used

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  • Natural anti-infection anti-tumor double-function polypeptide ECCT and gene and application thereof
  • Natural anti-infection anti-tumor double-function polypeptide ECCT and gene and application thereof
  • Natural anti-infection anti-tumor double-function polypeptide ECCT and gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of the gene encoding the natural anti-infection and anti-tumor bifunctional peptide ECCT:

[0039] 1) Extraction of total RNA from Chinese water snake (Enhydris chinensis) lung:

[0040] ① Take 250 mg of Chinese water snake lung tissue, put it into a mortar and add liquid nitrogen to grind it into powder, transfer it to an EP tube, add 1 ml of total RNA extraction reagent (Trizol, product of Invitrogen, USA), mix well, and then store at 4 °C, Centrifuge at 12000rpm for 10min.

[0041] 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.

[0042] ③ Add an equal volume of isopropanol to the supernatant, leave it 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 i...

Embodiment 2

[0069] Chemical synthesis of ECCT:

[0070] Ⅰ. Chemical synthesis method of ECCT: According to the mature peptide amino acid sequence deduced from the gene, its complete sequence was synthesized with an automatic peptide synthesizer (433A, Applied Biosystems), and desalted by HPLC reverse-phase column chromatography.

[0071] Ⅱ. Molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

[0072] Ⅲ. The purity of the purified ECCT was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence was determined by an automatic amino acid sequencer structure.

[0073] ECCT is a linear polypeptide encoded by the ECCT gene, containing 30 amino acid residues, with a molecular weight of 3...

Embodiment 3

[0076] ECCT antibacterial activity detection:

[0077] (1) Pick the test strains stored on the slope and evenly spread them on the MH solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) plate, and place the sterilized 0.5cm diameter filter paper on the medium On the surface, 10 μl of 2 mg / ml ECCT sample solution dissolved in sterilized deionized water was added dropwise, incubated upside down at 37°C for 18-20 hours, and observed whether the inhibition zone was formed or not. If the sample has antibacterial activity, a clear and transparent bacteriostatic zone will be formed around the filter paper, and the larger the bacteriostatic zone, the stronger the antibacterial activity of the sample.

[0078] (2) ECCT minimum inhibitory concentration (Minimum Inhibitory Concentration) determination (2-fold dilution method):

[0079] Select the strains with the inhibition zone in the previous experiment to carry out the MIC determination experiment. The test strains w...

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Abstract

The invention relates to natural anti-infection anti-tumor double-function polypeptide ECCT and a gene and application thereof. The ECCT is composed of 30 amino acid residues and is linear chain polypeptide, the molecular weight is 3,662.7 Da, the isoelectric point is 12.33, and the encoding gene of an ECCT precursor is a linear chain nucleotide sequence with the length of 720 basic groups. The polypeptide ECCT has the advantages of being small in molecular weight, simple in chemical synthetic method, efficient in antibacterial action broad spectrum, high in anti-inflammatory activity, efficient in anti-tumor activity broad spectrum and the like, and has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a natural anti-infection and anti-tumor bifunctional polypeptide ECCT and its gene and application. Background technique [0002] Microbial infections can cause severe local or systemic inflammatory diseases, such as pneumonia, gastritis, peritonitis, and sepsis. In recent years, the large-scale abuse of traditional antibiotics has led to more and more serious drug resistance of pathogenic microorganisms, which has brought great harm to human health. huge threat. The clinical response to drug-resistant microbial infections is to use new or alternative antibiotics, so this requires the continuous development of new antimicrobial infection drugs. [0003] Tumor is a new organism formed by the body under the action of various carcinogenic factors, the cells of local tissues lose the normal regulation of their growth at the gene level, resulting in abnormal clonal proliferation....

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/12A61K38/17A61K8/64A61P31/04A61P31/10A61P29/00A61P35/00A23K1/16
CPCA61K8/64A61K38/00C07K14/46Y02A50/30
Inventor 王义鹏于海宁许国强
Owner SUZHOU UNIV
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