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A kind of domestic pigeon cathelicidin-cl-cath2 peptide and its gene and application

A cl-cath2 and domestic pigeon technology, applied in the field of biomedicine, can solve problems such as aggravating psoriasis symptoms

Active Publication Date: 2018-01-23
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

LL-37 can combine with human's own DNA to form a DNA-LL-37 complex, which will not be recognized by the immune system of normal people, but can be recognized by the immune system of psoriasis patients and induce plasmacytoid dendritic cells Produces large amounts of IFN-α, and IFN-α production exacerbates psoriasis symptoms

Method used

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  • A kind of domestic pigeon cathelicidin-cl-cath2 peptide and its gene and application
  • A kind of domestic pigeon cathelicidin-cl-cath2 peptide and its gene and application
  • A kind of domestic pigeon cathelicidin-cl-cath2 peptide and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Gene Cloning and Sequencing of Cl-CATH2 Peptide Precursor Sequence

[0060] (1) Using RNeasy AxyPrep TM Multisource Total RNA Miniprep Kit (Qiagen, union city, CA, USA) was used to extract total RNA from domestic pigeon spleen, and all operations were performed according to the kit instructions.

[0061] (2) The mRNA was isolated and purified using the PolyATtract® mRNA IsolationSystems kit from PROMEGA, USA.

[0062] (3) The pigeon spleen cDNA library was constructed using the In-Fusion® SMARTer Directional cDNA Library Construction Kit. PowerScript Reverse Transcriptase reverse transcription to synthesize cDNA first strand.

[0063] Forward SMARTer V Oligonucleot Primer: 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3'

[0064] Reverse In-Fusion SMARTer CDSⅢ primer: 5’-ATTCTAGAGGCCGAGGCGGCCGA CATGT(30)N−1N-3’(N=A,G,C,orT;N−1=A, G, or C)

[0065] The second strand was synthesized by Advantage DNA Polymerase, the primers were: forward: 5'-AAGCAGTGGTATCAACGCA...

Embodiment 2

[0086] Example 2 Cl-CATH2 Peptide Inhibits LPS-Induced Expression of Inflammatory Cytokines and NO Synthase (INOS) Gene

[0087] Mouse macrophage RAW264.7 was cultured in DMEM medium containing 10% fetal bovine serum at 37˚C CO 2 Culture in an incubator. When the cells grow to the logarithmic growth phase in good condition, digest with 0.25% trypsin, discard the trypsin, and blow down the adherent cells with DMEM medium with 10% fetal bovine serum. After pipetting evenly, count the cells and adjust the cell concentration to 1x10 6 / ml. Add the cells evenly pipetted into a 24-well plate, 1ml per well, at 37˚C CO 2 Cultivate overnight in the incubator to allow the cells to adhere to the wall, then suck out the supernatant and discard it, add 960ul of fresh DMEM medium containing 10% fetal bovine serum, add 20ul of LPS at a certain concentration to the positive control group and the drug-dosed group, and then add 20ul was diluted with DMEM and incubated with different concen...

Embodiment 3

[0089] Example 3 Cl-CATH2 peptide inhibits LPS-induced secretion of inflammatory cytokines and NO in vitro

[0090] Mouse macrophages RAW264.7 were cultured in DMEM medium containing 10% fetal bovine serum to the logarithmic phase of growth, digested with trypsin, and seeded in 24-well plates at 1x106 cells per well. Cells were incubated overnight at 37 ˚C in a CO2 incubator. After the cells adhere to the wall, discard the supernatant, add 960ul of fresh DMEM medium containing 10% fetal bovine serum to each well, then add 20ul of different concentrations of Cl-CATH2 and 20ul of a certain concentration of LPS to each well, and add the blank 40ul serum-free DMEM medium, treated for 24h. The samples treated with drugs for 24 hours were used to collect the supernatant to detect the release of NO and inflammatory factors with Griess kit and ELISA kit, respectively, and 5 replicates were set for each experimental group. The Griess kit was purchased from Biyuntian Bioreagent Compan...

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Abstract

The invention relates to a domestic pigeon peptide Cathelicidin-Cl-CATH2 as well as a gene and an application thereof, and belongs to the technical field of biomedicine. The complete sequence of the domestic pigeon peptide Cathelicidin-Cl-CATH2 is as follows: Leu-Ile-glutamine-Arg-Gly-Arg-Hppr-Gly-Arg-Hppr-Leu-Gly-Arg-Ile-Arg-Arg-Hppr-Arg-Pro-Arg-Ile-asparagine-Hppr-aspartic-Ile-Arg-Ala-Arg-Gly-Ser-Ile-Arg-Leu-Gly. The gene of the domestic pigeon peptide Cathelicidin-Cl-CATH2 is composed of 471 nucleotides, wherein an encoded mature peptide is composed of No.367 to No.468 nucleotides. The RT-PCR shows that the peptide Cathelicidin-Cl-CATH2 has obvious functions of inhibiting the expressions of key inflammatory factors induced by lipopolysaccharide (LPS) and inducible NO synthase genes; based on a protein level, the peptide Cathelicidin-Cl-CATH2 has an obvious negative control effect on the inflammatory factors induced by the LPS; in addition, the peptide Cathelicidin-Cl-CATH2 is simple in structure and does not contain disulfide bonds and a cyclic structure, thereby bringing convenience for chemical synthesis and preparation of genetic engineering; and the peptide Cathelicidin-Cl-CATH2 has low cytotoxicity and hemolysis activity to normal cells, so that the peptide Cathelicidin-Cl-CATH2 can be applied to the preparation of anti-inflammatory medicines.

Description

technical field [0001] The invention relates to pigeon Cathelicidin-Cl-CATH2 peptide and its gene, and its application in the preparation of anti-inflammatory drugs, belonging to the technical field of biomedicine. Background technique [0002] Cathelicidin is found in almost all kinds of vertebrates and plays an extremely important role in the innate immune system of animals. Cathelicidin has broad-spectrum antibacterial properties, not only has very strong bactericidal activity against Gram-positive bacteria, Gram-negative bacteria, some fungi and viruses, but also has the same effect on many clinical drug-resistant bacteria. In addition, cathelicidin also has chemotactic effects on a variety of immune cells, stimulates mast cells to release histamine, regulates macrophage transcription, inhibits the secretion of inflammatory factors, stimulates the secretion of anti-inflammatory factors, induces angiogenesis, promotes wound healing, and induces tumor cells Apoptosis and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/465C12N15/12A61K38/17A61P29/00A61P31/00
CPCA61K38/00C07K14/465
Inventor 于海宁王义鹏鲁一灵卫林
Owner DALIAN UNIV OF TECH
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