CN104211794B - A kind of pigeon Cathelicidin-Cl CATH2 peptides and its gene, application - Google Patents

A kind of pigeon Cathelicidin-Cl CATH2 peptides and its gene, application Download PDF

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CN104211794B
CN104211794B CN201410412302.XA CN201410412302A CN104211794B CN 104211794 B CN104211794 B CN 104211794B CN 201410412302 A CN201410412302 A CN 201410412302A CN 104211794 B CN104211794 B CN 104211794B
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于海宁
王义鹏
鲁灵
鲁一灵
卫林
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Dalian University of Technology
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Abstract

A kind of pigeon Cathelicidin-Cl CATH2 peptides and its gene, application, belong to field of biomedicine technology.The peptide total order is classified as：The sweet different bright essence of silk of bright sweet different bright different bright different bright smart third essence of asparagine phenylpropyl alcohol asparagus fern of the third smart dried meat of refining benzene essence of essence of the sweet sweet refining benzene third of refining benzene third of bright different bright glutamine essence is bright sweet.Its gene is made up of 471 nucleotides, wherein encoding mature peptide moiety for the 367th 468 nucleotides.RT PCR show Cl CATH2 to bacteria lipopolysaccharide（LPS）The crucial pro-inflammatory cytokine of induction and the expression of Regulation of Inducible Nitric Oxide Synthase Gene are respectively provided with obvious inhibitory action.On protein level, the pro-inflammatory cytokine that Cl CATH2 are induced LPS also has the effect of significant negative regulation.In addition, Cl CATH2 are simple in construction, disulfide bond and cyclic structure are not contained, facilitates chemical synthesis and genetic engineering to prepare, and its cytotoxicity and hemolytic activity to normal cell is all very low, can be applied in anti-inflammatory medicaments are prepared.

Description

A kind of pigeon Cathelicidin-Cl-CATH2 peptides and its gene, application
Technical field
The present invention relates to a kind of pigeon Cathelicidin-Cl-CATH2 peptides and its gene, in anti-inflammatory medicaments are prepared Application, belong to field of biomedicine technology.
Background technology
Cathelicidin is found in the vertebrate body of almost all kinds, in animal innate immune system It is middle to play extremely important effect.Cathelicidin has the antibiotic property of wide spectrum, not only to gram-positive bacteria, gram-negative Property bacterium, some fungies and virus there is very strong bactericidal activity, and also there is same work to many clinical drug-resistant bacterium With.In addition, cathelicidin also has chemotaxis to panimmunity cell, stimulates mast cell to discharge histamine, regulation Macrophage transcription, suppress pro-inflammatory cytokine secretion, stimulate and press down scorching cytokine secretion, induction of vascular generation, promote wound healing, induction The various biological function such as apoptosis of tumor cells.Because cathelicidin has so numerous biological functions, and to height Almost there is no murder by poisoning ability Deng animal normal cell, therefore cathelicidin is always the focus that we study. Cathelicidin has very wide application prospect in field of biological pharmacy.
In addition, cathelicidin and the morbidity of some diseases are closely related.Recent research indicate that people source Cathelicidin, LL-37 played an important role in human autoimmune's dermatoses-psoriasic pathogenic process. LL-37 can combine to form DNA-LL-37 compounds with people itself DNA, and the compound will not be by the immune system institute of normal person Identification, but can be identified that simultaneously inducing plasma cell sample BMDC produces a large amount of IFN-αs by psoriatic's immune system, And the generation of IFN-α has aggravated psoriasic symptom.In addition, rheumatoid arthritis and Patients with SLE body Inside also detect that LL-37 expression quantity raises extremely, prompt the generation of LL-37 and these diseases that there is also certain relation.With Cathelicidin gos deep into the research of disease incidence mechanism correlation, and we will have brand-new understanding to many diseases, So as to provide brand-new idea and method for the treatment of disease.
The application of cathelicidin families small peptide medically at present achieves some gratifying achievements, has had Some related drugs enter clinical research.For example, MX-226（CPI-226）It is one to obtain from ox vivo clone Cathelicidin-indolicidin derivative, IIIb clinical trial phases are completed, in local prevention or reducing The related blood infection of cardiac vein conduit, it is shown that good application prospect.The Iseganan of source and pig (IB-367), it is Pig cathelicidin-protegrin-1 variant, it is made up of 17 amino acid residues, intramolecular contains 2 pairs of disulfide bond. Patients undergoing chemotherapy oral mucositis, have been enter into the clinical III phases.In addition, some are used for wound healing, suppress endotoxin infection, suppress The antibacterial peptide of tumour etc. also enters clinical experimental stage.
The content of the invention
In order to preferably expand applications of the cathelicidin in pharmaceutical field, the present invention provides a kind of pigeon Cathelicidin-Cl-CATH2 peptides and its gene, to utilize cathelicidin to body or the hemolytic activity of normal cell It is small with cytotoxicity；It is simple in construction, without disulfide bond and cyclic structure, the characteristics of being easy to chemical synthesis, it is applied to preparation In anti-inflammatory medicaments.
The technical scheme is that：A kind of pigeon（Columba livia ）Cathelicidin-Cl-CATH2 peptides, The Cl-CATH2 peptides are by a kind of straight-chain polypeptide of pigeon cathelicidin gene codes, rich in basic amino acid, Cl- The total order of CATH2 peptides is classified as：Leu-Ile-glutamine-arginine-glycine-Arg-Phe-sweet ammonia Acid-Arg-Phe-leucine-glycine-arginine-isoleucine-Arg-Arg-phenylalanine-smart ammonia Acid-Pro-Arg-Ile-Asn-phenylalanine-aspartic acid-isoleucine-Arg-Ala- Arginine-glycine-serine-ILE-ARG-LEU-glycine.
The gene of coding Cl-CATH2 peptides is made up of 471 nucleotides, is from 5 ' end to 3 ' terminal sequences：
1 ATGGCGGGGT GCTGGGTGCT GGTGCTGGCG CTGCTGGGGG GGGCCTGCGC CCTCCCGGCC
61 CCCCTGGCCT ACACCCAGGC GCTGGCTCAG GCCGTCGACT CCTACAACCA GCGCCCCGAG
121 GTGCCCAACG CCTTCCGGCT GCTCAGCGCC GACCCCGAGC CCGCCCCGGG CGTCGAGCTG
181 AGCTCCCTGC GGCTCCTCAA CTTCACCATG ATGGAGACCG AGTGCACCCC GAGCGCCCGC
241 GTGAACCCCG ATGACTGCGA CTTCAAGGAG AACGGGGTCA TCAAGGAGTG CTCGGGCCCG
301 GTGCAGTTTG GGCAGAGCTC CCCCGAGATC GACCTGCACT GCACCGACGC CTCCTCTGAT
361 CCGGTTCTCA TCCAGCGTGG CCGGTTCGGG CGCTTCCTGG GCAGAATCCG GCGCTTTCGG
421 CCCCGAATCA ACTTCGACAT CCGCGCCCGG GGCTCCATTC GCCTGGGCTG A
The mature peptide of coding Cl-CATH2 peptides is located at 367-468 positions nucleotides, encodes 34 amino acid residues, and it is ripe Peptide amino acid sequence is：Leu1Ile2Gln3Arg4 Gly5 Arg6 Phe7 Gly8 Arg9 Phe10 Leu11 Gly12 Arg13 Ile14 Arg15 Arg16 Phe17 Arg18 Pro19 Arg20 Ile21 Gln22 Phe23 Glu24 Ile25 Arg26 Ala27 Arg28 Gly29 Ser30 Ile31 Arg32 Leu33 Gly34。
The pigeon（Columba livia ）Cathelicidin-Cl-CATH2 peptides are applied to prepare controlling for anti-inflammatory Treat medicine.
The Cl-CATH2 peptides are applied to the medicine for preparing anti-inflammatory.
The Cl-CATH2 peptides synthesized according to the gene code of the coding Cl-CATH2 peptides are dissolved in sterilizing ultra-pure water, for pharmacology Activity determination.
1. the gene cloning of Cl-CATH2 peptides：
Pigeon spleen Total RNAs extraction, mRNA purifying, mRNA reverse transcriptions and cDNA library structure, according to the birds reported Cathelicidin sequences Design special primers, Cl-CATH2 genes are screened using the method for heminested PCR.The chains of cDNA first close It is Clontech companies In-Fusion into primerR SMARTerTMThere is provided in PCR cDNA synthesis Kit SMARTTMIV Oligonucleotide primers（5’-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGG
CCGGG-3’）With CDS III/3 ' PCR primers（5’-ATTCTAGAGGCCGAGGCGGCCGA CATGT(30)N− 1N-3’）；CDNA the second chain synthetic primers are（5’-AAGCAGTGGTATCAACGCAGAGT-3’）Draw with the PCR of CDS III 3 ' Thing.5 ' primers of the step of heminested PCR two are respectively：P1 (5 '-ATGGCGAGCTGCTG GGCTGCT-3 ') and P2 (5 '- AACGCCTTCCAGGCTGCTCAG-3 '), other end primer is to build in the kit of storehouse the PCR of CDS III 3 ' provided to draw Thing.Obtained positive monoclonal carries out gene nucleotide series measure, and sequencing result shows Cl-CATH2 gene order by 471 Nucleotides forms, and is from 5 ' ends to 3 ' terminal sequences：
1 ATGGCGGGGT GCTGGGTGCT GGTGCTGGCG CTGCTGGGGG GGGCCTGCGC CCTCCCGGCC
61 CCCCTGGCCT ACACCCAGGC GCTGGCTCAG GCCGTCGACT CCTACAACCA GCGCCCCGAG
121 GTGCCCAACG CCTTCCGGCT GCTCAGCGCC GACCCCGAGC CCGCCCCGGG CGTCGAGCTG
181 AGCTCCCTGC GGCTCCTCAA CTTCACCATG ATGGAGACCG AGTGCACCCC GAGCGCCCGC
241 GTGAACCCCG ATGACTGCGA CTTCAAGGAG AACGGGGTCA TCAAGGAGTG CTCGGGCCCG
301 GTGCAGTTTG GGCAGAGCTC CCCCGAGATC GACCTGCACT GCACCGACGC CTCCTCTGAT
361 CCGGTTCTCA TCCAGCGTGG CCGGTTCGGG CGCTTCCTGG GCAGAATCCG GCGCTTTCGG
421 CCCCGAATCA ACTTCGACAT CCGCGCCCGG GGCTCCATTC GCCTGGGCTG A
Coding Cl-CATH2 mature peptide is located at 367-468 positions nucleotides, and its ripe peptide amino acid sequence is： Leu1Ile2Gln3Arg4 Gly5 Arg6 Phe7 Gly8 Arg9 Phe10 Leu11 Gly12 Arg13 Ile14 Arg15 Arg16 Phe17 Arg18 Pro19 Arg20 Ile21 Gln22 Phe23 Glu24 Ile25 Arg26 Ala27 Arg28 Gly29 Ser30 Ile31 Arg32 Leu33 Gly34 (LIQRGRFGRFLGRIRRFRPRINFDIRARGSIRLG)。
2nd, Cl-CATH2 suppresses bacteria lipopolysaccharide（LPS）The expression of the pro-inflammatory cytokine of induction
Mouse macrophage RAW264.7 with the DMEM culture mediums containing 10% hyclone 37 C CO2 In incubator Culture, when cell grows to exponential phase in good condition, digested with 0.25% pancreatin, pancreatin is discarded, with 10% tire ox blood Clear DMEM culture mediums blow down attached cell.Cell count after piping and druming uniformly, 1x10 is adjusted to by cell concentration6 /ml.Will piping and druming Uniform cell is added in 24 orifice plates, per hole 1ml, in 37 C CO2 Overnight incubation makes cell attachment in incubator, then inhales Go out supernatant liquor to discard, add the fresh DMEM culture mediums containing 10% hyclone of 960ul, heliotropism control group and dosing Group adds the certain density LPS of 20ul, adds the Cl- of various concentrations that 20ul is diluted with DMEM and Jing Guo filtration sterilization CATH2 (final concentration of 5,10,20 ug/ml) is incubated 6h together, and blank control is added at 40ul plasma-free DMEM mediums 6h is managed, then extracts total serum IgE with RNA extracts kits, cDNA is synthesized with single-stranded cDNA synthetic agent box, and by fixed in real time Measure PCR（RT-PCR）To determine the cytokines Tumor Necrosis factor（TNF-α）, Interleukin -1β（IL-1β）And inducible NO-synthase （INOS）Expression quantity, each 5 repetitions of Setup Experiments.RNA extracts kits and cDNA synthetic agent box are all purchased from TaKaRa Company, operation by specification are carried out.
The upstream and downstream primer sequence of each cell factor is as follows：
5 ' end primers of TNF-α：5’-CGGTGCCTATGTCTCAGC CT-3’
3 ' end primers：5’-GAGGGTCTGGGCCATAGAAC-3’
IL-1 β 5 ' end primers：5’-AT GGCAACTGTTCCTGAACTC-3’
3 ' end primers：5’-GCCCATACTTTAGGAAGACA -3’
INOS 5 ' end primers be：5’-CTGCAGCACTTGGATCAGGAACCTG-3’
3 ' end primers：5’-GGAGTAGCCTGTGTGCACCTGGAA-3’
Internal reference GAPDH 5 ' end primers：5’-AAGCCCATCACCATCTTCCA-3’
3 ' end primers：5’-CCTGCTTCACCACCTTCT TG -3’.
3rd, the NO and pro-inflammatory cytokine of Cl-CATH2 negative regulators LPS inductions release
Mouse macrophage RAW264.7 is with the DMEM medium cultures containing 10% hyclone to growing logarithmic phase, pancreas After enzymic digestion, it is inoculated in 24 orifice plates, per hole 1x106It is individual.In 37 C CO2Being incubated overnight in incubator makes cell attachment.Cell After adherent, supernatant liquor is discarded, adds the fresh DMEM culture mediums containing 10% hyclone of 960ul per hole, then to each Kong Zhongjia Enter the certain density LPS of Cl-CATH2 and 20ul of 20ul various concentrations, blank adds 40ul plasma-free DMEM mediums, processing 24h.Supernatant is collected respectively with Griess kits and the burst size of ELISA kit detection NO and inflammatory factor, each experiment Group sets 5 repetitions.Griess kits are purchased from green skies biological reagent company, and ELISA kit is purchased from Xin Bosheng biology skills Art company, all operations operate according to kit specification.
4th, influences of the Western blot methods detection Cl-CATH2 to inflammatory signals path
Mouse macrophage RAW264.7, to logarithmic phase is grown, is used with the DMEM medium cultures containing 10% hyclone After pancreatin digestion, with 1x106Individual/ml is inoculated in 6 orifice plates, per hole 2ml.In 37 C CO2Being incubated overnight in incubator makes cell It is adherent, cell culture supernatants are then discarded, use phosphate buffer（PBS：0.8g NaCl ; 0.2g KCl; 2.9g Na2HPO4.12H2O; 0.27g KH2PO4Be dissolved in 800ml deionized waters, stirring and dissolving, be settled to 1L, with dense Hcl adjust PH to 7.4, autoclaving.）Wash cell 2-3 times, be changed to plasma-free DMEM medium Nature enemy 16h.Added in per hole different dense The sample 20ul of degree（Make final concentration of the 5,10,20ug/ml of every hole）, blank adds 20ul serum-free DMEM, after handling 1h, to 20ul LPS (final concentration of 100ng/ml) processing 3h is added in each hole beyond blank well.1000g centrifugations 5min collects cell, Cell is washed with the PBS of precooling twice.250ulRIPA cell pyrolysis liquids [50Mm Tris-HCl are added into the cell per hole (PH7.4), 1%Nonidet P-40,0.25% NaTDC, 150mM NaCl, 1mM EDTA, 1Mm PMSF, 1Mm NaF , 1mM Na3VO4, each 1ug/ml aprotinin, leupeptin and pepstatin], 30min is cracked on ice.4 C, 12000g centrifuge 20min, carefully draw supernatant, and supernatant is dispensed to new eppendorf pipes, draw 2ul and use Bradford methods determine protein concentration.40ug pyrolysis products are transferred on pvdf membrane after 12% SDS-PAGE electrophoresis, 5% ox blood Pure protein blocking 1h;Add special primary antibody, primary antibody（ERK、p-ERK、p38、p-p38、JNK、p-JNK）（1:2000）It is purchased from Cell Signaling Technology companies, GAPDH（1:3000）Purchased from ComWin Biotech companies, by specification behaviour Make, 4 C are incubated overnight；(39g Tris, 89gNaCl, 0.29g KCl, 0.5%Tween, are settled to 1000ml to TBST, use Dense HCl adjusts PH to 7.4) washing 3 times, each 5min;Plus HRPO then（HRP）Mark goat anti-rabbit antibody normal temperature 1h is incubated, secondary antibody is purchased from Cell Signaling Technology companies, and TBST is washed 3 times, each 5min;ECL reacts, Exposure imaging in darkroom.
The beneficial effects of the present invention are：A kind of pigeon（Columba livia ）Cathelicidin-Cl-CATH2 peptides And its gene, application, belong to field of biomedicine technology.Cl-CATH2 peptide total orders are classified as：Leu-Ile-glutamy Amine-arginine-glycine-Arg-Phe-glycine-Arg-Phe-leucine-glycine-arginine- Isoleucine-Arg-Arg-Phe-A taug-Pro-Arg-Ile-Asn-phenylpropyl alcohol ammonia Acid-aspartic acid-isoleucine-Arg-Ala-arginine-glycine-serine-isoleucine-arginine-bright ammonia Acid-glycine.Cl-CATH2 gene is made up of 471 nucleotides, wherein encoding mature peptide moiety for 367-468 positions core Thuja acid.Chemical synthesis Cl-CATH2,in vitroTo bacteria lipopolysaccharide（LPS）The inflammatory factor secretion of induction and induction type NO are closed The expression of enzyme is respectively provided with obvious inhibitory action.Research shows that Cl-CATH2 is to suppress scorching by suppressing MAPK signal paths The secretion of inflammation factor.Cl-CATH2 is simple in construction, does not contain disulfide bond and cyclic structure, facilitates chemical synthesis and genetic engineering system It is standby, and its cytotoxicity and hemolytic activity to normal cell is all very low, can be applied in anti-inflammatory medicaments are prepared.Cl- The pro-inflammatory cytokine of bacteria lipopolysaccharide (LPS) induction is expressed in vitro by CATH2 and secretion, and inducible NO-synthase expression（From And reduce NO emission levels）, all with apparent inhibitory action, and it is by suppressing in MAPK signal paths ERK's and JNK activates to suppress the secretion of inflammatory factor.MTT experiment also show Cl-CATH2 to normal cell- The cytotoxicity of RAW264.7 mouse macrophages is very weak, and hemolytic experiment shows that it is also very weak to people's globulolysis.
Brief description of the drawings：
Fig. 1 is the expression for the IL-1 β that RT-PCR detections Cl-CATH2 suppresses LPS inductions.
Fig. 2 is the expression for the TNF-α that RT-PCR detections Cl-CATH2 suppresses LPS inductions.
Fig. 3 is the expression for the induction type NO that RT-PCR detections Cl-CATH2 suppresses LPS inductions.
Fig. 4 is the release for the NO that Griess kits detection Cl-CATH2 suppresses LPS inductions.
Fig. 5 is the release for the TNF-α that ELISA kit detection Cl-CATH2 suppresses LPS inductions.
Fig. 6 is the release for the IL-1 β that ELISA kit detection Cl-CATH2 suppresses LPS inductions.
Fig. 7 is the release for the IL-6 that ELISA kit detection Cl-CATH2 suppresses LPS inductions.
Fig. 8 is the phosphorylation level that Western blot detections LPS suppresses ERK1/2.
Fig. 9 is the phosphorylation level that Western blot detections LPS suppresses JNK1/2.
Figure 10 is the phosphorylation level that Western blot detections LPS suppresses p-38.
Embodiment：
Illustrate the essentiality content of the present invention with example below, but present disclosure is not limited thereto.
The gene cloning of the Cl-CATH2 peptide precursor sequences of embodiment 1 and sequencing
（1）Using RNeasy AxyPrepTM Multisource Total RNA Miniprep Kit (Qiagen, Union city, CA, USA) pigeon spleen total serum IgE is extracted, all operations are carried out according to kit specification.
（2）MRNA isolates and purifies the PolyATtract mRNA Isolation using PROMEGA companies of the U.S. Systems kits.
（3）Utilize In-Fusion SMARTer Directional cDNA Library Construction Kit Build storehouse kit structure pigeon spleen cDNA library.PowerScript Reverse Transcriptase reverse transcriptions synthesize The chains of cDNA first.
Positive SMARTer V Oligonucleot primers：5’-AAGCAGTGGTATCAACGCAGAGTG GCCATTACGGCCGGG-3’
The reverse primers of In-Fusion SMARTer CDS III：5’-ATTCTAGAGGCCGAGGCGGCCGA CATGT(30)N −1N-3’(N=A,G,C,orT;N−1= A, G, or C)
The second chain is synthesized using Advantage DNA Polymerase, primer is：It is positive：5’-AAGCAGTGG TATCAACGCAGAGT-3 ', reverse primer are all In-Fusion SMARTer CDS III.The specification provided by kit comes Construction cDNA library.
（4）Design two specific forward primers（P1、P2）With a reverse non-specific universal primer（CDSⅢ）, with Pigeon spleen cDNA is template, and the gene gram of pigeon Cathelicidin-Cl-CATH2 peptides is carried out using the method for heminested PCR Grand screening.
Positive P1：5’-ATGGCGAGCTGCTGGGCTGCT-3’；
Positive P2：5’-AACGCCTTCCAGGCTGCTCAG-3’；
Reverse non-specific universal primer is In-Fusion SMARTer CDS III, and its sequence is：5’- ATTCTAGAGGCCGAGGCGGCCGACATGT(30)N−1N-3’(N=A,G,C,orT;N−1= A,G,orC)。
Concrete operations are：Using the cDNA library of structure as template, with P1 and reverse non-specific universal primer In-Fusion SMARTer CDS III carry out first step PCR as upstream and downstream primer, and reaction condition is：The min of 94 C pre-degenerations 5;26 are followed Ring：94 C are denatured 30 s, and 57.9 C annealing 30 s, 72 C extend 36 s;72 C extend 10 min; 4 ˚ C is preserved.Then using first step PCR primer as template, with P2 and reverse non-specific universal primer In-Fusion SMARTer CDS III is that upstream and downstream primer carries out heminested PCR second step, and reaction condition is arranged to：94 C pre-degenerations 5min;26 Circulation：94 C are denatured 30s, and 56.8 C annealing 30 s, 72 C extend 30 s；72 C further extend 7min; 4 C is preserved.Glue reclaim kit is used after the completion of amplification（Tiangeng biology）Carry out purpose fragment recovery.
（5）The target DNA fragment of recovery is connected with sequencing vector pMD19-T Vecter, is transformed into CaCl2-MgCl2Method The DH5 α competent cells prepared.100 μ l transformed bacteria solutions are taken to be uniformly coated on containing 100 μ lg/ml ampicillins （Amp）LB agar medium flat boards on；After surface is dried, it is placed in 37 C constant incubators and is inverted culture 12-16 h.Picking Single bacterium colony carries out bacterium colony PCR detection Insert Fragment sizes with M13 primers.Picking positive bacterium colony, bacterium extraction plasmid is shaken, is used Applied Biosystems DNA sequencer, model ABI PRISM 377 carry out nucleotide sequencing.
Universal primer is sequenced in pMD19-T Vecter：
Positive M13F：5'-CGCCAGGGTTTTCCCAGTCACGAC-3'；
Reverse M13R：5'-GAGCGGATAACAATTTCACACAGG-3'.
（6）Sequencing result
The pigeon cathelicidin of acquisition gene order, it is made up of 471 nucleotide sequences, from 5 ' ends to 3 ' ends For：
1 ATGGCGGGGT GCTGGGTGCT GGTGCTGGCG CTGCTGGGGG GGGCCTGCGC CCTCCCGGCC
61 CCCCTGGCCT ACACCCAGGC GCTGGCTCAG GCCGTCGACT CCTACAACCA GCGCCCCGAG
121 GTGCCCAACG CCTTCCGGCT GCTCAGCGCC GACCCCGAGC CCGCCCCGGG CGTCGAGCTG
181 AGCTCCCTGC GGCTCCTCAA CTTCACCATG ATGGAGACCG AGTGCACCCC GAGCGCCCGC
241 GTGAACCCCG ATGACTGCGA CTTCAAGGAG AACGGGGTCA TCAAGGAGTG CTCGGGCCCG
301 GTGCAGTTTG GGCAGAGCTC CCCCGAGATC GACCTGCACT GCACCGACGC CTCCTCTGAT
361 CCGGTTCTCA TCCAGCGTGG CCGGTTCGGG CGCTTCCTGG GCAGAATCCG GCGCTTTCGG
421 CCCCGAATCA ACTTCGACAT CCGCGCCCGG GGCTCCATTC GCCTGGGCTG A
The mature peptide of coding Cl-CATH2 peptides is located at 367-468 positions nucleotides, and its ripe peptide amino acid sequence is： Leu1Ile2Gln3Arg4 Gly5 Arg6 Phe7 Gly8 Arg9 Phe10 Leu11 Gly12 Arg13 Ile14 Arg15 Arg16 Phe17 Arg18 Pro19 Arg20 Ile21 Gln22 Phe23 Glu24 Ile25 Arg26 Ala27 Arg28 Gly29 Ser30 Ile31 Arg32 Leu33 Gly34 (LIQRGRFGRFLGRIRRFRPRINFDIRARGSIRLG)
The Cl-CATH2 peptides of embodiment 2 suppress the proinflammatory cytokine and NO synthase of LPS inductions（INOS）The expression of gene
Mouse macrophage RAW264.7 with the DMEM culture mediums containing 10% hyclone 37 C CO2 In incubator Culture, when cell grows to exponential phase in good condition, digested with 0.25% pancreatin, pancreatin is discarded, with 10% tire ox blood Clear DMEM culture mediums blow down attached cell.Cell count after piping and druming uniformly, 1x10 is adjusted to by cell concentration6 /ml.Will piping and druming Uniform cell is added in 24 orifice plates, per hole 1ml, in 37 C CO2 Overnight incubation makes cell attachment in incubator, then inhales Go out supernatant liquor to discard, add the fresh DMEM culture mediums containing 10% hyclone of 960ul, heliotropism control group and dosing Group adds the certain density LPS of 20ul, adds the Cl- of various concentrations that 20ul is diluted with DMEM and Jing Guo filtration sterilization CATH2 is incubated 6h together, and blank control adds 40ul plasma-free DMEM mediums processing 6h, then carried with RNA extracts kits Total serum IgE is taken, synthesizes cDNA with single-stranded cDNA synthetic agent box, and pass through real-time quantitative PCR（RT-PCR）Come determine cell because Son：TNF-α, IL-1 β and inducible NO-synthase（INOS）Expression quantity, each 5 repetitions of Setup Experiments.RNA extracts kits TaKaRa companies are all purchased from cDNA synthetic agent box.The upstream and downstream primer of each cell factor refers to above.
Test result indicates that Cl-CATH2 suppresses the pro-inflammatory cytokine of LPS inductions in a manner of dose-dependent in vitro, TNF-a, IL-6 and inducible NO-synthase gene expression.In the presence of 20ug/ml Cl-CATH2, almost it completely inhibit The expression of the above-mentioned GFP of 100ng/ml LPS inductions.
The Cl-CATH2 peptides of embodiment 3 suppress the secretion of the proinflammatory cytokine and NO of LPS inductions in vitro
Mouse macrophage RAW264.7 is with the DMEM medium cultures containing 10% hyclone to growing logarithmic phase, pancreas After enzymic digestion, it is inoculated in 24 orifice plates, it is individual per hole 1x106.Being incubated overnight in 37 C CO2 incubators makes cell attachment.Carefully After born of the same parents are adherent, supernatant liquor is discarded, the fresh DMEM culture mediums containing 10% hyclone of 960ul are added per hole, then into each hole The certain density LPS of Cl-CATH2 and 20ul of 20ul various concentrations is added, blank adds 40ul plasma-free DMEM mediums, place Manage 24h.Agent-feeding treatment 24h sample is used to collect supernatant respectively with Griess kits and ELISA kit detection NO and inflammation The burst size of inflammation factor, each experimental group set 5 repetitions.Griess kits are purchased from green skies biological reagent company, ELISA Kit is purchased from Xin Bosheng biotech companies, and all operations operate according to kit specification.As a result as shown in figs. 4-7. Fig. 4-7 is shown：In the presence of 20ug/ml Cl-CATH2, the NO and important pro-inflammatory cytokine of LPS inductions（TNF-a、IL-1β、IL- 6）Release significantly suppressed.
The Western blot methods of embodiment 4 detect influences of the Cl-CATH2 to MAPK inflammatory signals paths
Mouse macrophage RAW264.7, to logarithmic phase is grown, is used with the DMEM medium cultures containing 10% hyclone After pancreatin digestion, with 1x106Individual/ml is inoculated in 6 orifice plates, per hole 2ml.In 37 C CO2Being incubated overnight in incubator makes cell It is adherent, cell culture supernatants are then discarded, use phosphate buffer（PBS：0.8g NaCl ; 0.2g KCl; 2.9g Na2HPO4.12H2O; 0.27g KH2PO4Be dissolved in 800ml deionized waters, stirring and dissolving, be settled to 1L, with dense HCl adjust pH to 7.4, autoclaving.）Wash cell 2-3 times, be changed to plasma-free DMEM medium Nature enemy 16h.Added in per hole different dense The sample 20ul of degree（Make final concentration of the 5,10,20ug/ml of every hole）, blank adds 20ul serum-free DMEM, after handling 1h, to 20ul LPS (final concentration of 100ng/ml) processing 3h is added in each hole beyond blank well.1000g centrifugations 5min collects cell, Cell is washed with the PBS of precooling twice.250ulRIPA cell pyrolysis liquids [50Mm Tris-HCl are added into the cell per hole (PH7.4), 1%Nonidet P-40,0.25% NaTDC, 150mM NaCl, 1mM EDTA, 1Mm PMSF, 1Mm NaF , 1mM Na3VO4, each 1ug/ml aprotinin, leupeptin and pepstatin], 30min is cracked on ice.4 C, 12000g centrifuge 20min, carefully draw supernatant, and supernatant is dispensed to new eppendorf pipes, draw 2ul and use Bradford methods determine protein concentration.40ug pyrolysis products are transferred on pvdf membrane after 12% SDS-PAGE electrophoresis, 5% ox blood Pure protein blocking 1h;Add special primary antibody, primary antibody（ERK、p-ERK、p38、p-p38、JNK、p-JNK）（1:2000）It is purchased from Cell Signaling Technology companies, GAPDH（1:3000）Purchased from ComWin Biotech companies, by specification behaviour Make, 4 C are incubated overnight；(39g Tris, 89gNaCl, 0.29g KCl, 0.5%Tween, are settled to 1000ml to TBST, use Dense HCl adjusts PH to 7.4) washing 3 times, each 5min;Plus HRPO then（HRP）Mark goat anti-rabbit antibody normal temperature 1h is incubated, secondary antibody is purchased from Cell Signaling Technology companies, and TBST is washed 3 times, each 5min;ECL reacts, Exposure imaging in darkroom.As a result as seen in figs. 8-10.Fig. 8-10 shows that Cl-CATH2 suppresses to be lured by LPS in a manner of dose-dependent Lead caused ERK, p38 and JNK phosphorylation.It is 10ug/ml in Cl-CATH2 concentration from Western blot results When, the ERK and JNK of 100ng/ml LPS inductions phosphorylation are almost totally constrained, and also part is pressed down p38 phosphorylation System.It follows that Cl-CATH2 suppresses the MAPK signal activations of LPS inductions mainly by suppressing ERK and JNK signals, so as to The lower secretion for causing inflammatory factor of regulation.
Sequence 1：
<110>Dalian University of Technology
<120>A kind of pigeon Cathelicidin-Cl-CATH2 peptides and its gene, application
<160> 2
<210> 1
<211> 471
<212> DNA
<213>Pigeon（Columba livia ）
<400> 1
1 ATGGCGGGGT GCTGGGTGCT GGTGCTGGCG CTGCTGGGGG GGGCCTGCGC CCTCCCGGCC
61 CCCCTGGCCT ACACCCAGGC GCTGGCTCAG GCCGTCGACT CCTACAACCA GCGCCCCGAG
121 GTGCCCAACG CCTTCCGGCT GCTCAGCGCC GACCCCGAGC CCGCCCCGGG CGTCGAGCTG
181 AGCTCCCTGC GGCTCCTCAA CTTCACCATG ATGGAGACCG AGTGCACCCC GAGCGCCCGC
241 GTGAACCCCG ATGACTGCGA CTTCAAGGAG AACGGGGTCA TCAAGGAGTG CTCGGGCCCG
301 GTGCAGTTTG GGCAGAGCTC CCCCGAGATC GACCTGCACT GCACCGACGC CTCCTCTGAT
361 CCGGTTCTCA TCCAGCGTGG CCGGTTCGGG CGCTTCCTGG GCAGAATCCG GCGCTTTCGG
421 CCCCGAATCA ACTTCGACAT CCGCGCCCGG GGCTCCATTC GCCTGGGCTG A
<210> 2
<211> 34
<212> PRT
<213>Pigeon（Columba livia ）
<400> 2
Leu 1 Ile 2 Gln 3 Arg 4 Gly 5 Arg 6 Phe 7 Gly 8 Arg 9 Phe 10 Leu 11 Gly 12 Arg 13 Ile 14 Arg 15 Arg 16 Phe 17 Arg 18 Pro 19 Arg 20 Ile 21 Asn 22 Phe 23 Asp 24 Ile 25 Arg 26 Ala 27 Arg 28 Gly 29 Ser 30 Ile 31 Arg 32 Leu 33 Gly 34

Claims (1)

  1. A kind of 1. application of pigeon Cathelicidin-Cl-CATH2 peptides, it is characterised in that：Cl-CATH2 peptides in gene and The pro-inflammatory cytokine induced on protein level by suppressing bacteria lipopolysaccharide：IL-1 β, TNF-α, IL-6, NO generation are applied to system The medicine of standby anti-inflammatory；
    （1）Mouse macrophage RAW264.7 with the DMEM culture mediums containing 10% hyclone 37 C CO2 Trained in incubator Support, when cell grows to exponential phase in good condition, digested with pancreatin, discard pancreatin, cultivated with the DMEM of hyclone Base blows down attached cell；Cell count after piping and druming uniformly, 1x10 is adjusted to by cell concentration6 /ml；
    （2）Uniform cell will be blown and beaten to be added in 24 orifice plates, per hole 1ml, in 37 C CO2 Overnight incubation makes carefully in incubator Born of the same parents are adherent, then suction out supernatant liquor and discard, add the fresh DMEM culture mediums containing 10% hyclone of 960ul, heliotropism Control group and dosing group add 20ul LPS, add Cl-CATH2 mono- that 20ul is diluted with DMEM and Jing Guo filtration sterilization Rise and be incubated 6h；
    （3）RNA extracts kits extract total serum IgE, synthesize cDNA with single-stranded cDNA synthetic agent box, and pass through real-time quantitative PCR determines the cytokines Tumor Necrosis factor i.e. TNF-α, Interleukin -1β i.e. IL-1 β, interleukin-6 i.e. IL-6 and induction type NO synthase is INOS expression quantity；
    The Cl-CATH2 peptides be by a kind of straight-chain polypeptide of pigeon Cathelicidin gene codes, rich in basic amino acid, The total order of Cl-CATH2 peptides is classified as：Leu-Ile-glutamine-arginine-glycine-Arg-Phe- Glycine-Arg-Phe-leucine-glycine-arginine-isoleucine-Arg-Arg-phenylalanine- Arg-Pro-arginine-Ile-Asn-phenylalanine-aspartic acid-isoleucine-ammonia of arginine-the third Acid-arginine-glycine-serine-ILE-ARG-LEU-glycine.
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CN104725499B (en) * 2015-03-02 2017-12-26 大连理工大学 Application of the Shelled Turtle Trionyx Sinensis Cathelicidin-Ps CATH4 peptides in anti-inflammatory medicaments are prepared

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CN101265296A (en) * 2008-04-08 2008-09-17 中国科学院昆明动物研究所 Reptile cathelicidin antibiotic peptide and derivatives, and application thereof
CN102127549A (en) * 2010-12-22 2011-07-20 山东省农业科学院家禽研究所 Children antimicrobial peptides Cathelicidins and preparation method and applications thereof

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CN101265296A (en) * 2008-04-08 2008-09-17 中国科学院昆明动物研究所 Reptile cathelicidin antibiotic peptide and derivatives, and application thereof
CN102127549A (en) * 2010-12-22 2011-07-20 山东省农业科学院家禽研究所 Children antimicrobial peptides Cathelicidins and preparation method and applications thereof

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Novel Cathelicidins from Pigeon Highlights Evolutionary Convergence in Avain Cathelicidins and Functions in Modulation of Innate Immunity;Yu H.等;《Scientific Reports》;20150721;全文 *
The Cathelicidins – Structure, Function and Evolution;Tomasinsig L.等;《Current Protein and Peptide Science》;20051231;全文 *

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