CN106928334A - About pacify huge julid antithrombotic peptide Joannsin and its application - Google Patents

About pacify huge julid antithrombotic peptide Joannsin and its application Download PDF

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CN106928334A
CN106928334A CN201710144839.6A CN201710144839A CN106928334A CN 106928334 A CN106928334 A CN 106928334A CN 201710144839 A CN201710144839 A CN 201710144839A CN 106928334 A CN106928334 A CN 106928334A
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huge
julid
joannsin
antithrombotic
peptide
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赖仞
栾宁
段自磊
严秀文
莫国香
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Nanjing Agricultural University
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Nanjing Agricultural University
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract

The invention belongs to field of biomedicine technology, specifically related to about pacify huge julid antithrombotic peptide Joannsin and its gene and application, it is a kind of single chain polypeptide for about pacifying huge julid analgesia peptide gene coding, the dalton of molecular weight 8910.9, isoelectric point 7.60 about to pacify huge julid antithrombotic peptide Joannsin.About pacify huge julid antithrombotic peptide Joannsin by SEQ ID NO：Amino acid sequence composition shown in 1.Coding about pacifies the gene of huge julid antithrombotic peptide Joannsin, by SEQ ID NO：Nucleotide sequence composition shown in 2.About pacify applications of the huge julid antithrombotic peptide Joannsin in FXa, trypsin inhibitor and antithrombotic reagent is prepared.The present invention can suppress the activity of trypsin, FXa through the Joannsin recombinant polypeptides that prokaryotic expression is obtained from about pacifying the Joannsin sequences that are separated in huge julid defensive glands, and show extremely significant anti-thrombus function in vivo；The other polypeptide is obtained through prokaryotic expression, easy large-scale industrial production and antithrombotic acitivity is strong, as the reagent for suppressing FXa and can prepare antithrombotic reagent.

Description

About pacify huge julid antithrombotic peptide Joannsin and its application
Technical field
The present invention provides one kind and about pacifies huge julid (Prospirobolus joannsi) antithrombotic peptide Joannsin and its volume Code gene and application, belong to field of biomedicine technology.
Background technology
Protease is widely present in from virus, all biologies of bacterium to mammal, and in many physiological reactions Play an important role, for example food digestion, blood coagulation, tissue reconstruction, immune defense etc., in these physiology courses, protease Adjust a series of intracellulars and extracellular proteolytic cascade reaction.Protease is once activated, will cause it is a series of rapid and Irreversible specific cell reaction.Although protease is organism physiology, and reaction is necessary, the protease of excess is to body egg Environment is but a kind of potential danger in white matter, so protease inhibitors is indispensable to biological stable state, while In many venomous animals, protease inhibitors play an important role in predator as its predation or is resisted.
At present, four kinds of protease inhibitors are found that altogether in nature：Suppress trypsase, chymotrypsin, bullet Property protease and Stuart factor etc. serpin, suppress papain, ficin and pineapple egg The cystatin of white enzyme etc., suppresses the metalloproteinases suppression of clostridiopetidase A, aminopeptidase and thermolysin etc. Preparation, and inhibiting cathepsin D, retrotransposon protein enzyme and pepsin etc. asparaginic acid protease inhibitors, wherein, silk The most species of serine protease inhibitor.Disulfide bond and effect position according to sequence homology, cysteine number and in molecule Topographical relationships between point, serpin is divided into Serpin, TIL (trypsin inhibitor like again Cysteine rich domain), several families such as Kazal, Kunitz and Bowman-Birk.
Blood coagulation system includes endogenous pathway and exogenous cruor pathway.Intrinsic coagulation pathway：Refer to from the activation of the factor Ⅻ, To the process of tenase.When vascular wall is damaged, subendothelial tissue exposure, negatively charged subendothelial collagen fiber with Clotting factor is contacted, and the factor Ⅻ is i.e. in combination, and it is Ⅻ a to be activated in the presence of HK and PK.It is being independent of the bar of calcium ion Under part, a of the factor Ⅻ activates the factor Ⅺ.In the presence of calcium ion, Ⅺ a of activation have activated factor Ⅸ again.Single Ⅸ a swashs The effect of factor X living is at a fairly low, and it will combine to form 1 with VIII a：1 compound, is also called factor X multienzyme complexs.This reaction There must also be Ca2+Participated in jointly with PL；Exogenous cruor pathway：Refer to that the clotting factor of participation is not all present in blood In, also external clotting factor participates in hemostasis.This process is started exposed to blood from tissue factor, to F10 quilt The process of activation.Intrinsic coagulation pathway and exogenous cruor pathway are completed by the common coagulation pathway of Ⅹ a, fibrin ferment Last coagulation process；Coagulation pathway is made up of various clotting factor, and many clotting factor are all serine proteases, so silk Serine protease inhibitor may play certain function during anticoagulation.
Because extrinsic coagulation system and intrinsic coagulation system all meet at the common coagulation pathway of Ⅹ a, fibrin ferment, institute Suppress Ⅹ a, fibrin ferment to seem with the selection of anticoagulation antithrombotic reagent is an excellent selection, but clinical effectiveness shows, suppresses Ⅹ a, fibrin ferment these common coagulation pathways, long-time medication may result in coagulation disorders, cause bleeding, so logical It is perhaps a preferably selection to cross the other clotting factor of targeting.
Recent studies have shown that suppressing coagulation process by targetting FXII, influence will not be produced on the stable state of hematological system, Infestin-4 is the most strong FXII inhibitor for finding till now, and research shows that it will not cause bleeding as anticoagulation Deng；The antibody of FXII can also produce antithrombotic effect without producing the risk of bleeding simultaneously.
Protease inhibitors is also widely present in the organisms such as animal, plant, fungi, bacterium and virus, by excavating Biomass resource, and isolated and purified out from biology the blood coagulation that is directed in intrinsic coagulation pathway and exogenous cruor pathway because The protease inhibitors of son, plays an important role in the research and development of antithrombotic reagent.
Julid (millipede) is also named thousand-legger, thousand pin worms, arm beam of steelyard worm.Julid belongs to invertebrate, Myriapoda, times Sufficient subclass, body segment composition.It is about 20~35 centimetres, crineous, back side both sides and step limb helvolus.It is named at present to have Kind more than 12000, it has kind more than 80000 to estimate the whole world.It can be found that there is many protease inhibitors from the genome of julid, But the protide or polypeptide bioactive molecule in julid yet there are no research, so by transcript profile proteomics point Analysis perhaps it can be found that in julid individual defense function protein molecular, make it can also not only by smell when meeting predator It is enough that the effect resisted is played by these polypeptide toxins.It is very strong that we identify an effect from huge julid is about pacified first FXa and trypsin inhibitor Joannsin.
Polypeptide Joannsin of the present invention being capable of inhibition thrombosis.Confirm that it has by animal model very strong Antithrombotic acitivity.About pacify huge julid antithrombotic peptide Joannsin complete sequence amino acid structures through Protein Data Bank by of the invention Enter line search to compare, find no any phase homopolypeptide.About pacify huge julid antithrombotic peptide Joannsin codings base by of the invention Compare because entering line search through gene database, find no any homologous genes.
The content of the invention
For above-mentioned technical problem, the present invention proposes that one kind about pacifies huge julid antithrombotic peptide Joannsin and its encoding gene And application.
It is a kind of single chain polypeptide for about pacifying huge julid antithrombotic peptide gene coding about to pacify huge julid antithrombotic peptide Joannsin, The dalton of molecular weight 8910.9, about pacifies amino acid sequence (the EQ ID NO of huge julid antithrombotic peptide Joannsin：1) it is： QAWQNYYRPS RAGYSYCYDD YDIGPCRARF RQWYYNRRTG ECEIFFYGGCLGNDNKYESK EECEYICKKL LT。
The clone for about pacifying huge julid antithrombotic peptide gene includes：
About pacify huge julid defensive glands Total RNAs extraction, mRNA purifying, mRNA reverse transcriptions and cDNA library build, and design primer, About pacify huge julid antithrombotic peptide gene using PCR method screening.Amplimer length is 20 nucleotides, and its sequence is 5 ' Another amplimer of CGTACTGCCGTGGAACTTC 3 ', PCR is 3 ' PCR Primer primers, and its sequence is 5 ' CCGAGGTTTGGTGGCTCATT 3’.Obtained positive monoclonal carries out gene nucleotide series measure.Gene sequencing result shows Coding about pacifies being made up of 659 nucleotides for huge julid antithrombotic peptide, from 5 ' ends to 3 ' terminal sequences (nucleotide sequence SEQ ID NO：2) it is：
TCAGTCACAGGACTTAATATCTGTCGAACCTTAAGACGTACTGCCGTGGAACTTCCAAAATTTCTTCAT CTTTGCATGTACAGTAGCTCATATAAATTTTTCTGATTTTGAGACCGATTTAGGAAGGGAGAAAATAAATCAAGTCC AGTTTGTGAATTCTTAAAAACAAAGATGGACAATAGGTTTGTGGCACTGCTTACCGTCTTAGTCATCATGCACACAC TGACATTCAGCAGAGGTCAAGCATGGCAGAATTACTATAGGCCTAGCCGTGCCGGTTATTCATACTGCTACGATGAC TACGACATTGGGCCCTGCAGAGCCCGTTTTCGTCAGTGGTATTATAATAGACGTACCGGTGAATGTGAAATCTTTTT CTACGGGGGATGTTTGGGAAACGACAATAAATATGAGTCAAAAGAAGAATGCGAATATATCTGTAAGAAATTGCTCA CGTAGCAAGAGTGTTCACTAATTATGACTGTTGACGAACACGTGTTAATTTGTTCAAGCAGTAAACGGATCATGACA GCACAATGAAATATAAAATTGTTGGAACTGTTACTCATGATTGGATTTTGATAAGTGTTGCCTTTTCACGGTGTTTC ATAAACCATGTTTTAACAGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
The amino acid sequence of polypeptide of this nucleotide sequence transcription is：
MDNRFVALLTVLVIMHTLTFSRGQAWQNYYRPSRAGYSYCYDDYDIGPCRARFRQWYYNRRTGECEIFF YGGCLGNDNKYESKEECEYICKKLLT
Wherein the fragment of 24-95 is for about the huge julid antithrombotic peptide mature peptide of peace.About pacify huge julid antithrombotic peptide gene conduct Genetic engineering prepares the application for about pacifying huge julid antithrombotic peptide.
About pacify prokaryotic expression and the purifying of huge julid antithrombotic peptide：
The nucleotide sequence of gene chemical synthesis Joannsin mature peptides is simultaneously connected to Pet-32a expression vectors and is transferred to BL21 Bacterial strain, uses IPTG induced expressions, and by nickel affinity chromatography chromatogram, gel permeation chromatography, high pressure liquid chromatography etc. is purified, most Its molecular weight and purity are determined by MALDI-TOF-MS (MALDI-TOF).
The beneficial effects of the present invention are：From about pacifying the Joannsin sequences being separated in huge julid defensive glands through protokaryon table Can suppress trypsin, the activity of FXa up to the Joannsin recombinant polypeptides for obtaining, and show extremely significant anti-blood in vivo Bolt function；The other polypeptide is obtained through prokaryotic expression, easy large-scale industrial production and antithrombotic acitivity is strong, being capable of conduct Suppress the reagent of FXa and prepare antithrombotic reagent.
Brief description of the drawings
Fig. 1 is for about action effect figures of the huge julid antithrombotic peptide Joannsin of peace to various clotting factor；
Fig. 2 is for about the internal anti thrombotic action design sketch for pacifying huge julid antithrombotic peptide Joannsin：A：Joannsin is acted on Rat-tail thrombus length statistics after 12 hours；B：Joannsin acts on rat-tail thrombus length statistics after 24 lab scales；C：Locate after 24 hours Rat-tail thrombus measurement contrast between dead mouse, each group.
Specific embodiment
The present invention is further described below by embodiment, but present disclosure is not limited thereto.
About pacify huge julid antithrombotic peptide gene clone：
1. huge julid defensive glands Total RNAs extraction is about pacified：
(1) huge julid is about pacified in longitudinal direction dissection in an aseptic environment, has anti-in each section body segment both sides for about pacifying huge julid Imperial gland, defensive glands are taken out with tweezers, are put into the mortar for filling liquid nitrogen rapidly, are continuously added liquid nitrogen and are fully ground.Powder to be ground into After last shape, 1mL Trizol Extraction buffers (Invitrogen) are added, be co-mulled and made into liquid nitrogen.Treat that Trizol dissolves, by mortar Middle whole liquid are suctioned out into 1.5mL centrifuge tubes, and room temperature places 5min.
(2) 200 μ L chloroforms are added, acutely concussion mixes 15s, and room temperature places 5min；4 DEG C, 12000g centrifugations 10min；Inhale In taking upper strata colourless aqueous phase to new 1.5mL centrifuge tubes (it is as far as possible to inhale supernatant liquids more, but must avoid being drawn onto middle lower floor, in Layer white is albumen precipitation, and pink lower floor is phenol-chloroform).
(3) 500 μ L, 4 DEG C of pre- cold isopropanols are added, is gently overturned and is mixed, room temperature places 5min, 4 DEG C, 12000g centrifugations 10min, ttom of pipe microprecipitation is RNA, supernatant is slightly sucked with liquid-transfering gun and is discarded.
(4) precipitation adds the pre-cooled ethanol washings on ice of 1mL 75%, and 4 DEG C, 7500g centrifugation 5min abandon supernatant, repeat two It is secondary；5-10min is placed in super-clean bench, is dried to without ethanol flavor, that is, be for about to pacify huge julid defensive glands total serum IgE；Managed to the EP for drying 30 μ L 0.1%DEPC water of middle addition, slight concussion dissolving RNA.Therefrom draw 1 μ L plus 49 μ L DEPC water, in 230nm, Its light absorption value is examined at 260nm, 280nm wavelength.10 μ L are taken for 1% agarose gel electrophoresis.Remaining total serum IgE freezes in -80 DEG C.
2. the synthesis of huge julid defensive glands cDNA double-strands is about pacified：
According to CreatorTM SMARTTMCDNA Library Construction Kit (Clontech) specification is grasped Build and about pacify huge julid defensive glands cDNA library.Concrete operations are as follows：
(1) chains of cDNA first synthesis (mRNA reverse transcriptions)
(2) 2 μ L are added about to pacify huge julid defensive glands total serum IgE, 1 μ L SMART in without RNase PCR pipesTMⅣ Oligonucleotide, 1 μ L CDS III/3 ' Primer, plus 1 μ L DEPC water make cumulative volume reach 5 μ L, mix and are centrifuged 10s；72 DEG C of insulation 2min, are incubated 2min on ice；The chain buffer of 2 μ L 5 × the first, 1 μ L are continuously added in PCR pipe above 20mM DTT, 1 μ L 10mM dNTP mixtures, 1 μ L PowerScript reverse transcriptases, mix and are centrifuged 10s.42 in PCR instrument DEG C insulation 1h, on ice terminating reaction.
(3) chains of cDNA second are expanded using end polymeric PCR (LD-PCR) method long
By 1 the first chains of μ L cDNA (mRNA reverse transcriptions), 40 μ L deionized waters, 5 μ L 10 × buffer buffer solutions, 1 μ L50 × dNTP mixtures, the PCR primer of 1 μ L 5 ', 1 μ L CDS III/3 ' PCR primers and 1 μ L polymerases are mixed in PCR pipe It is even.Enter performing PCR by following procedure to expand：
①95℃ 1min
2. 20 circulations：
95℃ 15sec
65℃ 30sec
68℃ 6min
After amplification terminates, synthetic cDNA double-strand PCR pipes are distributed into 10 μ L and often manage, and taking out 5 μ L carries out 1% agarose Electrophoresis, remaining is placed in -80 DEG C of preservations at once.
3. the preparation of bacillus coli DH 5 alpha competent cell：
(1) the single DH5 α bacterium colonies of picking, are inoculated in LB fluid nutrient mediums of the 1mL without ampicillin, 37 DEG C of cultures Overnight, next day take above-mentioned bacterium solution in proportion 1：100 are inoculated in 1mL LB nutrient solutions, 37 DEG C of vibration 2h.(2) OD is worked as600Reach When 0.35, bacterium solution is placed into 10min on ice makes culture be cooled to 0 DEG C.
(3) 4 DEG C, 5000rpm centrifugation 5min, to reclaim cell.
(4) nutrient solution is poured out, pipe is inverted l min so that last trace nutrient solution flows to end.
(5) the 0.1M CaCl of 600 μ L precoolings are added per 1mL initial incubations liquid2-MgCl2Solution (80mM MgCl2,20mM CaCl2) resuspended every part of cell precipitation.
(6) 4 DEG C, 5000rpm centrifugation 5min, to reclaim cell.
(7) nutrient solution is poured out, pipe is inverted l min so that last trace of liquid is flow to end.
(8) the ice-cold 0.1M CaCl of 60 μ L are added per 1mL initial incubations thing2Re-suspended cell is precipitated.4 DEG C of refrigerators are put Put 10-18h.
The screening in 4.cDNA libraries：
(1) synthesis of specific primer
Two primers are designed using primer blast.
(2) target sequence is cloned from julid defensive glands cDNA
20 μ L systems：Each 0.4 μ L, dNTP0.4 μ L, buffer2 μ L, Mg of Tag enzymes 0.1 μ L, CDSIII, SMART42+1.2μ The μ L of L, PCR water 16
PCR conditions：1 predegeneration：95 DEG C, 5min
(3) connection of sequence, conversion and detection.0.2 μ L Takara PMD19-T carriers are added in microcentrifugal tube, 2.3 μ L DNA double chains；Add the ligase buffer solution mixture (dissolving on ice) of 2.5 μ L equivalent；16 DEG C of reactions are overnight；Whole 5 μ L connection products are added in 60 μ L DH5 α competent cells, and 30min is placed on ice；42 DEG C of heat shock 90s, put down gently in being incubated 3- on ice 5min, the LB culture mediums for adding warm bath to cross, 37 DEG C, 80rpm shakes bacterium 45min；Take 100 μ L coatings and contain 100ug/ml AMP's In LB culture mediums, 37 DEG C of culture 16h；After bacterium colony grows, choosing monoclonal carries out 10 μ L bacterium solutions PCR.
5. picking monoclonal is sequenced and sequence screening：
20 single bacterium colonies close with target sequence nucleotides size of picking send the sequencing company to carry out DNA sequencing.Using ABI3730 sequencers.Sequencing primer is M13 (+)：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’,M13(-)：5’- GAGCGGATAACAATTTCACACAGG-3’.Sequencing result carries out sequence screening.
6. huge julid antithrombotic peptide Joannsin gene sequencings are about pacified
Extract DNA dideoxy and determine nucleotide sequence, the use of instrument is U.S. Applied The full-automatic nucleotide sequencing instrument of Biosystems373A, sequencing primer is BcaBESTTM Sequencing Primer RV- M and BcaBESTTMSequencing Primer M13-47, BcaBESTTMSequencing Primer RV-M sequences：5` GAGCGGATAACAATTTCACACAGG 3 ', BcaBESTTMSequencing Primer M13-47：5’ CGCCAGGGTTTTCCCAGTCACGAC 3’.Gene sequencing result is from 5 ' ends to 3 ' terminal sequences (nucleotide sequence SEQ ID NO： 2) it is：
TCAGTCACAGGACTTAATATCTGTCGAACCTTAAGACGTACTGCCGTGGAACTTCCAAAATTTCTTCAT CTTTGCATGTACAGTAGCTCATATAAATTTTTCTGATTTTGAGACCGATTTAGGAAGGGAGAAAATAAATCAAGTCC AGTTTGTGAATTCTTAAAAACAAAGATGGACAATAGGTTTGTGGCACTGCTTACCGTCTTAGTCATCATGCACACAC TGACATTCAGCAGAGGTCAAGCATGGCAGAATTACTATAGGCCTAGCCGTGCCGGTTATTCATACTGCTACGATGAC TACGACATTGGGCCCTGCAGAGCCCGTTTTCGTCAGTGGTATTATAATAGACGTACCGGTGAATGTGAAATCTTTTT CTACGGGGGATGTTTGGGAAACGACAATAAATATGAGTCAAAAGAAGAATGCGAATATATCTGTAAGAAATTGCTCA CGTAGCAAGAGTGTTCACTAATTATGACTGTTGACGAACACGTGTTAATTTGTTCAAGCAGTAAACGGATCATGACA GCACAATGAAATATAAAATTGTTGGAACTGTTACTCATGATTGGATTTTGATAAGTGTTGCCTTTTCACGGTGTTTC ATAAACCATGTTTTAACAGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
The sequence table for about pacifying huge julid antithrombotic peptide Joannsin gene nucleotides is：Sequence length is 659 bases, sequence Row type：Nucleic acid, chain number：It is single-stranded, topology：Straight-chain, sequence species：CDNA, source：About pacify huge julid defensive glands.
That coding about pacifies huge julid maturation antithrombotic peptide Joannsin is nucleotide sequence SEQ ID NO：2 241- 456 nucleotides.About pacify amino acid sequence (the SEQ ID NO of huge julid antithrombotic peptide Joannsin：1) it is：QAWQNYYRPS RAGYSYCYDD YDIGPCRARF RQWYYNRRTG ECEIFFYGGC LGNDNKYESK EECEYICKKL LT。
About pacify huge julid antithrombotic peptide Joannsin genes and about pacify answering for huge julid antithrombotic peptide as genetic engineering preparation With.
About pacify huge julid antithrombotic peptide Joannsin, the application in FXa inhibitor and antithrombotic reagent is prepared.
About pacify prokaryotic expression and the purifying of huge julid antithrombotic peptide Joannsin
1. the structure of prokaryotic expression carrier
2. the prokaryotic expression of huge julid antithrombotic peptide Joannsin is about pacified
(1) microorganism collection
After the carrier bacterium solution that will be obtained stroke flat board obtains monoclonal, the sequencing of picking monoclonal, checking carrier correct sequence, And monoclonal thalline is frozen under certain glycerol concentration.A small amount of induced expression is carried out first, in different IPTG concentration, temperature And under the conditions of the time, grope most suitable inductive condition.After preliminary experiment terminates, a large amount of induced expressions are carried out using optimal conditions. Expression terminates, centrifugation, discards supernatant, collects thalline, and with binding buffer that thalline is resuspended.
(2) soluble analysis of recombinant protein
Thalline re-suspension liquid is carried out into ultrasonic (on ice) with smudge cells, full cell, supernatant is taken after being centrifuged again respectively and is sunk Forming sediment, it is soluble to verify it to run SDS-PAGE glue.
3. the purifying of huge julid antithrombotic peptide Joannsin is about pacified in restructuring
(1) affinity chromatography of recombinant protein and formic acid cut
Will it is broken after thalline supernatant with 0.45 μm of membrane filtration after, be splined on and balanced with Binding Buffer His Bind Resin affinity columns, and run the sample under SDS-PAGE glue verifies different buffer solution elutions.
Formic acid cuts：Fusion protein under eluting adds the formic acid cutting of 50% (v/v) to cut off fusion protein label. Sample drying after cutting is verified with removing formic acid with SDS-PAGE glue.
(2) Sephadex G-50 sephadexes purifying
Formic acid cleaved products are taken into 2ml and is splined on the equilibrated Sephadex of PBS (0.1mol/L, pH 6.0) buffer solution G-50 (Amersham Bioscience) gel column (26mm × 100cm).Eluted with the level pad of same concentration, flow velocity It is 0.3ml/min, 3ml/ pipes collect (Qingpu Shanghai Hu Xi instrument plants) using CBS-A program control automatics fraction collector.Use Μ Ltrospec 2100pro spectrophotometers (Amersham Biosciences) determine 280nm and 215nm values.Collect each peak Component be stored in -20 DEG C it is standby, and verified with SDS-PAGE.
(3) RP-HPLC purifying
The purpose peak that G50 sephadexes are collected into uses reversed phase high-pressure chromatogram (Waters 1525Binary HPLC Pump)C4Post (10 × 250mm of Lichrospher) continues to separate；Solvent orange 2 A：The ultra-pure water solution of 0.1%TFA, solvent B： The acetonitrile solution of 0.1%TFA.Wash-out uses linear concentration gradient：0-10min, B：0%；10-70min, B：0-60%；60- 70min, B：70-100%, flow velocity is 1.5mL/min.Peak collect using Waters 2489 it is visible/UV-detector detect (215nm/280nm), each peak is a collection unit.
(4) MALDI-TOF Mass Spectrometric Identifications
The voltage crest high collected is carried out into Mass Spectrometric Identification.
4. the pharmacological evaluation of huge julid antithrombotic peptide Joannsin is about pacified
(1) enzyme kinetics：The FXIIa of 10 μ L samples and the 10 final concentration of 0.1nM of μ L is mixed in 40 μ L and delays in 96 orifice plates In fliud flushing (0.05M Tris-HCl, pH 7.8).After room temperature is placed 5 minutes, add 30 μ L buffer solutions final concentration of with 10 μ L The mixed liquor of 0.05mM chromophoric substrates, final volume is 100 μ L.The dynamics of Coagulation test using Epoch (BioTek) ELIASA, The software detection OD of GEN CHS 1.09405, 20min, interval 30s.Sample concentration is 0.3 μM.Joannsin to FXIIa, The unrestraints such as kallikrein are acted on.FXa and trypsin can be suppressed.It is computed suppressions of the Joannsin to FXa and trypsin Constant processed is respectively 29.5 ± 4.7nM, 182.7 ± 14.6nM.
(2) APTT and PT is tested：
APTT：APTT reagents are balanced to room temperature, is gently inverted and is mixed APTT reagents.50 μ L APTT reagents, 40 μ L remove blood After platelet-poor plasma (PPP) and 10 μ L samples are mixed, it is incubated 3 minutes in 37 DEG C of water-bath, adds the 0.025M of 50 μ L preheatings CaCl2Solution, is mixed immediately, and detection OD is remembered with ELIASA650。
PT：37 DEG C preheat factor reagent (PT) 15 minutes.40 μ L PDPs (PPP) are with 10 μ L samples 37 It is incubated 3 minutes in DEG C water-bath, adds the μ L of factor reagent 50 of preheating, mixed immediately, OD is recorded with ELIASA650。
Joannsin has effect to APTT and PT, shows that Joannsin can suppress external source and intrinsic coagulation pathway, Be FXa inhibitor result it is consistent.
(3) carragheen causes rat-tail thrombus model：Experimental animal kunming mice, male, body weight 20-25g (unming Medical College Experimental Animal Center is provided), after raising one week, random packet (n=8).Respectively saline control group, sample sets：2mg/ Kg, 4mg/kg, 6mg/kg, positive controls：0.2mg/kg Apixaban(MedChem Express).Tail vein administration is processed After 30min, with the dosage of 60mg/kg, from mouse peritoneal injection carragheen, (carrageenan, type I, Sigma, use physiology salt Concentration of the water dissolves into 1%), due at low ambient temperatures, thrombosis rate>90%, so raising temperature is 17.5 DEG C.12、 Thrombotic average length is determined according to tail skin color change after 24h.Increase over time, each group thrombus it is average Length is both increased, and sample sets length is slightly increased.In each time of measuring point, relative to control group, 2mg/kg's Joannsin and 0.2mg/kg Apixaban influences thrombotic on rat-tail are all meaningful；And relative to 0.2mg/kg Apixaban groups, the Joannsin of 4mg/kg can reach approximate equal level, and the Joannsin of 6mg/kg can then be formed More preferable inhibition, as shown in Figure 2.Data processing is Graphpad Prime5, statistical average ± SD, t-test (* p< 0.05)。
Sequence table
<110>Agricultural University Of Nanjing
<120>About pacify huge julid antithrombotic peptide Joannsin and its application
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<170> PatentIn version 3.5
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<212> PRT
<213>About pacify huge julid antithrombotic peptide Joannsin
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Gln Ala Trp Gln Asn Tyr Tyr Arg Pro Ser Arg Ala Gly Tyr Ser Tyr
Cys Tyr Asp Asp Tyr Asp Ile Gly Pro Cys Arg Ala Arg Phe Arg Gln
Trp Tyr Tyr Asn Arg Arg Thr Gly Glu Cys Glu Ile Phe Phe Tyr Gly
Gly Cys Leu Gly Asn Asp Asn Lys Tyr Glu Ser Lys Glu Glu Cys Glu
Tyr Ile Cys Lys Lys Leu Leu Thr 72
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<211> 659
<212> DNA
<213>About pacify huge julid antithrombotic peptide Joannsin genes
<400> 2
tcagtcacaggacttaatatctgtcgaaccttaagacgtactgccgtggaacttccaaaatttcttcatcttt gcatgtacagtagctcatataaatttttctgattttgagaccgatttaggaagggagaaaataaatcaagtccagtt tgtgaattcttaaaaacaaagatggacaataggtttgtggcactgcttaccgtcttagtcatcatgcacacactgac attcagcagaggtcaagcatggcagaattactataggcctagccgtgccggttattcatactgctacgatgactacg acattgggccctgcagagcccgttttcgtcagtggtattataatagacgtaccggtgaatgtgaaatctttttctac gggggatgtttgggaaacgacaataaatatgagtcaaaagaagaatgcgaatatatctgtaagaaattgctcacgta gcaagagtgttcactaattatgactgttgacgaacacgtgttaatttgttcaagcagtaaacggatcatgacagcac aatgaaatataaaattgttggaactgttactcatgattggattttgataagtgttgccttttcacggtgtttcataa accatgttttaacagacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 659

Claims (9)

1. huge julid antithrombotic peptide Joannsin is about pacified, it is characterised in that：Following polypeptide, the ammonia of polypeptide are included in the antithrombotic peptide Base acid sequence is SEQ ID NO：1.
It is 2. according to claim 1 about to pacify huge julid antithrombotic peptide Joannsin, it is characterised in that：The antithrombotic peptide molecule Measure 8910.9 dalton, isoelectric point 7.60.
3. the pact described in claim 1 pacifies the encoding gene of huge julid antithrombotic peptide Joannsin, it is characterised in that comprising following Nucleotide sequence SEQ ID NO：2.
4. the pact described in claim 1 pacifies the preparation method of the encoding gene of huge julid antithrombotic peptide Joannsin：About pacify huge horse Land defensive glands Total RNAs extraction, mRNA purifying, mRNA reverse transcriptions and cDNA library build, and design primer, are screened using PCR method About pacify huge julid antithrombotic peptide gene；Amplimer length is 20 nucleotides, and its sequence is 5 ' Another amplimer of CGTACTGCCGTGGAACTTC 3 ', PCR is 3 ' PCR Primer primers, and its sequence is 5 ' CCGAGGTTTGGTGGCTCATT 3’；Obtained positive monoclonal carries out gene nucleotide series measure and obtains final product.
5. the pact described in claim 1 pacifies huge julid antithrombotic peptide Joannsin in preparation FXa, trypsin or thrombin suppression Application in preparation.
6. the huge julid antithrombotic peptide Joannsin of pact peace described in claim 1 is preparing antithrombotic, anti-acute cerebral hemorrhage medicine In application.
7. a kind of pharmaceutical composition, it is characterised in that：Pact described in claim 1 containing the upper effective dose for the treatment of pacifies huge julid Antithrombotic peptide Joannsin, or simultaneously contain its pharmaceutically acceptable excipients or additional dose.
8. application of the pharmaceutical composition described in claim 7 in FXa, trypsin or thrombin inhibitor is prepared.
9. application of the pharmaceutical composition described in claim 7 in antithrombotic, anti-acute cerebral hemorrhage medicine is prepared.
CN201710144839.6A 2017-03-13 2017-03-13 About pacify huge julid antithrombotic peptide Joannsin and its application Pending CN106928334A (en)

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CN102199584A (en) * 2011-03-07 2011-09-28 中国科学院昆明动物研究所 Antithrombotic enzyme Eupolytinl from Eupolyphaga sinensis Walker and gene and application thereof
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Application publication date: 20170707