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CN105646702B - Hiruto Kazal type trypsin inhibitor Bdellin-HM and its encoding gene and application - Google Patents

Hiruto Kazal type trypsin inhibitor Bdellin-HM and its encoding gene and application Download PDF

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Publication number
CN105646702B
CN105646702B CN201610226789.1A CN201610226789A CN105646702B CN 105646702 B CN105646702 B CN 105646702B CN 201610226789 A CN201610226789 A CN 201610226789A CN 105646702 B CN105646702 B CN 105646702B
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hiruto
bdellin
trypsin inhibitor
kazal type
application
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CN105646702A (en
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赖仞
龙承波
赖艳梅
李博文
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Kunming Institute of Zoology of CAS
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Kunming Institute of Zoology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8135—Kazal type inhibitors, e.g. pancreatic secretory inhibitor, ovomucoid

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Abstract

A kind of hiruto Kazal type trypsin inhibitor Bdellin-HM and its encoding gene and application, hiruto Kazal type trypsin inhibitor Bdellin-HM, 17432.8 dalton of molecular weight, isoelectric point 4.84, complete sequence is as shown in SEQ ID NO:2, its the 4th with the 29th, the 6th with the 25th, the 14th with the 40th cysteine 3 pairs of intramolecular disulfide bonds of formation.The gene of the coding as shown in SEQ ID NO:1 is made of 504 nucleotide, and it is nucleotide that wherein encoding mature peptide moiety, which is 55-501,.Beneficial effect is: isolating and purifying to obtain hiruto Kazal type trypsin inhibitor Bdellin-HM, and clones and obtain its cDNA sequence；The inhibitor show significant trypsin inhibition activity (K i ~ 8.45nM), but the inhibiting effect of elastoser, chymotrypsin, kallikrein, fibrin ferment, plasmin, FXIIa, FXIa and FXa is not observed, it can be as the application for preparing trypsin inhibitor.

Description

Hiruto Kazal type trypsin inhibitor Bdellin-HM and its encoding gene and Using
Technical field:
The present invention provides a kind of hiruto (Hirudinaria manillensis) Kazal type trypsin inhibitor Bdellin-HM and its encoding gene and application, belong to field of biomedicine technology.
Background technique:
Leeches is a kind of traditional Chinese medicine in China, and the record for leech blood coagulation resisting function is " you of B.C. 2nd century It is refined ", the Compendium of Material Medica of Ming Dynasty's Li Shizhen (1518-1593 A.D.) and Shennong's Herbal.Compendium of Material Medica calls it: " salty flavor acting on blood, hardship victory blood.Leech it Salty hardship, to be Liver Channel blood system medicine, therefore the poly- blood of Liver Channel can be led to except blood is stored." Chinese medicine thinks that leech is a kind of traditional stasis-breaking drug, have The effect of by the stasis of blood, qualcomm meridian, dredging water passages, is mainly used for treating the diseases such as blood stasis, hemiplegia, traumatic injury.Leeches is treated Method has very long history, just there is the picture treated using leeches in B.C. 1500 Egyptian Pharaoh's mausoleums.2005, Europe Official approval leeches therapy in continent is legal treatment means.Often be only just has 350,000 leech to be used for medical treatment in Germany.
Hiruto (Hirudinaria manillensis), the quasi- doctor leech of alias horse Buddhist nun, is commonly called as phnom penh leech.Hirudinea cures leech Section's Poecilobdella is the biggish kind of individual in current common leeches of sucking blood.The type locality of the species is in Luzon, Philippine.Point It is distributed in Indonesia, Philippine, India, Bangladesh, Sri Lanka, Burma, Thailand, Vietnam, the island of Taiwan and China's Mainland The ground such as Fujian, Guangdong, Guangxi, Hainan.In the paddy field, ditch or pond that mainly inhabit faintly acid (pH value 5.5~7.0). When people and animals pass through, blood is sucked with regard to attachment.Hiruto can secrete anticoagulant substance, destroy coagulation function, therefore by luxuriant and rich with fragrance ox The wound that leech stung often is bled incessantly.Civil also to utilize this property, the regional flow that patient is treated with hiruto is unsmooth, but It is that people are also knows about seldom the material base and molecular mechanism of these therapeutic effects.
Serpin (serine protease inhibitor) is distributed widely in animals and plants and microorganism In, existing more than 500 family members are found.By adjusting the activity of serine protease, serpin It participates in adjusting many important vital movement processes in vivo, there are the physiological activity such as anticoagulant, anti-inflammatory, anti-infective, antitumor, It is widely used in the disease treatments such as acute pancreatitis, surgical operation, tumour, cranial vascular disease.In addition, in blood coagulation system and complement The protease to play a role in system also can by serpin regulation to avoid its proteinase activity to surrounding Tissue damages.Kunitz, Kazal, Bowman-Birk, a-2-macroglobulin and serpin are that research is relatively broad Serpin type.
Kazal type serpin LDTI from Hirudo medicinalis is first man class mast cell class pancreas egg The protease inhibitors that white enzyme is combined closely may roar as a pharmacology probe to assess mast cell tryptase Pathophysiological role in asthma, arthritis, periodontal disease, skin disease and blood coagulation disorder.In addition it is sent out from Hirudo medicinalis The Bdellin-KL found in existing Bdellin B-3 and Hirudo japonica is shown to trypsase, plasmin With the inhibiting effect of acrosin, imply that they may work during biological self reproducing.
Currently, both at home and abroad there is not yet the related research of hiruto Kazal type serpin is reported
Inventor is by hiruto Kazal type trypsin inhibitor Bdellin-HM complete sequence amino acid structure of the invention Search comparison is carried out through Protein Data Bank, finds no any phase homopolypeptide.Inventor is by hiruto Kazal type of the invention Trypsin inhibitor Bdellin-HM encoding gene carries out search comparison through gene database, finds no any identical base Cause.
Summary of the invention:
The purpose of the present invention is being based on above-mentioned theory research and prior art basis, provide a kind of new with significant pancreas egg The hiruto Kazal type serpin Bdellin-HM and its encoding gene of white enzyme inhibition activity and application.
By the present invention in that with anion-exchange column DEAE Sephadex A-50, reversed high performance liquid chromatography (RP- HPLC)C18Column and Matrix-Assisted Laser Desorption Ionization Time of Flight, which isolate and purify, obtains hiruto trypsin inhibitor Bdellin-HM.The N- end part amino acid of coding hiruto Kazal type trypsin inhibitor is measured according to Edman edman degradation Edman Sequence, then by building hiruto head cDNA library and cDNA library screening is carried out, obtain hiruto Kazal type tryptose The complete sequence and encoding gene of enzyme inhibitor.Hiruto Kazal type trypsin inhibitor is having of finding for the first time The hiruto serine protease of Kazal type serpin feature and significant trypsin inhibition activity inhibits Agent.
Main technical schemes of the invention are as follows:
The isolation and purification method of hiruto:
The hiruto head crude extract of collection crosses anion-exchange column DEAE Sephadex A-50 first, and collection has The peak of trypsin inhibition activity, freeze-drying, excessively reversed high performance liquid chromatography (RP-HPLC) C18Column will finally have trypsase Inhibitory activity peak is detected with Matrix-Assisted Laser Desorption Ionization Time of Flight, and purifying obtains the inhibition of hiruto trypsase Agent.
The clone of hiruto trypsin inhibitor gene includes:
Hiruto head Total RNAs extraction, mRNA purifying, mRNA reverse transcription and cDNA library building, design primer utilize PCR method screens hiruto trypsin inhibitor gene.Two pairs of amplimers are as follows: Primer 15 '- 25 '-AAGCAGTGGTATCAACGCAGAGT-3 ' of GAYWSNGARTGYGTNTGYAC-3 ' and Primer；Primer 3 5'- 45 '-ATTCTAGAGGCCGAGGCGGCCGA-3 ' (R=A/G of CTYACRCANACRTGNTTY-3 ' and Primer；Y=C/T；S =C/G；W=A/T；N=A/C/G/T).Obtained positive monoclonal carries out gene nucleotide series measurement.Gene sequencing result table Bright coding hiruto trypsin inhibitor gene is made of 504 nucleotide, from 5 ' end to 3 ' terminal sequences be (SEQ ID NO: 1):
ATGAAGCTGTTGCTTGCTTTGGCCCTTTTCGGTGCATTTGTCACAATCAACGCCGACTCAGAATGTGTC TGCACCAAAGAATTGAACCAGGTTTGCGGAAGTGATGGACATACCTACGACAACCCTTGTCTAGCTACGTGCCACGG GGCGTCTGTCGCCCACGAACACGCCTGCGAAGGACACGAAGAGGAACATCACGAAGAAGACCACCATGACGGTGAGC ACAATAATGGTGACCACCATGAAGGTGAGCACAATAATGGTGAACACCATGACGACGAGCACAAGGATGACCACCAT GGAGAGGACCACCACGAAGACGGGGAACACAATGAAGAGCACAAGAATGACCATCACGATGATGGTCATGATGATCA TCACAACAATGGCGACCACAAGGATGACCACCATGAAGAAGAACACCATGAAGAAGAACACCACGAAGGGGACCACC ACGAAGAGGAGCATCACGAAGAAGAGCACAAAGACGACCATCACGATTAA
The wherein fragment coding hiruto trypsin inhibitor mature peptide of 55-501.Hiruto trypsin inhibitor base Because preparing the application of hiruto trypsin inhibitor as genetic engineering.
The beneficial effects of the present invention are:
It isolates and purifies to obtain hiruto trypsin inhibitor Bdellin-HM, and its cDNA sequence is obtained by clone； The hiruto trypsin inhibitor shows significant trypsin inhibition activity (Ki~8.45nM), but does not observe elasticity The inhibition of protease, chymotrypsin, kallikrein, fibrin ferment, plasmin, FXIIa, FXIa and FXa is made With can be as the application for preparing trypsin inhibitor.
Detailed description of the invention:
Fig. 1 shows the protease inhibiting activity of hiruto trypsin inhibitor Bdellin-HM.
Fig. 2 shows that the trypsase of hiruto trypsin inhibitor Bdellin-HM inhibits type and inhibition constant.
Specific embodiment:
Below by embodiment, property content is described in further detail for the essence of the present invention.
Hiruto trypsin inhibitor Bdellin-HM is isolated and purified:
I, DEAE Sephadex A-50 anion-exchange column:
The 3g hiruto head crude extract being lyophilized is dissolved in 20ml 50mM Tris hydrochloride buffer (pH 8.9), 12000rpm be centrifuged 10 minutes, take supernatant be splined on balanced DEAE Sephadex A-50 anion-exchange column (5 × 60cm), with the Tris hydrochloride buffer gradient elution of the sodium chloride containing various concentration, 200 pipe of elution is (with automatic fraction collector It is collected, every pipe 15ml, flow velocity 1.5ml/min).Specific sodium chloride concentration are as follows: 0-0.2M NaCl (0,0.2M NaCl it is each 1L), 0.2-0.4M NaCl (0.2, each 1L of 0.4M NaCl), 0.4-0.6M NaCl (0.4, each 1L of 0.6M NaCl), 1M NaCl.The concentration of protein or polypeptide in 280nm and 215nm ultraviolet detection collection liquid, merging, there is trypsase to inhibit to live Property part, freeze-drying, -20 DEG C save backup.
II, reverse phase HPLC chromatography (RP-HPLC):
By DEAE Sephadex A-50 anion-exchange column obtained Peak Activity 2ml Tris hydrochloride buffer (pH 8.9) it re-dissolves, 4 DEG C, 12000rpm is centrifuged 15 minutes, takes supernatant, with 0.45 μm of membrane filtration, is collected filtrate and is splined on Reversed phase high-pressure liquid phase C18Column, with water (contain 0.1% trifluoroacetic acid): the elution system that acetonitrile (containing 0.1% trifluoroacetic acid) is constituted into Row gradient elution, elution speed 1ml/min.Collect the peak with trypsin inhibition activity.
III, Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF-MS) detects
10ul is taken to carry out Matrix-Assisted Laser Desorption Ionization Time of Flight from trypsin inhibition activity peak (MALDI-TOF-MS) detection molecules amount, remaining freeze-drying, -20 DEG C of preservations.
IV, Edman edman degradation Edman is sequenced
By Edman edman degradation Edman to purify resulting trypsin inhibitor sterling carry out the sequencing of the end N- (model491, ABI, the U.S.).
Hiruto trypsin inhibitor gene clone:
I, hiruto head Total RNAs extraction:
A. living body hiruto washes with water completely, is put into liquid nitrogen quick-frozen 4 hours, takes head tissue, and weighing takes 300mg Tissue is added 10ml Total RNAs extraction buffer (Trizol solution, U.S.'s life technologies Products), in 20ml It is homogenized 30 minutes in glass homogenizer.
B. isometric chloroformic solution is added, concussion mixes, and is placed at room temperature for 10 minutes, and 4 DEG C, 12000rpm is centrifuged 10 minutes, Reject precipitating.
C. isometric isopropanol is added in supernatant, is placed at room temperature for 10 minutes, and 4 DEG C, 12000rpm is centrifuged 10 minutes, and precipitating is used 75% ethanol washing three times, dry by room temperature, and tube bottom sediment is hiruto head total serum IgE.
II, the purifying of hiruto head mRNA:
Hiruto head mRNA is isolated and purified using PROMEGA company, the U.S.mRNA Isolation Systems kit.
A. it takes total serum IgE 500 μ g in hiruto head to be dissolved in 500 μ l DEPC water, is put into 65 DEG C of water-baths 10 minutes, adds 3 μ of people Oligo (dT) probe of l and 13 μ l 20 × SSC solution mix, and place room temperature cooling, referred to as A liquid.
B. the washing of magnetic bead (SA-PMP): flicking mixing for magnetic bead, until magnetic frame adsorbs 30 seconds, abandons supernatant, add 0.5 × SSC 0.3m1, until magnetic frame adsorbs 30 seconds, finally plus 0.5 × SSC of 0.1ml suspends, referred to as B liquid.
C. A liquid is added in B liquid, is placed at room temperature for 10 minutes, until magnetic frame adsorbs 30 seconds, abandoned supernatant, washed with 0.1 × SSC It washs 4 times, finally abandons supernatant, add 0.lml DEPC aqueous suspension, until adsorbing 30 seconds on magnetic frame, supernatant is moved to new test tube, then 0.15m1DEPC water is added to suspend again, until magnetic frame adsorbs 30 seconds, moves supernatant to above-mentioned test tube, is then the phenanthrene of purifying in supernatant Ox leech head mRNA.
D. it is added 1/10 volume 3M sodium acetate, pH5.2, isometric isopropanol places 30 minutes in -70 DEG C, and 4 DEG C, 12000rpm is centrifuged 10 minutes, is abandoned supernatant, is precipitated and dissolved in 10 μ l DEPC water.
III, hiruto head cDNA library constructs: in strict accordance with CLONTECH company SMARTTM PCR cDNA Library Construction Kit operational manual.
The first chain of A.cDNA synthesizes (mRNA reverse transcription):
1. 1 μ l hiruto head mRNA, 1 μ l SMART IV oligonucleotide, 1 μ l is added in 0.5ml sterile centrifuge tube CDS III/3 ' PCR primer adds 2 μ l deionized waters that total volume is made to reach 5 μ l.
2. mixing the reagent in centrifuge tube and centrifugation, 72 DEG C are incubated for 2 minutes.Centrifuge tube is incubated on ice 2 minutes.
3. 2 μ l, 5 × the first chain buffer, 1 μ l 20mM dithiothreitol (DTT), 1 μ l are and then added in above-mentioned centrifuge tube 10mM dNTP mixture, 1 μ l PowerScript reverse transcriptase.
4. mixing and being centrifuged, it is incubated for 1 hour at 42 DEG C.Centrifuge tube is placed in stopped reaction on ice.
5. taking the first chain of cDNA synthesized by 2 μ l spare from centrifuge tube.
B. the second chain is expanded using long end polymeric enzyme chain reaction (LD-PCR) method
1.95 DEG C of preheating PCR instruments.
2. by 2 the first chains of μ l cDNA, 80 μ l deionized waters, 10 μ 10 × Advantage of l 2PCR buffering, 2 μ l 50 × DNTP mixture, 2 μ l, 5 ' PCR primer, 2 μ l CDS III/3 ' PCR primers and 2 μ l Escherichia coli polymerases are mixed in centrifuge tube It is even to be expanded.
3. being expanded in PCR instrument by following procedure:
1. 95 DEG C 1 minute
2. 30 circulations:
95 DEG C 15 seconds
65 DEG C 30 seconds
68 DEG C 3 minutes
4. after circulation terminates, the cDNA double-strand synthesized in centrifuge tube is stripped.
C.PCR product PROMEGA companySV Gel and PCR Clean-Up System kit into Row extracting and reclaiming, steps are as follows:
1. isometric film is added in the cDNA double-strand obtained by PCR to be mixed by inversion in conjunction with buffering, then by mixed liquor It is transferred to centrifugal purification column, is stored at room temperature 5 minutes, makes DNA sufficiently in conjunction with pellosil.16,000g centrifugations 1 minute, outwell collection Waste liquid in pipe.
2. the eluent (containing ethyl alcohol) of 700 μ l is added in centrifugal purification column, collecting pipe is outwelled in 16,000g centrifugations 1 minute In waste liquid.
3. repeating step 2.
4.16,000g is centrifuged 5 minutes.
5. centrifugal purification column is placed in new centrifuge tube.
6. 30 μ l ultrapure waters are added, 5 minutes are stood at room temperature.
7.16,000g centrifugations 1 minute, tube bottom solution is the cDNA double-strand of purified mistake.
IV, hiruto trypsin inhibitor gene colony screening:
A. the preparation of bacillus coli DH 5 alpha competent cell:
1. the single DH5 α bacterium colony of picking is inoculated in 1ml LB culture medium not with ampicillin, 37 DEG C of overnight incubations, Next day takes above-mentioned bacterium solution, and 1:100 is inoculated in 1ml LB culture solution in proportion, 37 DEG C shaken cultivation 2-3 hours.Work as OD600Value When reaching 0.5, bacterial cultures is harvested.
2. centrifuge tube is placed 10min on ice, culture is made to be cooled to 0 DEG C.
3. being centrifuged 5min in 4 DEG C with 4100r/min, cell is recycled.
4. pouring out culture solution, pipe is inverted in 1min on filter paper so that last culture solution is flow to end.
5. the CaCl that every 1ml initial incubation object is pre-chilled with 600 μ l2-MgCl2Solution (80mmol/L MgCl2,20mmol/L CaCl2), every part of cell precipitation is resuspended.
6. 5min is centrifuged in 4 DEG C with 4100r/min, to recycle cell.
7. pouring out culture solution, pipe is inverted in 1min on filter paper so that last culture solution is flow to end.
8. the 0.1mol/L CaCl that every 1ml initial incubation object is pre-chilled with 100 μ l2Every part of cell precipitation is resuspended, is placed in 4 It is DEG C spare.
B. target sequence is cloned from hiruto cDNA
Using the cDNA of synthesis as template, two pairs of amplimers: 15 '-GAYWSNGARTGYGTNTGYAC-3 ' of Primer and Primer 2 5'-AAGCAGTGGTATCAACGCAGAGT-3'；35 '-CTYACRCANACRTGNTTY-3 ' of Primer and 45 '-ATTCTAGAGGCCGAGGCGGCCGA-3 ' (R=A/G of Primer；Y=C/T；S=C/G；W=A/T；N=A/C/G/ ), T wherein Primer 1 and Primer 3 is designed according to the N- terminal sequence that Edman edman degradation Edman measures.PCR is reacted in following condition Lower progress: 95 DEG C 30 seconds, 60 DEG C 30 seconds and 72 DEG C 60 seconds, 30 circulation.
C. the conversion of digestion, connection and connection product:
1. 1 μ l Promega company is added in microcentrifugal tubeCarrier, 4 μ l hiruto cDNA double-strands are molten Liquid, full dose are 5 μ l.
2. the ligase buffer mixture of 5 μ l (equivalent) is added.
3.16 DEG C are reacted 2 hours.
4. 5 μ l is taken to be added into 100 μ l DH5 α competent cells, placed 30 minutes in ice.
5.42 DEG C after heating 90 seconds, then place 2 minutes in ice.
6. the LB culture medium 890 μ l for being added that 37 DEG C of warm bath cross, 37 DEG C 100rpm shaken cultivation 60 minutes.
7. taking 200 μ to be coated on the LB culture medium containing X-Gal, IPTG, Amp to cultivate 16 hours for 37 DEG C, form single bacterium It falls.
8. picking monoclonal carries out bacterium solution PCR.
V, hiruto trypsin inhibitor Bdellin-HM gene sequencing and result:
With the full-automatic nucleotide sequencing instrument of U.S. Applied Biosystems 377, sequencing primer M13R: CAGGAAACAGCTATGACC；M13F:TGTAAAACGACGGCCAGT.
It is (SEQ ID NO:1) that gene sequencing result, which is held from 5 ' to 3 ' terminal sequences:
ATGAAGCTGTTGCTTGCTTTGGCCCTTTTCGGTGCATTTGTCACAATCAACGCCGACTCAGAATGTGTC TGCACCAAAGAATTGAACCAGGTTTGCGGAAGTGATGGACATACCTACGACAACCCTTGTCTAGCTACGTGCCACGG GGCGTCTGTCGCCCACGAACACGCCTGCGAAGGACACGAAGAGGAACATCACGAAGAAGACCACCATGACGGTGAGC ACAATAATGGTGACCACCATGAAGGTGAGCACAATAATGGTGAACACCATGACGACGAGCACAAGGATGACCACCAT GGAGAGGACCACCACGAAGACGGGGAACACAATGAAGAGCACAAGAATGACCATCACGATGATGGTCATGATGATCA TCACAACAATGGCGACCACAAGGATGACCACCATGAAGAAGAACACCATGAAGAAGAACACCACGAAGGGGACCACC ACGAAGAGGAGCATCACGAAGAAGAGCACAAAGACGACCATCACGATTAA
Coding hiruto Kazal type serpin Bdellin-HM mature sequence is 55-501 nucleosides Acid, amino acid sequence are (SEQ ID NO:2): NH2-DSECVCTKELNQVCGSDGHTYDNPCLATCHGASVAHEHACE GHEEEHHEEDHHDGEHNNGDHHEGEHNNGEHHDDEHKDDHHGEDHHEDGEHNEEHKNDHHDDGHDDHHNNGDHKDD HHEEEHHEEEHHEGDHHEEEHHEEEHKDDHHD-COOH。
Hiruto trypsin inhibitor Bdellin-HM gene prepares hiruto trypsase as genetic engineering and inhibits The application of agent.
The protease Inhibition test of hiruto trypsin inhibitor Bdellin-HM:
A. reagent and material
1. buffer (pH 7.4)
10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05%v/v Surfactant P20
2. protease and corresponding chromophoric substrate:
400nM trypsase (SIGMA) and 0.2mM Gly-Arg-p-nitroanilide dihydrochloride (SIGMA)
400nM plasmin (SIGMA) and 0.2mM Gly-Arg-p-nitroanilide dihydrochloride(SIGMA)
400nM elastoser (SIGMA) and 0.2mM N-Methoxysuccinyl-Ala-Ala-Pro-Val-p- nitroanilide(SIGMA)
400nM chymotrypsin (SIGMA) and 0.2mM N-Succinyl-Gly-Gly-Phe-p-nitroanilide (SIGMA)
400nM kallikrein, 10nM FXIIa, 400nM FXIa (Enzyme Research Laboratories, USA) with 0.2mM H-D-Pro-Phe-Arg-pNA2HCl (Hyphen Biomed, France)
10nM fibrin ferment (SIGMA) and 0.2mM H-D-Phe-Pip-Arg-pNA.2HCl (Hyphen Biomed, France)
10nM FXa (Enzyme Research Laboratories, USA) and 0.2mM CH3OCO-D-CHA-Gly- Arg-pNA-AcOH(SIGMA)。
B. protease Inhibition test
1. 10 μ L Bdellin-HM (final concentration of 11.5 μM), 10 μ L protease and 40 μ L buffering are added in 96 orifice plates Liquid, 37 DEG C are incubated for 5 minutes.
2. the mixed liquor of 30 μ L buffers and 10 μ L chromophoric substrates is added immediately, final volume is 100 μ L.
3. the dynamics of reaction uses Epoch (Bio-Tek) microplate reader, 1.09 software detection OD405nm of GEN CHS, 30min is spaced 30s.
C. inhibit the measurement of type and inhibition constant to trypsase
Dixon plot curve: the trypsase (0,2.3,4.6,6.9,9.2, and 11.5nM) of various concentration from it is different The Gly-Arg-p-nitroanilide dihydrochloride (131.99 263.98 μM of and) of concentration, for example preceding institute of step It tells.Using inhibitor concentration as abscissa, ordinate is that the reciprocal of reaction rate draws Dixon plot curve.2 straight lines extend The abscissa value of line intersection point is inhibition constant of the inhibitor to trypsase.Intersection point just indicates that it is competing for inhibiting type on the horizontal scale Striving property inhibits, and indicates that Combination inhibits below abscissa.
SEQUENCE LISTING
<110>Kunming Institute of Zoology, Chinese Academy of Sciences
<120>hiruto Kazal type trypsin inhibitor Bdellin-HM and its encoding gene and application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 504
<212> DNA
<213> Hirudinaria manillensis
<220>
<222> (1)..(504)
<400> 1
atgaagctgt tgcttgcttt ggcccttttc ggtgcatttg tcacaatcaa cgccgactca 60
gaatgtgtct gcaccaaaga attgaaccag gtttgcggaa gtgatggaca tacctacgac 120
aacccttgtc tagctacgtg ccacggggcg tctgtcgccc acgaacacgc ctgcgaagga 180
cacgaagagg aacatcacga agaagaccac catgacggtg agcacaataa tggtgaccac 240
catgaaggtg agcacaataa tggtgaacac catgacgacg agcacaagga tgaccaccat 300
ggagaggacc accacgaaga cggggaacac aatgaagagc acaagaatga ccatcacgat 360
gatggtcatg atgatcatca caacaatggc gaccacaagg atgaccacca tgaagaagaa 420
caccatgaag aagaacacca cgaaggggac caccacgaag aggagcatca cgaagaagag 480
cacaaagacg accatcacga ttaa 504
<210> 2
<211> 149
<212> PRT
<213> Hirudinaria manillensis
<220>
<222> (1)..(149)
<400> 2
Asp Ser Glu Cys Val Cys Thr Lys Glu Leu Asn Gln Val Cys Gly Ser
1 5 10 15
Asp Gly His Thr Tyr Asp Asn Pro Cys Leu Ala Thr Cys His Gly Ala
20 25 30
Ser Val Ala His Glu His Ala Cys Glu Gly His Glu Glu Glu His His
35 40 45
Glu Glu Asp His His Asp Gly Glu His Asn Asn Gly Asp His His Glu
50 55 60
Gly Glu His Asn Asn Gly Glu His His Asp Asp Glu His Lys Asp Asp
65 70 75 80
His His Gly Glu Asp His His Glu Asp Gly Glu His Asn Glu Glu His
85 90 95
Lys Asn Asp His His Asp Asp Gly His Asp Asp His His Asn Asn Gly
100 105 110
Asp His Lys Asp Asp His His Glu Glu Glu His His Glu Glu Glu His
115 120 125
His Glu Gly Asp His His Glu Glu Glu His His Glu Glu Glu His Lys
130 135 140
Asp Asp His His Asp
145

Claims (3)

1. hiruto Kazal type serpin Bdellin-HM, it is characterised in that as shown in SEQ ID NO:2 Amino acid sequence composition.
2. encoding the gene of hiruto Kazal type serpin Bdellin-HM, it is characterised in that by SEQ ID The composition of nucleotide sequence shown in NO:1.
3. hiruto Kazal type serpin Bdellin-HM described in claim 1 is preparing trypsase Application in inhibitor.
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