CN105646702A - Hirudinaria manillensis Kazal trypsin inhibitor Bdellin-HM, and encoded gene and application thereof - Google Patents

Abstract

The invention relates to a Kazal-type trypsin inhibitor Bdellin-HM of Hirudinaria? manillensis and its encoding gene and application, belonging to the technical field of biomedicine. The phenanthrene Kazal type trypsin inhibitor Bdellin-HM has a molecular weight of 17432.8 Daltons and an isoelectric point of 4.84. The full sequence is shown as SEQ? ID? As shown in NO:1, the cysteines at the 1st and 5th, 2nd and 4th, and 3rd and 6th positions form 3 pairs of intramolecular disulfide bonds. Such as SEQ? ID? The encoded gene represented by NO:2 consists of 504 nucleotides, wherein the 54th-501st nucleotides encode the mature peptide part. The beneficial effect of the present invention is: separate and purify and obtain Bdellin-HM of phenanthrene Kazal type trypsin inhibitor, and clone and obtain its cDNA sequence; This inhibitor shows remarkable trypsin inhibitory activity (K _i ～8.45nM), but The inhibitory effect of elastase, chymotrypsin, kallikrein, thrombin, plasmin, FXIIa, FXIa and FXa is not observed, and can be used as an application for preparing trypsin inhibitors.

Description

Bdellin-HM and its coding gene and application

Technical field:

The invention provides a Kazal-type trypsin inhibitor Bdellin-HM of Hirudinaria manillensis and its encoding gene and application, belonging to the technical field of biomedicine.

Background technique:

Leeches are a kind of traditional Chinese medicine in my country. The anticoagulant effect of leeches is recorded in "Erya" in the 2nd century BC, "Compendium of Materia Medica" and "Shen Nong's Materia Medica" by Li Shizhen in the Ming Dynasty. "Compendium of Materia Medica" said: "Salty to remove blood, bitterness is better than blood. The salty and bitterness of leeches is used to remove blood accumulation, and it is the liver meridian blood to divide the medicine, so it can pass the liver meridian and gather blood." Chinese medicine believes that leeches are a traditional broken medicine. Blood medicine has the effects of dispelling blood stasis, dredging meridians, and benefiting water channels. It is mainly used to treat blood stasis amenorrhea, stroke hemiplegia, bruises and other diseases. Leech therapy has a long history, and there are pictures of the use of leech therapy in the tombs of Egyptian pharaohs in 1500 BC. In 2005, leech therapy was officially approved as a legal treatment in Europe. Every year 350,000 leeches are used medically in Germany alone.

Filipino leech (Hirudinaria manillensis), also known as Mani leech, commonly known as Phnom Penh leech. Hirudophylum, belonging to the genus Hirudophyllidae, is the largest species among the common blood-sucking leeches. The type locality of this species is Luzon, Philippines. Distributed in Indonesia, Philippines, India, Bangladesh, Sri Lanka, Myanmar, Thailand, Vietnam, Taiwan Island and Fujian, Guangdong, Guangxi, Hainan and other places in mainland China. It mainly inhabits paddy fields, ditches or ponds with weak acidity (pH value 5.5-7.0). When people and animals pass by, they attach and suck blood. The leeches can secrete anticoagulant substances and destroy the blood coagulation function, so the wounds bitten by the leech often bleed profusely. Folks also use this property to treat patients with poor local blood flow, but people still know little about the material basis and molecular mechanism of these therapeutic effects.

Serine protease inhibitors (serine protease inhibitor) are widely distributed in animals, plants and microorganisms, and more than 500 family members have been discovered. By regulating the activity of serine proteases, serine protease inhibitors participate in the regulation of many important life activities in the body, and have physiological activities such as anticoagulation, anti-inflammation, anti-infection, and anti-tumor, and have been widely used in acute pancreatitis, surgery, tumors, etc. , cerebrovascular diseases and other diseases. In addition, proteases that function in the coagulation and complement systems are also regulated by serine protease inhibitors to prevent their protease activity from causing damage to surrounding tissues. Kunitz, Kazal, Bowman-Birk, a-2-macroglobulin and serpin are widely studied types of serine protease inhibitors.

The Kazal-type serine protease inhibitor LDTI from European leech is the first tightly bound protease inhibitor of human mast cell tryptase and may serve as a pharmacological probe to evaluate mast cell tryptase in asthma, arthritis, periodontal Pathophysiological roles in disease, skin disorders and blood coagulation disorders. In addition, BdellinB-3 found in European leech and Bdellin-KL found in Japanese leech both showed inhibitory effects on trypsin, plasmin and sperm head grain protein, suggesting that they may be involved in biological reproduction kick in.

At present, there are no relevant research reports on the Kazal-type serine protease inhibitors

The inventor searched and compared the amino acid structure of the complete sequence of Bdellin-HM, a Kazal-type trypsin inhibitor of the present invention, through protein databases, and found no identical polypeptides. The inventor searched and compared the gene encoding the Bdellin-HM Kazal-type trypsin inhibitor Bdellin-HM of the present invention through a gene database, and found no identical gene.

Invention content:

The object of the present invention is to provide a new Bdellin-HM, a Kazal-type serine protease inhibitor with significant trypsin inhibitory activity, and its coding gene and application based on the above theoretical research and prior art.

In the present invention, Bdellin-HM, an inhibitor of phenanthrene trypsin, is obtained by separating and purifying by using anion exchange column DEAESephadexA-50, reverse high performance liquid chromatography (RP-HPLC) C ₁₈ column and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. According to the Edman degradation method, the N-terminal partial amino acid sequence encoding the Kazal type trypsin inhibitor of Hirudo phenanthae was determined, and then by constructing the cDNA library of Hirudo phenanthae head and screening the cDNA library, the Kazal type trypsin inhibitor of Hirudo phenanthae was obtained. The full sequence of the agent and the coding gene. The Hiruba phenanthrene Kazal-type trypsin inhibitor is the first discovered serine protease inhibitor of Hirudo phenanthrene with the characteristics of a Kazal-type serine protease inhibitor and significant trypsin inhibitory activity.

Main technical scheme of the present invention is as follows:

Separation and purification method of Hirudo phenantha:

The crude extract of the head of Hirudo phenanthae that collects at first passes through anion-exchange column DEAESephadexA-50, collects the peak that has trypsin inhibitory activity, lyophilizes, crosses reverse high-performance liquid chromatography (RP-HPLC) C ₁₈ post, finally will have The peak of trypsin inhibitory activity was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and the trypsin inhibitor of P. phenanthrene was purified.

The cloning of the phenanthrene trypsin inhibitor gene includes:

The total RNA was extracted from the head of Hirudo phenanthae, the mRNA was purified, the mRNA was reverse transcribed and the cDNA library was constructed, the primers were designed, and the trypsin inhibitor gene was screened by PCR method. Two pairs of amplification primers are: Primer15'-GAYWSNGARTGYGTNTGYAC-3' and Primer25'-AAGCAGTGGTATCAACGCAGAGT-3'; Primer35'-CTYACRCANACRTGNTTY-3' and Primer45'-ATTCTAGAGGCCGAGGCGGCCGA-3' (R=A/G; Y=C/T ; S=C/G; W=A/T; N=A/C/G/T). The obtained positive single clones were subjected to gene nucleotide sequence determination. Gene sequencing results show that the gene encoding the phenanthrene trypsin inhibitor consists of 504 nucleotides, and the sequence from the 5' end to the 3' end is (SEQ ID NO: 1):

ATGAAGCTGTTGCTTGCTTTGGCCCTTTTCGGTGCATTTGTCACAATCAACGCCGACTCAGAATGTGTCTGCACCAAAGAATTGAACCAGGTTTGCGGAAGTGATGGACATACCTACGACAACCCTTGTCTAGCTACGTGCCACGGGGCGTCTGTCGCCCACGAACACGCCTGCGAAGGACACGAAGAGGAACATCACGAAGAAGACCACCATGACGGTGAGCACAATAATGGTGACCACCATGAAGGTGAGCACAATAATGGTGAACACCATGACGACGAGCACAAGGATGACCACCATGGAGAGGACCACCACGAAGACGGGGAACACAATGAAGAGCACAAGAATGACCATCACGATGATGGTCATGATGATCATCACAACAATGGCGACCACAAGGATGACCACCATGAAGAAGAACACCATGAAGAAGAACACCACGAAGGGGACCACCACGAAGAGGAGCATCACGAAGAAGAGCACAAAGACGACCATCACGATTAA

The 54-501 fragment encodes the mature peptide of the phenanthrene trypsin inhibitor. The application of the phenanthrene trypsin inhibitor gene as a genetic engineering preparation of the phenanthrene trypsin inhibitor.

The beneficial effects of the present invention are:

The trypsin inhibitor Bdellin-HM was isolated and purified, and its cDNA sequence was obtained by cloning; the trypsin inhibitor Bdellin-HM showed significant trypsin inhibitory activity (Ki ~ 8.45nM), but no elasticity was observed The inhibition of protease, chymotrypsin, kallikrein, thrombin, plasmin, FXIIa, FXIa and FXa can be used as a preparation of trypsin inhibitors.

Description of drawings:

Figure 1 shows the protease inhibitory activity of Bdellin-HM, a trypsin inhibitor of B. phenanthrene.

Fig. 2 shows the type of trypsin inhibition and the inhibition constant of Bdellin-HM, a trypsin inhibitor of B. phenanthrene.

detailed description:

The substantive content of the present invention will be described in further detail below by way of examples.

Separation and purification of phenanthrene trypsin inhibitor Bdellin-HM:

Ⅰ. DEAESephadexA-50 anion exchange column:

Dissolve 3 g of the lyophilized crude extract of Hirudo phenanthae head in 20 ml of 50 mM Tris hydrochloric acid buffer (pH8.9), centrifuge at 12000 rpm for 10 minutes, take the supernatant and load it on a well-balanced DEAESephadexA-50 anion exchange column (5 × 60 cm ), eluted with Tris hydrochloric acid buffer gradient containing different concentrations of sodium chloride, eluted 200 tubes (collected with an automatic part collector, 15 ml per tube, flow rate 1.5 ml/min). The specific sodium chloride concentration is: 0-0.2MNaCl (0, 0.2MNaCl each 1L), 0.2-0.4MNaCl (0.2, 0.4MNaCl each 1L), 0.4-0.6MNaCl (0.4, 0.6MNaCl each 1L), 1MNaCl. The concentration of protein or polypeptide in the collected solution was detected by ultraviolet light at 280nm and 215nm, and the fractions with trypsin inhibitory activity were combined, freeze-dried, and stored at -20°C for later use.

Ⅱ. Reverse High Pressure Liquid Chromatography (RP-HPLC):

Redissolve the active peak obtained from the DEAESephadexA-50 anion exchange column with 2ml Tris hydrochloric acid buffer (pH8.9), centrifuge at 12000rpm for 15 minutes at 4°C, take the supernatant, filter it with a 0.45μm filter membrane, collect the filtrate and load it on Reversed-phase high-pressure liquid phase _C18 column, gradient elution was performed with an elution system composed of water (containing 0.1% trifluoroacetic acid): acetonitrile (containing 0.1% trifluoroacetic acid), and the elution rate was 1 ml/min. Peaks with trypsin inhibitory activity were collected.

Ⅲ. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) detection

10 ul was taken from the peak with trypsin inhibitory activity to detect the molecular weight by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the rest was freeze-dried and stored at -20°C.

Ⅳ. Edman degradation sequencing

N-terminal sequencing (model491, ABI, USA) was performed on the purified trypsin inhibitor by Edman degradation method.

Cloning of the phenanthrene trypsin inhibitor gene:

Ⅰ. Extraction of total RNA from the head of Hirudo phenanthae:

A. The living body of A. phenanthrene was cleaned with water, put into liquid nitrogen and quick-frozen for 4 hours, got head tissue, weighed, got 300mg tissue, added 10ml total RNA extraction buffer (Trizol solution, American lifetechnologies company product), in 20ml Homogenize in a glass homogenizer for 30 minutes.

B. Add an equal volume of chloroform solution, vortex to mix, let stand at room temperature for 10 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C, and discard the precipitate.

C. Add an equal volume of isopropanol to the supernatant, leave it at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, wash the precipitate three times with 75% ethanol, and dry it at room temperature. .

Ⅱ. Purification of the head mRNA of Hirudo phenanthae:

The mRNA separation and purification of the head of Hirudo phenanthae was carried out by the American PROMEGA company. mRNAIsolationSystems kit.

A. Dissolve 500 μg of total RNA in 500 μl of DEPC water in 500 μl of DEPC water, put it in a water bath at 65°C for 10 minutes, add 3 μl of Oligo(dT) probe and 13 μl of 20×SSC solution, mix well, leave it at room temperature to cool, and call it A solution .

B. Washing of magnetic beads (SA-PMP): Flick and mix the magnetic beads until the magnetic stand is absorbed for 30 seconds, discard the supernatant, add 0.5×SSC0.3m1, wait for 30 seconds to absorb on the magnetic stand, and finally add 0.1ml0. Suspend in 5×SSC and call it liquid B.

C. Add liquid A to liquid B, let stand at room temperature for 10 minutes, let the magnetic stand absorb for 30 seconds, discard the supernatant, wash 4 times with 0.1×SSC, finally discard the supernatant, add 0.1ml DEPC water to suspend, and put it on the magnetic stand Adsorb for 30 seconds, move the supernatant to a new test tube, add 0.15m1 DEPC water to re-suspend, absorb on the magnetic stand for 30 seconds, transfer the supernatant to the above test tube, and the supernatant contains the purified head mRNA of B.

D. Add 1/10 volume of 3M sodium acetate, pH 5.2, equal volume of isopropanol, place at -70°C for 30 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C, discard the supernatant, and dissolve the precipitate in 10 μl DEPC water.

Ⅲ. Construction of the cDNA library of the head of Hirudo phenanthae: strictly follow the operating manual of SMART ^TM PCRcDNALibraryConstruction Kit of CLONTECH Company.

A. cDNA first-strand synthesis (mRNA reverse transcription):

1. Add 1 μl phenanthrene head mRNA, 1 μl SMARTIV oligonucleotide, 1 μl CDSIII/3’PCR primer to a 0.5 ml sterile centrifuge tube, and add 2 μl deionized water to make the total volume reach 5 μl.

2. Mix the reagents in the centrifuge tube and centrifuge, and incubate at 72°C for 2 minutes. Incubate the centrifuge tube on ice for 2 minutes.

3. Immediately add 2 μl of 5× first-strand buffer, 1 μl of 20 mM dithiothreitol, 1 μl of 10 mM dNTP mixture, and 1 μl of PowerScript reverse transcriptase to the above-mentioned centrifuge tube.

4. Mix and centrifuge, incubate at 42°C for 1 hour. Place the centrifuge tube on ice to stop the reaction.

5. Take 2 μl of the synthesized cDNA first strand from the centrifuge tube for later use.

B. Amplification of the Second Strand Using the Long-Term Polymerase Chain Reaction (LD-PCR) Method

Preheat the PCR instrument at 1.95°C.

2. Mix 2 μl first strand cDNA, 80 μl deionized water, 10 μl 10×Advantage2 PCR buffer, 2 μl 50×dNTP mixture, 2 μl 5’ PCR primer, 2 μl CDSIII/3’ PCR primer and 2 μl E. coli polymerase in a centrifuge tube for amplification.

3. Amplify in the PCR instrument according to the following procedures:

①1 minute at 95°C

②30 cycles:

95°C for 15 seconds

65°C for 30 seconds

68°C for 3 minutes

4. After the cycle is over, extract the double-strand cDNA synthesized in the centrifuge tube.

C.PCR product with PROMEGA company SVGelandPCClean-UpSystem kit for extraction and recovery, the steps are as follows:

1. Add the double-strand cDNA obtained by PCR into an equal volume of membrane-binding buffer and mix by inversion, then transfer the mixture to a centrifugal purification column, and let it stand at room temperature for 5 minutes to fully bind the DNA to the silica gel membrane. Centrifuge at 16,000g for 1 minute and discard the waste in the collection tube.

2. Add 700 μl of eluent (containing ethanol) to the centrifugal purification column, centrifuge at 16,000 g for 1 minute, and discard the waste liquid in the collection tube.

3. Repeat step 2.

4. Centrifuge at 16,000 g for 5 minutes.

5. Place the spin column into a new centrifuge tube.

6. Add 30 μl of ultrapure water and let stand at room temperature for 5 minutes.

7. Centrifuge at 16,000g for 1 minute, and the solution at the bottom of the tube is the purified cDNA double strand.

Ⅳ. Cloning and Screening of the Trypsin Inhibitor Gene of Hirudo phenanthrene:

A. Preparation of Escherichia coli DH5α competent cells:

1. Pick a single DH5α colony, inoculate it in 1ml of LB medium without ampicillin, and incubate overnight at 37°C. The next day, take the above-mentioned bacteria solution and inoculate it in 1ml of LB medium at a ratio of 1:100, and culture it with shaking at 37°C for 2 -3 hours. Bacterial cultures were harvested when the _OD600 value reached 0.5.

2. Place the centrifuge tube on ice for 10 min to cool the culture to 0°C.

3. Centrifuge at 4100r/min for 5min at 4°C to recover the cells.

4. Pour out the culture solution, and place the tube upside down on the filter paper for 1 min to drain the last culture solution.

5. Resuspend each cell pellet with 600 μl pre-cooled CaCl ₂ -MgCl ₂ solution (80 mmol/LMgCl ₂ , 20 mmol/LCaCl ₂ ) per 1 ml of initial culture.

6. Centrifuge at 4100r/min for 5min at 4°C to recover the cells.

7. Pour out the culture medium and place the tube upside down on the filter paper for 1 min to drain the last culture medium.

8. Resuspend each cell pellet with 100 μl pre-cooled 0.1mol/LCaCl ₂ for each 1 ml of initial culture, and place it at 4°C for use.

B. Cloning of the target sequence from the phenanthrene cDNA

Using the synthesized cDNA as a template, two pairs of amplification primers: Primer15'-GAYWSNGARTGYGTNTGYAC-3' and Primer25'-AAGCAGTGGTATCAACGCAGAGT-3'; Primer35'-CTYACRCANACRTGNTTY-3' and Primer45'-ATTCTAGAGGCCGAGGCGGCCGA-3' (R=A/G ; Y=C/T; S=C/G; W=A/T; N=A/C/G/T), wherein Primer1 and Primer3 were designed according to the N-terminal sequence determined by Edman degradation method. The PCR reaction was performed under the following conditions: 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 60 seconds, 30 cycles.

C. Digestion, ligation and conversion of ligation products:

1. Add 1 μl of Promega’s Carrier, 4 μl phenanthrene cDNA double-strand solution, the total volume is 5 μl.

2. Add 5 [mu]l (equal volume) of ligase buffer mix.

3. React at 16°C for 2 hours.

4. Add 5 μl to 100 μl DH5α competent cells and place on ice for 30 minutes.

After heating at 5.42°C for 90 seconds, place in ice for 2 minutes.

6. Add 890 μl of LB medium warmed at 37°C, and shake at 100 rpm at 37°C for 60 minutes.

7. Take 200μ and spread it on the LB medium containing X-Gal, IPTG, and Amp and culture it at 37°C for 16 hours to form a single colony.

8. Pick a single clone for bacterial liquid PCR.

Ⅴ. Sequence determination and results of Bdellin-HM gene sequence of Bdellin-HM trypsin inhibitor:

U.S. Applied Biosystems 377 automatic nucleotide sequencer was used, and the sequencing primers were M13R: CAGGAAACAGCTATGACC; M13F: TGTAAAACGACGGCCAGT.

The gene sequencing result from the 5' end to the 3' end sequence is (SEQ ID NO: 1):

ATGAAGCTGTTGCTTGCTTTGGCCCTTTTCGGTGCATTTGTCACAATCAACGCCGACTCAGAATGTGTCTGCACCAAAGAATTGAACCAGGTTTGCGGAAGTGATGGACATACCTACGACAACCCTTGTCTAGCTACGTGCCACGGGGCGTCTGTCGCCCACGAACACGCCTGCGAAGGACACGAAGAGGAACATCACGAAGAAGACCACCATGACGGTGAGCACAATAATGGTGACCACCATGAAGGTGAGCACAATAATGGTGAACACCATGACGACGAGCACAAGGATGACCACCATGGAGAGGACCACCACGAAGACGGGGAACACAATGAAGAGCACAAGAATGACCATCACGATGATGGTCATGATGATCATCACAACAATGGCGACCACAAGGATGACCACCATGAAGAAGAACACCATGAAGAAGAACACCACGAAGGGGACCACCACGAAGAGGAGCATCACGAAGAAGAGCACAAAGACGACCATCACGATTAA

编码菲牛蛭Kazal型丝氨酸蛋白酶抑制剂Bdellin-HM成熟序列为第54-501位核苷酸，其氨基酸序列为(SEQIDNO:2)：NH ₂ -DSECVCTKELNQVCGSDGHTYDNPCLATCHGASVAHEHACEGHEEEHHEEDHHDGEHNNGDHHEGEHNNGEHHDDEHKDDHHGEDHHEDGEHNEEHKNDHHDDGHDDHHNNGDHKDDHHEEEHHEEEHHEGDHHEEEHHEEEHKDDHHD-COOH。

The application of the Bdellin-HM gene, which is an inhibitor of phenanthrene trypsin, as a gene engineering preparation of an inhibitor of phenanthrene trypsin.

Protease inhibition experiment of Bdellin-HM trypsin inhibitor:

A. Reagents and materials

1. Buffer (pH7.4)

10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% v/v SurfactantP ₂₀

2. Protease and corresponding chromogenic substrate:

400nM trypsin (SIGMA) and 0.2mMGly-Arg-p-nitroanilidedihydrochloride (SIGMA)

400nM plasmin (SIGMA) and 0.2mMGly-Arg-p-nitroanilidedihydrochloride (SIGMA)

400nM elastase (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)

400 nM Kallikrein, 10 nMFXIIa, 400 nMFXIa (Enzyme Research Laboratories, USA) and 0.2 mM H-D-Pro-Phe-Arg-pNA·2HCl (HyphenBiomed, France)

10 nM thrombin (SIGMA) and 0.2 mM H-D-Phe-Pip-Arg-pNA.2HCl (HyphenBiomed, France)

10 nMFXa (Enzyme Research Laboratories, USA) and 0.2 mM CH3OCO-D-CHA-Gly-Arg-pNA-AcOH (SIGMA).

B. Protease Inhibition Assay

1. Add 10 μL of Bdellin-HM (final concentration 11.5 μM), 10 μL of protease and 40 μL of buffer in a 96-well plate, and incubate at 37°C for 5 minutes.

2. Immediately add 30 μL buffer solution and 10 μL chromogenic substrate mixture, the final volume is 100 μL.

3. Kinetics of the reaction Use Epoch (Bio-Tek) microplate reader, GENCHS1.09 software to detect OD405nm, 30min, interval 30s.

C. Determination of trypsin inhibition type and inhibition constant

Dixonplot curve: different concentrations of trypsin (0, 2.3, 4.6, 6.9, 9.2, and 11.5nM) and different concentrations of Gly-Arg-p-nitroanilidedihydrochloride (131.99and263.98μM), the steps are as described above. The Dixonplot curve is drawn with the inhibitor concentration as the abscissa and the ordinate as the reciprocal of the reaction rate. The abscissa value of the intersection point of the two extended straight lines is the inhibition constant of the inhibitor on trypsin. The intersection above the abscissa indicates competitive inhibition, and the intersection below the abscissa indicates mixed inhibition.

SEQUENCELISTING

<110> Kunming Institute of Zoology, Chinese Academy of Sciences

<120>Bdellin-HM and its coding gene and application

<130> Kunming Institute of Zoology

<160>1

<170>PatentInversion3.5

<210>1

<211>504

<212> DNA

<213> Hirudinaria manillensis

<220>

<221>1-504

<222>(1)..(504)

<400>1

atgaagctgttgcttgctttggcccttttcggtgcatttgtcacaatcaacgccgactca60

gaatgtgtctgcaccaaagaattgaaccaggtttgcggaagtgatggacatacctacgac120

aacccttgtctagctacgtgccacggggcgtctgtcgcccacgaacacgcctgcgaagga180

cacgaagaggaacatcacgaagaagaccaccaccatgacggtgagcacaataatggtgaccac240

catgaaggtgagcacaataatggtgaacaccatgacgacgagcacaaggatgaccaccat300

ggagaggacccacgaagacggggaacacaatgaagagcacaagaatgaccatcacgat360

gatggtcatgatgatcatcacaacaatggcgaccacaaggatgaccaccatgaagaagaa420

caccatgaagaagaacacccacgaaggggaccaccacgaagaggagcatcacgaagaagag480

cacaaagacgaccatcacgattaa504

SEQUENCELISTING

<110> Kunming Institute of Zoology, Chinese Academy of Sciences

<120>Bdellin-HM and its coding gene and application

<130>Kunming Institute of Zoology

<160>1

<170>PatentInversion3.5

<210>1

<211>149

<212>PRT

<213> Hirudinaria manillensis

<220>

<221>1-149

<222>(1)..(149)

<400>1

AspSerGluCysValCysThrLysGluLeuAsnGlnValCysGlySer

151015

AspGlyHisThrTyrAspAsnProCysLeuAlaThrCysHisHisGlyAla

202530

SerValAlaHisGluHisAlaCysGluGlyHisGluGluGluHisHis

354045

GluGluAspHisHisAspGlyGluHisAsnAsnGlyAspHisHisGlu

505560

GlyGluHisAsnAsnGlyGluHisHisAspAspGluHisLysAspAsp

65707580

HisHisGlyGluAspHisHisGluAspGlyGluHisAsnGluGluHis

859095

LysAsnAspHisHisAspAspGlyHisAspAspHisHisAsnAsnGly

100105110

AspHisLysAspAspHisHisGluGluGluHisHisGluGluGluHis

115120125

HisGluGlyAspHisHisGluGluGluHisHisGluGluGluHisLys

130135140

AspAspHisHisAsp

145

Claims (3)

1. Bdellin-HM, a Kazal-type serine protease inhibitor, is characterized in that it consists of the amino acid sequence shown in SEQ ID NO:1. 2. A gene encoding Bdellin-HM, a Kazal-type serine protease inhibitor Bdellin-HM, characterized in that it consists of the nucleotide sequence shown in SEQ ID NO:2. 3. the application of Bdellin-HM, the Kazal-type serine protease inhibitor Bdellin-HM described in claim 1, in the preparation of trypsin inhibitors.