CN101705218A - Protease, preparation method of same, as well as application and pharmaceutical formulation thereof - Google Patents

Abstract

The invention relates to a protease, a preparation method of same, as well as application and pharmaceutical formulation thereof. A protease is separated and extracted from a spitting liquid secreted before the emergence of an insect with capabilities of spinning and cocooning. The protease with fibrinolysis activity has the characteristics of high bioactivity, excellent stability and the like. The protein has strong arginine esterase activity and can degrade Aa Bb gamma chain in fibrinogen so as to have degradation function to the fibrin and the fibrinogen. As the protease has functions of degrading thrombus of animal and human beings and has the characteristics of not damaging the released blood cells and the like, the protease becomes a new thrombolysis type of medicine for treating diseases related with thrombus.

Description

The preparation method of a kind of proteolytic enzyme, this proteolytic enzyme and application thereof and pharmaceutical dosage form

Technical field

The present invention relates to insect, zymetology, medicine and other fields, be the sequence and the amino acid sequence coded of a kind of extracting method with proteolytic enzyme of plasmin activity of excretory, thrombus dissolving effect, gene when sprouting wings, and treat application of human thrombotic diseases etc. clinically from cocoon chrysalis.

Background technology

Thrombotic disease has become the first cause of developed country's human diseases death at present.Along with the improving constantly of people's living standard, the sickness rate of cardiovascular and cerebrovascular diseases is ascendant trend year by year, according to incompletely statistics, has every year 2000000 people to die from cardiovascular and cerebrovascular diseases approximately in China.

At present, the main treatment means of thrombotic disease has three kinds: the first, and adopt the blood vessel at surgical operation removing thrombus or excision embolism position and use " artificial blood vessel " bridge joint or replacement etc., as myocardial infarction.This method complicated operation, the expense height, and postoperative is prone to embolism again; The second, adopt and take antithrombotics for a long time, reduce the conservative treatment of blood coagulation tendency.This is owned by France in auxiliary treating method; The 3rd, medicine thrombolysis method is promptly injected thrombolytics and is made revascularization.Compare with conservative treatment, treatment back interior mortality ratio of two weeks significantly reduces, and expense is more much lower than surgical operation.Therefore, treat the methods of treatment that human thrombus disease is tool application prospect with thrombolysis ruling by law.

The medicine main source of treatment thrombotic diseases comprises animal, plant, microorganism etc.For example, nineteen fifty-one Willams extracts urokinase first (Urokinase UK), belongs to plasminogen activator from people urine and nephridial tissue culture.The Lumbrukinase that from coelomic fluid of earthworm, obtains (Limbrokinase).These enzyme majorities have direct solution fibrin and Profibrinolysin activated double effects; Nineteen fifty-five, the first was applied to clinical thrombolytic drug---and (Streptokinase SK) derives from microorganism to streptokinase, is a kind of strand extracellular protein of Hemolytic streptococcus excretory; And the fucosterol that the phytohaemagglutinin of some marine alga can suppress in hematoblastic gathering, the brown alga endotheliocyte can produce plasminogen activator etc.

At present, thrombolytic drug has developed the three generations, has reduced the mortality ratio and the disability rate of cardiovascular and cerebrovascular diseases effectively.Yet existing thrombolytic drug still exists some shortcomings: 1. lack the thrombolysis specificity; 2. hemorrhage side effect is strong; 3. the transformation period is short; 4. dosage is difficult to control; 5. be prone to embolism again; 6. the resistance reaction appears.Therefore, be badly in need of seeking a kind of more effective, safe, cheap novel thrombolytic drug in the clinical treatment.

Some have the cocoon insect (as tussah) of ability of battalion silk can synthesize and secrete a proteinoid enzyme when sprouting wings, in order to the sericin in the dissolving cocoon shell, finish eclosion process.Therefore infer that this proteinoid enzyme may have the activity of plasmin, might become the resource of the new antithrombotic reagent of exploitation.In tussah, the separation and purification of this proteolytic enzyme, enzyme activity assay are identified and report is not still studied in the effect on thrombus dissolving.

Summary of the invention

For developing new antithrombotic reagent, the objective of the invention is to study and confirm the liquid that spues when cocoon chrysalis is sprouted wings into moth that purifying obtains a kind of proteolytic enzyme and its new purposes on thrombus dissolving with fibrin degradation effect first, the sequence of separation purification method, gene of this proteolytic enzyme and amino acid sequence coded also are provided simultaneously, treat the application of human thrombotic diseases clinically.

Technical solution of the present invention comprises: the excretory liquid that spues when collecting cocoon chrysalis and sprouting wings, adjust pH to 6.0 with damping fluid 0.02mol/L HAC-NaAC (pH6.0), and the centrifugal 10min of 12000r/min, it is standby to get supernatant, during purifying, gets supernatant with cation seperation column (HiTrap ^TMSP-FF) carry out purifying.The amount of each purifying is generally 2ml, if the not enough 2ml of supernatant, available damping fluid 0.02mol/L HAC-NaAC, pH6.0 complements to 2ml.Gel electrophoresis of protein (SDS-PAGE) detected result shows that the molecular weight of the proteolytic enzyme of purifying is about 24000 dalton.With the fibrin plate method proteolytic enzyme plasmin activity of purifying is measured, the result shows that this proteolytic enzyme has higher plasmin vigor, optimal pH=8.0 in pH value 7～9.5 scopes; And when pH＜7.0, its enzyme activity is dropped rapidly to below 50%.

The Fibrinogen hydrolytic activity is measured, and the result shows that the proteolytic enzyme of purifying can the direct hydrolysis Fibrinogen, former A α-chain and the B beta chain of hydrolysis of fibrin effectively, hatch 4 hours after, γ-chain is also had tangible Degradation.

Utilize Kunming mouse external thrombus model to carry out the external thrombolysis test of proteolytic enzyme, the result shows that this proteolytic enzyme has thrombolysis activity preferably, and the thrombolysis rate reaches 92.9%, and microscopically is observed the not damage of hemocyte to discharging.

We have carried out the analysis of N terminal amino acid sequence to this proteolytic enzyme, and the result shows that this proteolytic enzyme contains the IVGGYSVTIDKAPYQ aminoacid sequence.Further gene clone and dna sequence analysis show, this proteolytic enzyme is by a genes encoding that contains 786bp.Complete gene is encoded altogether one and contained 261 amino acid whose protein, infers that the proteolytic enzyme have the enzymic activity part is made up of 228 amino acid, and theoretic molecular weight is 23858.91 dalton, and is approaching with the molecular weight of the proteolytic enzyme of purifying; Wherein comprise the IVGGYSVTIDKAPYQ aminoacid sequence, consistent with proteinic N terminal amino acid sequencing result.The claimed interior complete gene order of proteolytic enzyme of extracting of tussah (Antheraeapernyi) body of the present invention is:

ATGTACAAGTATTATTTTTTACTATTGGCTTGTGCCATTTTTTGGAAAGATGGTAGC

TGTAAGAAAAGTAGTCAAAGAACGAATGATGTTGGGGGGAAAATTGTTGGCGGATAC

AGCGTTACAATCGATAAAGCTCCTTACCAAGCGTACTTGCTTTTAGAAAAGGGTAAG

GAGTATTACCAATGTGGGGGATCCATCATCAGCAAAAGGCATATTTTAACCGCTGCT

CATTGTCTTGTTGATAATATATCACAAGTACACGCAAGAGTTGGTAGTACGAATAAC

GATAAAGGCGGTAAAGTGTATTCGACCAAATCGATGACTATACACCCCAATTATAAT

TCTCGAACTTACGATTATGATGTGGCTATTTTGACATTCTCACAAGATATAGCAATC

GACGGTGTCAACACTAAAATCGTAACACTACCGAGTGAAAACAATGGGGCTGTACCT

GCGAAGACAACATTATTTGTCACCGGTTGGGGAGCTACATCGGAAGGCGGTGATTCC

TCAAAAACATTGTTGGCAGTTAATGTTCCTACTCTATCCGTAGCAGAATGCAAAAAA

AGTGTGTCAAACTTTTCTGACAGAATGCTTTGTGCAGGCGTCTCTCAAGGAGGAAAA

GATGCTTGCCAGGGAGACTCTGGTGGTCCGGCTGTTAGCAATAATGTACAATTGGGA

GTTGTCTCATTTGGAAGTGGCTGTGCCCGTCCTGGAAACCCTGGTGTATATGCAAAT

GTCACAGCACTCCGTGGTTGGATACGAAAGACTGCAGGAGTTTAA

Infer the aminoacid sequence that this proteolytic enzyme is complete according to the said gene sequence:

mykyyflllacaifwkdgsckkssqrtndvggk

Bold race capital partly is the aminoacid sequence of the proteolytic enzyme of the purifying inferred.

The acquisition of this proteolytic enzyme had both comprised by the direct separation and Extraction from insect of separation means, also comprises by the Protocols in Molecular Biology means and in plant, animal and microbe, expresses the product that produces, and the product by the chemical process synthetic.For example also can extend to the albumen of separation and Extraction from the lepidopterous insects of other kind.

Its form that fibrinous biological action of thrombus can various pharmaceutical preparations in external, body is applied to the prevention and the treatment of human thrombus disease, and these preparations mainly comprise powder injection, formulation such as oral.

By formulation, vehicle are studied, are screened, obtained clinically to use and be powder injection in pharmaceutically acceptable optimal formulation, every bottle contains 0.2mg tussah proteolytic enzyme, 90mg dextran-40.

The present invention first the liquid that spues when cocoon chrysalis is sprouted wings separation and purification a kind of proteolytic enzyme, supposition is made up of 228 amino acid, theoretical molecular is 23858.91 dalton.This proteolytic enzyme has plasmin activity, and external can thrombus.The present invention also provides the separation purification method of this proteolytic enzyme, the analytical procedure of plasmin activity, the analytical procedure of thrombus dissolving effect, the method for gene clone.The complete sequence and the amino acid sequence coded of gene also are provided simultaneously, and application clinically and at pharmaceutically acceptable preparation.This proteolytic enzyme with fibrinolytic, characteristics such as possess the biological activity height, have good stability.This albumen has intensive arginine ester enzyme activity, the Aa Bb r chain in can fibrin degradation former, thereby to scleroproein and the original Degradation of scleroproein.Because such proteolytic enzyme has the effect of degraded animal and human thrombus, and does not cause characteristics such as damage to the hemocyte that discharges, will become a kind of new thrombolysis class medicine that is used for the treatment of the thrombus relative disease.

Description of drawings

The gel electrophoresis result of the proteolytic enzyme of accompanying drawing 1 purifying, molecular weight is about 24000 dalton

The fibrinogenic gel electrophoresis analysis of accompanying drawing 2 proteasome degradations

The optimal pH of accompanying drawing 3 proteolytic enzyme action activities is measured

Accompanying drawing 4 inhibitor and metal ion are to the gel electrophoresis analysis that influences of protease activity

The external thrombus dissolving effect of accompanying drawing 5 proteolytic enzyme

The microscopic examination of accompanying drawing 6 proteolytic enzyme pair cells influence

Embodiment

Below be described further by specific embodiment:

Embodiment one: the separation preparation of proteolytic enzyme

Tussah (Antheraea pernyi) belongs to lepidopteran (Lepidoptera) Saturniidae (Saturniidae), is a kind of economic insects of mainly putting in a suitable place to breed in the northern area of China field, and its silk is used for the filature weaving, and silkworm chrysalis is edible.Employed experiment material is the cocoon chrysalis alive that sell in market among the present invention.Get cocoon chrysalis, under 25 ℃ of conditions, hatch and moth to sprouting wings, collect the maxillary gland liquid that spues.Get the liquid that spues, place centrifuge tube, adjust pH to 6.0 with damping fluid 0.02mol/L HAC-NaAC (pH6.0), the centrifugal 10min of 12000r/min, get supernatant standby (sample such as not enough 2ml, available damping fluid 0.02mol/LHAC-NaAC, pH6.0 complements to 2ml). clean cationic exchange coloum (HiTrap with distilled water earlier ^TMSP-FF), (0.02mol/L HAC-NaAC pH6.0) carries out balance, and flow velocity is 2ml/min to use the Binding Buffer of 5 times of column volumes again.Sample thief 2ml goes up sample, uses Elution Buffer (0.02mol/LHAc-NaAC, 1mol/L NaC L pH6.0) to carry out linear gradient elution then, and flow velocity is 2ml/min.When the post internal conductance reaches 8.8, collect elution peak, carry out the check of SDS-PAGE electrophoresis, the result shows that the molecular weight of purified product is about 24000 dalton, sees accompanying drawing 1.

Embodiment two: the plasmin activity analysis of proteolytic enzyme and zymologic property are measured

1, to the mensuration of fibrinogenic degraded and plasmin activity thereof

(1), to fibrinogenic degraded

Get 0.2% human fibrinogen (0.1mol/L Tris-HCL, pH 8.0) each 10 microlitre of proteolytic enzyme behind solution and the purifying, mixing the back cultivated 0,0.5,1,2,3,4,8,16,24 hour at 37 ℃, add denaturing agent (10M urea, 4%SDS, the 4%2-mercaptoethanol) stopped reaction is measured with SDS-PAGE.As Fig. 2, shown in, tussah proteolytic enzyme is former A α-chain and the B beta chain of hydrolysis of fibrin effectively, and to the hydrolytic activity of γ-chain a little less than.Calculate the result of gained shown in Fig. 3 .4 with Bandscan software process, tussah proteolytic enzyme is the former A α-chain of hydrolysis of fibrin at first, be that hydrolyzable is more than 90% in 2 hours, hydrolysis for the B beta chain needed 4 hours to reach 90%, degrade 90% after then will waiting until 6h to the hydrolysis of γ chain, see accompanying drawing 2.

(2), the mensuration of plasmin activity (fibrin plate method)

Carry out the mensuration of fibrinolytic according to Astrup and Deogny method.Detailed process is as follows, with 1% sepharose (50mM Tris-HCL, pH7.5,100mMNaCL) 9ml dissolves, and treats that liquid adds 0.2% human fibrinogen (0.1mol/L Tris-HCL when cooling to 37-42 ℃, pH 8.0) 1ml, pour in the 10-cm Petri plate, add 0.2ml human thrombin (100NIH U/mL, 0.1mol/L Tris-HCL again, pH8.0), room temperature leaves standstill 1h it is condensed.Add protein enzyme solution behind the 10 μ l purifying in dull and stereotyped aperture, hatch 24h for 37 ℃, measure the diameter of hydrolysis circle.With the standard urinary kinases of different concns gradient, with its solusphere area (cm ²) as the vigor size, the drawing standard curve.The fibrinolytic vigor of tussah proteolytic enzyme is compared with typical curve according to its solusphere size on fibrin plate, calculates the fibrinolytic vigor.The result shows that the fibrinolytic vigor of tussah proteolytic enzyme is about 487.2U/mg (UK) with respect to the standard urinary kinases.

2, the mensuration of proteolytic enzyme optimum temperuture, pH

(1), the proteolytic enzyme optimum temperuture is measured

Preparation tussah proteolytic enzyme extracting solution, concentration is 0.1mg/ml (0.1mol/L Tris-HCL, pH 8.0), place 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ waters bath with thermostatic control to hatch, take a sample successively every 30min, measure its enzyme activity remaining under condition of different temperatures respectively with above-mentioned fibrin plate method.Each sample is all measured three times, averages then.Find that this tussah proteolytic enzyme is stronger to the adaptability of temperature, subject range in this temperature range, remains the vigor of higher hydrolysis substrate between 25-35 ℃.When temperature is lower than 20 ℃ or when being higher than 40 ℃, its enzyme activity is reduced to below 50% rapidly; When temperature is higher than 50 ℃, basic inactivation.This shows that this tussah proteolytic enzyme has tolerance preferably to thermal distortion, is a kind of thermophilic enzyme.

(2), the proteolytic enzyme optimal pH is measured

Getting 20 microlitre concentration is 0.1mg/ml (0.1mol/L Tris-HCL, pH 8.0) liquid of protease, join 180 microlitre 0.1mol/L Tris-HCL respectively, pH in 4.0,5.0,6.0,7.0,8.0,8.5,9.0,9.5 the solution, was hatched 30 minutes at 37 ℃.Get 10 μ l and be added in the dull and stereotyped aperture of determination of activity, measure its enzyme activity respectively with above-mentioned fibrin plate method, find that this enzyme all maintains higher enzyme activity in pH7.0～9.5 scopes, its optimal pH is 8.0.Illustrate that tussah proteolytic enzyme is a kind of proteolytic enzyme that adapts to weakly alkaline pH, sees accompanying drawing 3.

3, proteinase inhibitor and metal ion are to the influence of protease activity

(1), the metal ion inhibitor is for the influence of protease activity.

In 2% human fibrinogen solution of 100 microlitres, add 10 microlitre 0.1mg/ml protein enzyme solutions (0.1mol/L Tris-HCL, pH 8.0) respectively, add 7 metal ion species (Fe simultaneously respectively ⁺, Ca ²⁺, Mg ²⁺, K ⁺, Cu ²⁺, Mn ²⁺, Zn ²⁺), ultimate density is 10mM, is contrast not add metal ion, the sample that each group is mixed is 37 ℃ of cultivations, gets at 4 hours and 24 hours respectively respectively to organize sample and carry out the SDS-PAGE cataphoretic determination.The result carries out analysis revealed, Fe through Bandscan software ²⁺And Cu ²⁺Ion is the potent inhibitor of this enzyme, can obviously reduce the molten fine vigor of proteolytic enzyme, and Ca ²⁺, K ⁺Then slight inhibitory enzyme is lived Mg ²⁺, Mn ²⁺The influence that ion is lived for enzyme is not remarkable, sees accompanying drawing 4.

(2), proteinase inhibitor is for the influence of protease activity

In 2% human fibrinogen solution of 100 microlitres, add 10 microlitre 0.1mg/ml protein enzyme solution (0.1mol/L Tris-HCL respectively, pH 8.0), add 4 kinds of proteinase inhibitor 10 microlitres (0.1mM PMSF simultaneously respectively, 1mM DTT, 10mM β-ME, 10mM EDTA), unconstrained dose is contrast, and the sample that each group is mixed is 37 ℃ of cultivations.Took a sample at 4 hours and 24 hours respectively and carry out the SDS-PAGE cataphoretic determination.The result shows that the tussah protease activities can significantly be suppressed by serpin PMSF, shows that this enzyme is the serine stretch protein enzyme family.This enzyme can significantly be suppressed by halfcystine reductibility denaturing agent DTT, but reductive agent β-mercaptoethanol etc. do not have obvious restraining effect to its enzymic activity, and metal ion chelation agent EDTA is not obvious to its restraining effect, illustrates that this enzyme is not a metal-containing enzyme.

Embodiment three: the external thrombus dissolving function analysis of proteolytic enzyme

1, thrombolytic effect detects

With Kunming mouse with etherization after, heart puncturing extracting blood.After treating blood coagulation, cut into uniform fritter, weigh.Blood clotting being joined in tussah proteolytic enzyme (0.02mmol/L NaAC-HAC) solution of 0.5mL, is contrast with 0.02mmol/L NaAC-HAC, puts 37 ℃ of waters bath with thermostatic control.Every 10min, slight vibration is regularly observed, writes down, is taken pictures.Found that contrast top solution is more clear, thrombolysis is not obvious, contains the red corpuscle of minute quantity in the solution, in the tussah proteolytic enzyme group, and top solution muddiness, thrombolysis is obvious, contains a large amount of cells that dissolving discharges from thrombus in the solution, sees accompanying drawing 5.

2, thrombolysis efficient detects

With Kunming mouse with etherization after, heart puncturing extracting blood is made experimental thrombosis.Thrombus is cut into equal 3 parts, and (0.173mg/mL, 0.02mmol/LNaAC-HAC) tussah proteolytic enzyme are contrast with the 0.02mmol/L NaAC-HAC with volume, carry out the thrombolysis experiment to add 500 μ L after weighing.Calculate the thrombolysis rate in the formula below the removal of thromboses carefully behind 37 ℃ of water bath with thermostatic control 4h, and after blotting the thrombus surface-moisture gently with the filter paper bar, weighing, substitution.

The thrombolysis rate=(W-G)/W*100%

Thrombus weight before the W test

G test back thrombus weight

The dissolution rate that found that thrombus in the tussah protease treatment is 92.9%, and contrast is 5.4%.

3, the microscopic examination of enzyme pair cell damage

Liquid behind the removal of thromboses is examined under a microscope hemocyte and is discharged and the metamorphosis situation behind the smear.The result shows that tussah protease treatment group cellular form is complete, compares indifference with control group.Illustrate that pair cell had no adverse effects when tussah proteolytic enzyme had solvency action to the mouse thrombus, see accompanying drawing 6.

Embodiment four: the N terminal amino acid sequence mensuration of proteolytic enzyme and gene clone and sequential analysis

1, the N terminal amino acid sequence of proteolytic enzyme is analyzed

With isolating sample after the SDS-PAGE gel electrophoresis separates, transfer to pvdf membrane, carry out proteinic amino acid sequence analysis according to the Edman edman degradation Edman. by comparing with standard substance, 15 aminoacid sequences of measured protein N terminal are: IVGGYSVTIDKAPYQ

2, the clone of gene and sequential analysis

Gather the preceding 2-3 days maxilla tissues (about 200mg) of ripe tussah moth emergence, extract test kit with RNA

Reagent (Invitrogen) extracts total RNA, and carries out quantitatively with ultraviolet-visible pectrophotometer.The total RNA that is about 360 μ g is used for separating mRNA, with mRNA separating kit (Oligotex ^TM-dT30＜Super〉mRNA Purification Kit, TaKaRa) carry out the purifying of mRNA and quantitatively.The mRNA that gets 5 μ g is a template, and (cDNA LibraryConstruction Kit TaKaRa) carries out the foundation in cDNA library with cDNA library test kit.The specification sheets of method reference reagent box, final positive transformation efficiency is 78% in the library.The positive colony that 10 of picked at random contain the above clip size of 700bp carries out sequential analysis, and the result has in the sequence of 4 genes and contains the IVGGYSVTIDKAPYQ sequence.Further sequence comparative analysis result confirms, this proteolytic enzyme is by the genes encoding of a 786bp, and 261 amino acid of encoding altogether infer that having enzymic activity proteolytic enzyme partly is made up of 228 amino acid, and theoretic molecular weight is 23858.91 dalton.See attached list 1.

Subordinate list 1:

ATGTACAAGTATTATTTTTTACTATTGGCTTGTGCCATTTTTTGGAAAGATGGTAGCTGTAAGAAAAGTAGTCAA

m??y??k??y??y??f??l??l??l??a??c??a??i??f??w??k??d??g??s??c??k??k??s??s??q

AGAACGAATGATGTTGGGGGGAAAATTGTTGGCGGATACAGCGTTACAATCGATAAAGCTCCTTACCAAGCGTAC

r??t??n??d??v??g??g??k

A??Y

TTGCTTTTAGAAAAGGGTAAGGAGTATTACCAATGTGGGGGATCCATCATCAGCAAAAGGCATATTTTAACCGCT

L??L??L??E??K??G??K??E??Y??Y??Q??C??G??G??S??I??I??S??K??R??H??I??L??T??A

GCTCATTGTCTTGTTGATAATATATCACAAGTACACGCAAGAGTTGGTAGTACGAATAACGATAAAGGCGGTAAA

A??H??C??L??V??D??N??I??S??Q??V??H??A??R??V??G??S??T??N??N??D??K??G??G??K

GTGTATTCGACCAAATCGATGACTATACACCCCAATTATAATTCTCGAACTTACGATTATGATGTGGCTATTTTG

V??Y??S??T??K??S??M??T??I??H??P??N??Y??N??S??R??T??Y??D??Y??D??V??A??I??L

ACATTCTCACAAGATATAGCAATCGACGGTGTCAACACTAAAATCGTAACACTACCGAGTGAAAACAATGGGGCT

T??F??S??Q??D??I??A??I??D??G??V??N??T??K??I??V??T??L??P??S??E??N??N??G??A

GTACCTGCGAAGACAACATTATTTGTCACCGGTTGGGGAGCTACATCGGAAGGCGGTGATTCCTCAAAAACATTG

V??P??A??K??T??T??L??F??V??T??G??W??G??A??T??S??E??G??G??D??S??S??K??T??L

TTGGCAGTTAATGTTCCTACTCTATCCGTAGCAGAATGCAAAAAAAGTGTGTCAAACTTTTCTGACAGAATGCTT

L??A??V??N??V??P??T??L??S??V??A??E??C??K??K??S??V??S??N??F??S??D??R??M??L

TGTGCAGGCGTCTCTCAAGGAGGAAAAGATGCTTGCCAGGGAGACTCTGGTGGTCCGGCTGTTAGCAATAATGTA

C??A??G??V??S??Q??G??G??K??D??A??C??Q??G??D??S??G??G??P??A??V??S??N??N??V

CAATTGGGAGTTGTCTCATTTGGAAGTGGCTGTGCCCGTCCTGGAAACCCTGGTGTATATGCAAATGTCACAGCA

Q??L??G??V??V??S??F??G??S??G??C??A??R??P??G??N??P??G??V??Y??A??N??V??T??A

CTCCGTGGTTGGATACGAAAGACTGCAGGAGTTTAA

L??R??G??W??I??R??K??T??A??G??V??＊

Embodiment five: the clinical application formulation

Get 0.1mg/ml protein enzyme solution (0.1mol/L NaAC-HAC, pH 8.0), add dextran-40 respectively and make into different concns (15,30,45,60mg/ml), be divided in the cillin bottle respectively, every bottle of 2ml, highly be about 0.8cm, place in the freeze drying box, pre-freeze was to-20 ℃ of maintenances 3 hours, and when treating that sample temperature reaches-30 ℃, the beginning vacuum pump vacuumizes sublimation drying, about 15h, vacuum tightness≤20Pa, 30 ℃ of dry again 5h, capping is promptly. in the freeze-drying process, vacuum tightness remains unchanged, distiller keeps-40 ℃, is dried to residual moisture≤5.0%. and carries out the freeze-drying operation, and sample after the freeze-drying is checked appearance character and preparation solvability. and test-results shows, the consumption of dextran-40 is when 45mg/ml, the moulding and the solvability of preparation are best, thereby the usage quantity of determining dextran-40 in the prescription is 45mg/ml, i.e. 90mg/ bottle (subordinate list 2).

Table 2 proteolytic enzyme freeze-dried excipient dextran-40 consumption shaker test result

Dextran-40 (mg/ml)	??15	??30	??45	??60
					Appearance character	Subside	Loose bulk	Loose block	Block
The preparation solvability	Dissolving	Yi Rong	Yi Rong	Dissolving

SEQUENCE?LISTING

＜110〉Dalian Biological Tech. Inst., Liaoning Academy of Agricultural Science

＜120〉preparation method of a kind of proteolytic enzyme, this proteolytic enzyme and application thereof and pharmaceutical dosage form

<160>2

<170>PatentIn?version?3.3

<210>1

<211>786

<212>DNA

＜213〉tussah (Antheraea pernyi)

<400>1

atgtacaagt?attatttttt?actattggct?tgtgccattt?tttggaaaga?tggtagctgt?????60

aagaaaagta?gtcaaagaac?gaatgatgtt?ggggggaaaa?ttgttggcgg?atacagcgtt????120

acaatcgata?aagctcctta?ccaagcgtac?ttgcttttag?aaaagggtaa?ggagtattac????180

caatgtgggg?gatccatcat?cagcaaaagg?catattttaa?ccgctgctca?ttgtcttgtt????240

gataatatat?cacaagtaca?cgcaagagtt?ggtagtacga?ataacgataa?aggcggtaaa????300

gtgtattcga?ccaaatcgat?gactatacac?cccaattata?attctcgaac?ttacgattat????360

gatgtggcta?ttttgacatt?ctcacaagat?atagcaatcg?acggtgtcaa?cactaaaatc????420

gtaacactac?cgagtgaaaa?caatggggct?gtacctgcga?agacaacatt?atttgtcacc????480

ggttggggag?ctacatcgga?aggcggtgat?tcctcaaaaa?cattgttggc?agttaatgtt????540

cctactctat?ccgtagcaga?atgcaaaaaa?agtgtgtcaa?acttttctga?cagaatgctt????600

tgtgcaggcg?tctctcaagg?aggaaaagat?gcttgccagg?gagactctgg?tggtccggct????660

gttagcaata?atgtacaatt?gggagttgtc?tcatttggaa?gtggctgtgc?ccgtcctgga????720

aaccctggtg?tatatgcaaa?tgtcacagca?ctccgtggtt?ggatacgaaa?gactgcagga????780

gtttaa???????????????????????????????????????????????????????????????786

<210>2

<211>261

<212>PRT

＜213〉tussah (Antheraea pernyi)

<400>2

MYKYYFLLLA?CAIFWKDGSC?KKSSQRTNDV?GGKIVGGYSV?TIDKAPYQAY

LLLEKGKEYY????60

QCGGSIISKR?HILTAAHCLV?DNISQVHARV?GSTNNDKGGK?VYSTKSMTIH

PNYNSRTYDY????120

DVAILTFSQD?IAIDGVNTKI?VTLPSENNGA?VPAKTTLFVT?GWGATSEGGD

SSKTLLAVNV????180

PTLSVAECKK?SVSNFSDRML?CAGVSQGGKD?ACQGDSGGPA

VSNNVQLGVV?SFGSGCARPG????240

NPGVYANVTA?LRGWIRKTAG?V????261

Claims (5)

1. proteolytic enzyme, its complete dna sequence dna is:

ATGTACAAGTATTATTTTTTACTATTGGCTTGTGCCATTTTTTGGAAAGATGGTAGCTGTAAGAAAAG

TAGTCAAAGAACGAATGATGTTGGGGGGAAAATTGTTGGCGGATACAGCGTTACAATCGATAAAGCTC

CTTACCAAGCGTACTTGCTTTTAGAAAAGGGTAAGGAGTATTACCAATGTGGGGGATCCATCATCAGC

AAAAGGCATATTTTAACCGCTGCTCATTGTCTTGTTGATAATATATCACAAGTACACGCAAGAGTTGG

TAGTACGAATAACGATAAAGGCGGTAAAGTGTATTCGACCAAATCGATGACTATACACCCCAATTATA

ATTCTCGAACTTACGATTATGATGTGGCTATTTTGACATTCTCACAAGATATAGCAATCGACGGTGTC

AACACTAAAATCGTAACACTACCGAGTGAAAACAATGGGGCTGTACCTGCGAAGACAACATTATTTGT

CACCGGTTGGGGAGCTACATCGGAAGGCGGTGATTCCTCAAAAACATTGTTGGCAGTTAATGTTCCTA

CTCTATCCGTAGCAGAATGCAAAAAAAGTGTGTCAAACTTTTCTGACAGAATGCTTTGTGCAGGCGTC

TCTCAAGGAGGAAAAGATGCTTGCCAGGGAGACTCTGGTGGTCCGGCTGTTAGCAATAATGTACAATT

GGGAGTTGTCTCATTTGGAAGTGGCTGTGCCCGTCCTGGAAACCCTGGTGTATATGCAAATGTCACAG

CACTCCGTGGTTGGATACGAAAGACTGCAGGAGTTTAA。

2. the preparation method of proteolytic enzyme as claimed in claim 1 comprises:

The excretory liquid that spues is adjusted pH to 6.0 with damping fluid 0.02mol/L HAC-NaAC (pH6.0) when collecting cocoon chrysalis and sprouting wings, and the centrifugal 10min of 12000r/min gets supernatant, with cation seperation column (HiTrap ^TMSP-FF) carry out purifying.

3. proteolytic enzyme as claimed in claim 1 is used for the prevention and the treatment of human thrombus disease.

4. the formulation of proteolytic enzyme as claimed in claim 1 is powder injection.

5. the formulation of proteolytic enzyme as claimed in claim 4, described powder injection contains 0.2mg tussah proteolytic enzyme, 90mg dextran-40 for every bottle.

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