CN109679941B

CN109679941B - Cordyceps militaris fibrinolytic enzyme and preparation method and application thereof

Info

Publication number: CN109679941B
Application number: CN201910170774.1A
Authority: CN
Inventors: 刘英丽; 王静; 丁立; 万真; 孙宝国
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2019-03-07
Filing date: 2019-03-07
Publication date: 2021-08-03
Anticipated expiration: 2039-03-07
Also published as: CN109679941A

Abstract

Cordyceps militaris fibrinolytic enzyme and a preparation method and application thereof, belonging to the technical field of bioengineering. The invention provides a cordyceps militaris fibrinolytic enzyme, which has an amino acid sequence shown as SEQ ID No.1 and a gene sequence shown as SEQ ID No.2 and can be applied to thrombolysis. The enzyme is proved to have better thrombolytic activity through a series of preliminary researches on enzymology properties and in-vitro and in-vivo thrombolytic properties, and the in-vivo thrombolytic mechanism of the enzyme is disclosed by constructing other animal thrombus models, and the safety of clinical application of the enzyme is explored by means of toxicological tests, so that scientific proofs and data support are provided for the development of new drugs or functional foods for treating thrombus in future.

Description

Cordyceps militaris fibrinolytic enzyme and preparation method and application thereof

Technical Field

The invention relates to a cordyceps militaris fibrinolytic enzyme and a preparation method and application thereof, and belongs to the technical field of bioengineering.

Background

Cardiovascular and cerebrovascular diseases are one of the leading causes of human death in the world, and intravascular thrombosis is one of the leading causes of cardiovascular and cerebrovascular disease. Intravascular thrombosis is caused by the conversion of fibrinogen to fibrin by thrombin, followed by the formation of insoluble fibrin clots (the clotting reaction), which are the major components of the thrombus. To date, commercially used clinical thrombolytic agents such as tissue plasminogen activator (t-PA), urokinase and streptokinase have some unavoidable side effects such as bleeding and clogging complications, low specificity for fibrin degradation, allergic reactions and high price.

Cordyceps militaris is a traditional Chinese precious edible fungus resource used as both medicine and food, can be artificially produced in large batches, and is considered as an ideal substitute of Cordyceps sinensis. The protease with fibrinolytic activity in the cordyceps militaris is used as the fibrinolytic enzyme from edible fungi, and compared with the fibrinolytic enzyme from other sources and the existing medicines for treating thrombus, the protease has the advantages of wide raw materials, low price, high safety, good specificity and the like. However, the research on the cordyceps militaris fibrinolytic enzyme still stays at the downstream stage of the biological process at present, the gene research on the fibrinolytic enzyme is not reported, and the gene sequence for coding the fibrinolytic enzyme is obtained by utilizing the molecular biological means for amplification. Heterologous expression is carried out on the cordyceps militaris fibrinolytic enzyme gene, so that the yield and the enzyme activity are improved.

Disclosure of Invention

The invention aims to overcome the defects and provides the cordyceps militaris fibrinolytic enzyme and the preparation method and the application thereof, wherein a fibrinolytic enzyme gene in cordyceps militaris is obtained for the first time and is cloned and then expressed in escherichia coli BL21, so that a high-enzyme-activity engineering bacterium of the cordyceps militaris fibrinolytic enzyme is obtained, and the expression yield is improved.

According to the technical scheme, purified cordyceps militaris fibrinolytic enzyme is subjected to N-terminal sequencing and mass spectrum sequencing, the Protein is identified to be alkaline serine protease Alp1 (NCBI Protein id = EGX 91264.1), a fibrinolytic enzyme gene in cordyceps militaris is obtained for the first time, the gene is cloned and then expressed in escherichia coli BL21, high-enzyme-activity engineering bacteria of cordyceps militaris fibrinolytic enzyme are obtained, and the expression yield is improved.

The prepared cordyceps militaris fibrinolytic enzyme has the enzymological properties that: the optimum temperature of the enzyme is 60 ℃, but the enzyme is placed at the temperature above room temperature, and the temperature exceeds 30 ℃, and the enzyme activity begins to decline rapidly after the enzyme is placed for 1 h; the optimum reaction pH is 7.0-11.0, and is stable in a wide pH range (5.0-11.0).

Low concentration of Fe³⁺Has significant inhibition effect on the enzyme activity of the cordyceps militaris fibrinolytic enzyme, basically completely loses the enzyme activity, and is Fe²⁺And Cu²⁺Has slight inhibitory effect on enzyme activity, and other ions such as Na⁺、Zn²⁺、Mg²⁺、Co²⁺、K⁺The influence on enzyme activity is not particularly remarkable, Ca²⁺And Mn²⁺The ions have a remarkable enhancement effect on the enzyme activity of the cordyceps militaris fibrinolytic enzyme; the serine protease inhibitor and the metalloprotease inhibitors PMSF, pepstatin A, EDTA-2Na, leupeptin and TPCK have obvious inhibition effect on enzyme activity, and can prove that the enzyme is serine metalloprotease and Ca²⁺And Mn²⁺The ions participate in the catalysis of the active center of the enzyme.

According to the comparison with a heated fibrin flat plate, the cordyceps militaris fibrinolytic enzyme thrombolytic mode is proved to be the mode of directly degrading fibrin, has higher fibrin specificity and does not have fibrinogen catalytic activity. The protease has a certain degradation effect on three chains of fibrinogen, the content of alpha chain can be degraded to a certain degree within a certain reaction time, the beta chain can be completely degraded within 60min, the degradation degree of gamma chain is inferior to that of beta chain, and the content of three chains is less and less along with the extension of the reaction time.

A method for constructing Cordyceps militaris fibrinolytic enzyme engineering bacteria comprises using Cordyceps militaris CM01 (Cordyceps militaris CM 01) strain as strain, wherein amino acid sequence of Cordyceps militaris fibrinolytic enzyme is shown as SEQ ID NO.1, and gene sequence is shown as SEQ ID NO. 2.

The method comprises the following specific steps:

(1) according to the results of protein N-terminal sequencing and mass spectrum identification, a primer is designed by analyzing the coding gene sequence of Alp1 protein in NCBI database. Through CDS annotation and signalP4.1 signal peptide prediction and N-terminal sequencing results, the protein is found to be composed of a signal peptide, a leader peptide and a mature peptide. Therefore, the three parts are amplified separately when designing primers, which are shown in Table 1, wherein P1, P2 and P3 are upstream primers and P4 is a downstream primer. The amplification products of P1 and P4 are full-length gene g1, namely preproenzyme containing signal peptide, leader peptide and mature peptide; the amplification products of P2 and P4 are zymogen gene g2 containing leader peptide and mature peptide; p3 and P4 are the mature peptide only enzyme gene g 3.

(2) Extracting and reverse transcribing the RNA of the cordyceps militaris, and performing PCR amplification by taking cDNA obtained by reverse transcription as a template.

(3) The amplification product purified in step (2) was ligated into pEASY-Blunt E2 vector, transformed into E.coli DH 5. alpha. and the correct recombinant plasmid was transferred into E.coli BL21 (DE 3), and then confirmed by sequencing to obtain E.coli containing the recombinant plasmid.

TABLE 1 primers used for E.coli expression

Primer name	Primer sequences	Length of amplification
			P1	5'-ATGGTCGGCT TCAAAAGCTT CG-3'	1187 bp
P2	5'-AAGTACATTG TCACGCTCAA-3’	1133 bp
			P3	5'-GCCCTGACCA CGCAGTCCAA-3'	867 bp
P4	5'-CTGGTTACCG TTGTACGAGA GCA-3'	-

An electrophoresis chart of PCR amplification products of Cordyceps militaris fibrinolytic enzyme gene is shown in FIG. 1, an electrophoresis chart of recombinant plasmid extraction is shown in FIG. 2, and an electrophoresis chart of PCR bacteria liquid of Cordyceps militaris fibrinolytic enzyme gene is shown in FIG. 3. Wherein M1 corresponds to P1 and P4 primer pairs, M2 corresponds to P2 and P4 primer pairs, and M3 corresponds to P3 and P4 primer pairs.

The optimal method for applying the genetic engineering bacteria to the induction expression of the heterologous expression cordyceps militaris fibrinolytic enzyme comprises the following steps:

transferring the Escherichia coli cells containing the recombinant plasmids into an LB liquid culture medium containing ampicillin (50-100 mu g/mL), and culturing overnight in a shake flask, wherein the Escherichia coli cells are used as a primary seed solution. Then transferring the cells to 50mL of LB liquid culture medium containing ampicillin according to the inoculation amount of 1% -10%, under the same culture conditions, when monitoring the OD600 of the bacterial liquid to be 0.1-1, adding IPTG to the final concentration of 10-50 mug/mL so as to induce the generation of enzyme, and continuing to culture for 10-20 h. Centrifuging the induced bacteria liquid, washing with sterile water, metering the volume of each 50mL of original bacteria liquid to 10 mL, performing wall breaking treatment by using an ultrasonic crushing extractor, centrifuging the sample after wall breaking treatment, taking supernatant, centrifuging at 5000-10000 rpm for 20min at 4 ℃, and purifying the recombinant expression enzyme again by using an affinity chromatography method for subsequent enzymology property research and thrombolysis activity analysis.

Provides the thrombolytic activity analysis of the cordyceps militaris fibrinolytic enzyme in vivo and in vitro:

(1) in vitro thrombolysis of recombinant enzymes: taking blood from heart of a healthy SD rat, numbering a centrifuge tube in advance, weighing empty tubes, recording the mass as m1, naturally placing the blood for coagulation, centrifuging at 3000rpm for 10min to remove supernatant serum, and weighing the residual blood clot and the mass of the centrifuge tube as m 2. Setting 5 groups, each group comprising three tubes, adding 1 XPBS buffer solution as negative control for group one, adding urokinase and streptokinase as positive control for group two and three, adding 10 μ g purified Cordyceps militaris fibrinolytic enzyme for group four, and adding 30 μ g purified Cordyceps militaris fibrinolytic enzyme for group five. After incubation for 3h, 6h, 9h, 12h and 24h at 37 ℃, the micro centrifuge briefly centrifuges and takes out the supernatant to another clean centrifuge tube, and the mass of the residual blood clot and the mass of the empty tube are weighed and recorded as m 3. And after weighing is finished, adding the liquid into the blood clot again for continuous incubation, and finally calculating the dissolution rate of the blood clot according to a formula.

(2) In vivo thrombolytic action of the recombinase: the thrombolytic activity of the cordyceps militaris fibrinolysin is researched by utilizing a rat tail vein thrombosis model. Wistar male rats were selected and injected with rat tail veins with different concentrations of k-carrageenan dissolved in 1 × PBS to induce thrombosis. The 42 male mice were divided into 7 groups of 6 mice each. Group 1 was a negative control group injected with 1 × PBS.

Groups

2, 3, 4, and 5 are experimental groups, and cordyceps militaris fibrinolytic enzyme with different concentrations (dissolved by 1 × PBS) is injected respectively. Groups 6 and 7 are experimental positive control groups, and a certain dose of urokinase and streptokinase are injected respectively. After 30min of injection of the enzyme solution, the tail vein of the mouse was ligated and the K-carrageenan was injected intravenously to induce thrombosis. Removing the ligation after 15min, measuring the thrombosis length after 24h, taking a picture, and calculating the in vivo thrombolysis rate according to a formula.

The invention has the beneficial effects that: the invention splices the obtained sequences in a mode of combining N-terminal sequencing and mass spectrometry sequencing of purified cordyceps militaris fibrinolytic enzyme, obtains a fibrinolytic enzyme gene in cordyceps militaris for the first time, and performs cloning expression. The large intestine expression quantity of the cordyceps militaris fibrinolytic enzyme can reach 91.14mg/L, the unit enzyme activity is 1779U/mL, the direct fermentation expression quantity is improved by 32.7 times compared with the natural bacterial strain, the unit enzyme activity is improved by 35%, the fermentation time is greatly shortened, and the production efficiency is improved.

The enzyme is proved to have better thrombolytic activity through a series of preliminary researches on enzymology properties and in-vitro and in-vivo thrombolytic properties, and the in-vivo thrombolytic mechanism of the enzyme is disclosed by constructing other animal thrombus models, and the safety of clinical application of the enzyme is explored by means of toxicological tests, so that scientific proofs and data support are provided for the development of new drugs or functional foods for treating thrombus in future.

Drawings

FIG. 1 is an electrophoresis diagram of PCR amplification products of a gene of a fibrinolytic enzyme of Cordyceps militaris.

FIG. 2 recombinant plasmid extraction scheme.

FIG. 3 is a PCR electrophoresis chart of the gene bacterial liquid of the fibrinolysin of Cordyceps militaris.

Wherein M is Marker, Lane 1 is PCR amplification product or recombinant plasmid of g1, Lane 2 is PCR amplification product or recombinant plasmid of g2, and Lane 3 is PCR amplification product or recombinant plasmid of g 3.

FIG. 4 SDS-PAGE of purified recombinant protein.

FIG. 5 comparison of the in vitro thrombolytic activities of different fibrinolysins.

FIG. 6 the effect of different fibrinolytic enzymes on tail vein thrombosis in rats.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments. It should be understood that the following specific examples are illustrative only and are not limiting upon the invention.

Example 1 screening of high enzyme Activity Cordyceps militaris strains

Activating 10 Cordyceps militaris strains with solid plate culture medium, cutting a piece from the inclined plane, and placing on solid improved PDA plate (adding 2% peptone and 0.3% KH peptone based on common PDA solid culture medium)₂PO₄，0.15% MgSO₄，0.002% VB₁) And (4) performing constant-temperature dark culture at 24 ℃ for 10 days in the center, wherein the mycelium grows over the plate and is not changed in color. Liquid culture fermentation was performed on 10 activated cordyceps militaris strains.

The culture conditions were: 50mL of culture medium is filled in a 250mL triangular flask, a bacterium block with the side length of about 1cm is added, and shaking bottles are cultured in a dark place at 24 ℃ and 150 r/min. Sampling 1mL of cordyceps militaris every day from the 5 th day, centrifuging for 5min at 4 ℃ and 10000r/min, taking the supernatant as a crude enzyme solution to perform enzyme activity detection, monitoring for 20 days, and finally selecting the optimal high-enzyme-activity cordyceps militaris strain for subsequent separation and purification by comprehensively considering the enzyme activity and the enzyme production speed.

Example 2: identification of cordyceps militaris fibrinolytic enzyme

(1) N-terminal sequencing of plasmin: taking the high-enzyme-activity cordyceps militaris strain obtained in the example 1 as a research object, carrying out liquid fermentation on the cordyceps militaris strain, separating and purifying a crude enzyme solution, carrying out gel running on a protein sample according to an electrophoresis method shown in the table 2, and transferring the protein to a PVDF (polyvinylidene fluoride) membrane after the gel running is finished. Before transfer, the glue and filter paper with proper size are soaked in CAPS buffer solution for 5-10 min. The PVDF membrane was soaked in methanol for several seconds and then also placed in CAPS electroblotting buffer. During installation, the filter paper, the PVDF film, the glue and the filter paper are sequentially arranged from bottom to top, and air bubbles are completely removed.

Adopting a semidry method, wherein the transfer printing conditions are as follows: constant pressure of 25V, 1A, 60 min. And (3) after the transfer printing is finished, dyeing the PVDF membrane by Coomassie brilliant blue, drying after dyeing and decoloring, cutting off a target band, and determining the first 10 amino acid sequences of the N end of the plasmin by using an Shimadzu protein sequencer PPSQ-31B by using a standard method, wherein data results are analyzed according to mass spectrometry instructions.

The first 10 amino acid sequences of the N-terminal of the cordyceps militaris plasmin are detected to be ALTTQSNVPH by using a protein sequencer through the Edman degradation principle. Performing Blast search on the protein by using NCBI protein database to find the protein and cordyceps militaris (II)Cordyceps militarisCM 01) was completely matched with amino acids 108-117 of Alp1 (NCBI Protein id = EGX 91264.1), and had very low similarity to plasmin purified from Cordyceps militaris by other researchers and plasmin derived from other fungi. The cordyceps militaris can secrete a plurality of proteases, and the plasmin separated from the CM01 is probably a novel protease and has high research and development potential and value.

TABLE 2 SDS-PAGE formulation Table

(2) Mass spectrum sequencing and identification of plasmin: and (4) cutting off the protein band which runs out of the SDS-PAGE after dyeing and decoloring in a clean 1.5mL centrifuge tube, and performing secondary mass spectrometry identification. The mass spectrum is Q-active (thermo scientific), the secondary mass spectrum data analysis is identified by searching a library by Mascot and Proteome resolver, and the protein database is uniprot-musca flytrap.

After a band of a target Protein is obtained through Protein electrophoresis, a peptide fragment mixture with smaller molecular weight is obtained after the target Protein is subjected to enzymolysis through trypsin, mass spectrum analysis is carried out on the peptide fragment mixture to obtain a secondary mass spectrum and fragment information, and then the obtained Score is compared through a Protein database, the alkaline serine protease Alp1 (NCBI Protein id = EGX 91264.1) in a Cordyceps militaris CM01 strain with the largest Score is obtained, and the database is scored into 9232.55 (the Score is 100, namely the identification is considered to be successful, and the Score is more credible). The sequences obtained by sequence analysis of the secondary peptide end-spectrum are shown in Table 3, the read amino acid sequence fragments are respectively compared with the database, and the sequence is found to be completely consistent with the amino acids 126-298 and 369-395 of Alp1 after splicing. Thus, it is believed that the protease with fibrinolytic activity in cordyceps militaris is likely to have high homology and sequence similarity with the alkaline serine protease.

TABLE 3 protein sequence Mass Spectrometry identification results

Example 3: induced expression, separation and purification of recombinant cordyceps militaris fibrinolytic enzyme

Coli cells containing the recombinant plasmid were cultured in 250mL shake flasks containing 50mL LB broth supplemented with 100. mu.g/mL ampicillin, and grown at 37 ℃ and 200rpm until an optical density of 0.6 was reached at 600 nm. The culture was then added to 50. mu.g/mL of IPTG to induce enzyme production, and incubation was continued for 20h at 20 ℃. After the culture, the culture was centrifuged at 10000rpm for 10min at 4 ℃. The cells were then resuspended in cell culture medium. Lysis buffer (50 mM NaH)₂PO₄300mM NaCl, 10mM Tris, 20mM imidazole, pH 8.0) and disrupted by sonication. The crude extract was then centrifuged at 10000rpm for 20min at 4 ℃. Since the C end of the pEASY Blunt E2 vector is provided with a 6 XHis tag, the expression product is purified by nickel column affinity chromatography. Fermenting and centrifuging the bacterial liquid, taking the precipitate, washing twice with sterile water, and then using an equilibrium buffer (50 mM NaH)₂PO₄300mM NaCl, 10mM Tris, 20mM imidazole, pH 8.0) and then made up to 1/5. Breaking cell wall by ultrasonic crusher to extract protein, performing ultrasonic treatment with the same ultrasonic parameters as above, centrifuging at 10000rpm and 4 deg.C for 10min, and collecting supernatant. Load pre-equilibrated Ni-NTA (5 mL) column, flow rate5 mL/min. At the end of the loading, the column was washed for 20 additional volumes, after which elution buffer (50 mM NaH) was used₂PO₄300mM NaCl, 10mM Tris, 500mM imidazole, pH 8.0) was linearly eluted for 10 column volumes. Collecting by using an automatic collector, collecting 2mL of the solution in each tube, and carrying out enzyme activity detection and electrophoresis detection according to the peak position. The finally optimized large intestine expression level of the fibrinolytic enzyme is 91.14mg/L, the unit enzyme activity is 1779U/mL, the expression level is 32.7 times higher than that of the natural bacterial strain in direct fermentation, the unit enzyme activity is improved by 35 percent, the fermentation time is greatly shortened, and the enzyme production efficiency is greatly improved. And (3) centrifuging the bacterial lysate after wall breaking to obtain supernatant, loading the supernatant into a Ni-NTA column, and finding that the supernatant can specifically adsorb target protein through electrophoresis detection, wherein the obtained purity is high, as shown in figure 4.

Example 4: determination of in vitro thrombolytic Activity of recombinant enzymes

Blood is taken from heart of a healthy SD rat, and the blood is subpackaged into 1.5mL centrifuge tubes, each tube is about 0.6mL, the centrifuge tubes are numbered in advance, and the mass of each empty tube is weighed and recorded as m 1. After the blood is naturally placed and coagulated, centrifuging at 3000rpm for 10min to remove supernatant serum, sucking the liquid by using filter paper, and weighing the residual blood clot and the mass of a centrifuge tube, wherein the mass is recorded as m 2. Setting 5 groups, each group comprising three tubes, adding 200 μ L of 1 XPBS buffer solution as negative control into one group, adding 10 μ g urokinase and 10 μ g streptokinase 200 μ L as positive control into two groups and three groups, respectively, adding 200 μ L of 10 μ g purified Cordyceps militaris fibrinolytic enzyme into four groups, and adding 200 μ L of 30 μ g purified Cordyceps militaris fibrinolytic enzyme into five groups. After incubation for 3h, 6h, 9h, 12h and 24h at 37 ℃, the micro centrifuge briefly centrifuges and takes out the supernatant to another clean centrifuge tube, and the mass of the residual blood clot and the mass of the empty tube are weighed and recorded as m 3. After weighing, adding the liquid into the blood clot again for continuous incubation. Finally, the clot dissolution rate was calculated as follows:

thrombolysis rate (%) = [ (m 2-m 3)/(m 2-m 1) ] × 100%

The remaining clot mass at different time intervals was recorded and the results are shown in figure 5. The urokinase and the streptokinase are used as positive controls, and the recombinant fibrinolysin expressed by the method has the thrombolytic activity equivalent to that of urokinase and higher than that of streptokinase within 6h before reaction. Within 12h, both urokinase and streptokinase reach the highest thrombolysis activity, the urokinase thrombolysis rate reaches 24 percent, the streptokinase is only 12 percent, and the thrombolysis rate is not obviously different from that after 24 h. However, the recombinant fibrinolytic enzyme expressed by the method shows obvious thrombolysis activity from reaction for 9 hours to 24 hours, the thrombolysis activity is increased all the time, the thrombolysis rate after 24 hours is obviously higher than that of urokinase and streptokinase, and the thrombolysis rate of two dose groups reaches more than 40%.

Example 5 determination of in vivo thrombolytic Activity of recombinant enzymes-Effect on rat Tail vein thrombosis

(1) Optimizing the induction concentration of the k-carrageenan: the thrombolytic activity of the cordyceps militaris fibrinolysin is researched by utilizing a rat tail vein thrombosis model. Wistar male rats were selected, first optimized for the induction concentration of k-carrageenan, and then injected with rat tail veins to induce thrombosis by dissolving k-carrageenan (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg/kg) in 1 × PBS at different concentrations. Six of each group are parallel, the tail thrombus formation condition is observed after 24 hours, and the formed wine red thrombus length is measured. And (5) carrying out significance analysis and selecting the optimal induction concentration.

(2) In vivo thrombolytic activity of recombinant fibrinolytic enzyme: and carrying out a rat tail vein thrombosis model test according to the optimized k-carrageenan induction concentration. The 42 male mice were divided into 7 groups of 6 mice each. Group a was a negative control group injected with 1 × PBS. Groups b, c, d, e are experimental groups, and 200, 600, 1800, 3000 μ g/kg (1 × PBS dissolved) of Cordyceps militaris fibrinolytic enzyme are injected respectively. Group f and group g are experimental positive control groups, injected with 600. mu.g/kg dose urokinase, 600. mu.g/kg dose streptokinase, respectively. After the enzyme solution is injected for 30min, the tail vein of the mouse is ligated, and the optimized concentration of the k-carrageenan is used for intravenous injection to induce thrombosis. Removing the ligation after 15min, measuring the thrombus formation length after 24h, taking a picture, and calculating the in vivo thrombolysis rate according to the following formula:

after injecting purified fibrinolysin with different dose concentrations and each control medicament, the tail vein thrombosis of rats is induced, and the thrombus length of the tail vein thrombosis of each group is measured after 24h, and the result is shown in figure 6. The enzyme activity thrombolysis rate of different dose groups of the recombinant plasmin purified by the method is different from 83-289U/cm by taking the enzyme activity thrombolysis rate of thrombus unit length, urokinase reaches up to 1883U/cm, more enzyme activity is needed for dissolving tail vein thrombus with the same length, and streptokinase is 131U/cm, so that the thrombolytic effect of the recombinant plasmin purified by the method is larger than that of urokinase and is inferior to that of streptokinase, but is basically equivalent. Moreover, according to the set dosage relationship, the thrombolysis rate of the pupal cellulase has an obvious dosage-dependent relationship.

Sequence listing

<110> Beijing university of Industrial and commercial

<120> cordyceps militaris fibrinolytic enzyme, and preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 395

<212> PRT

<213> Cordyceps militaris (cordyces militaris)

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Met Val Gly Phe Lys Ser Phe Ala Ala Leu Phe Leu Ala Ala Leu Gly

1 5 10 15

Thr Ala Ala Pro Ala Pro Ser Gly Gly Lys Tyr Ile Val Thr Leu Lys

20 25 30

Asp Gly Ala Thr Ala Ser Lys Val Glu Ser His Leu Gln Trp Val Asn

35 40 45

Asp Val His Ala Arg Ser Val Gly Arg Arg Asp Leu Asn Leu Asn Gly

50 55 60

Val Glu Lys Thr Tyr Gly Ile Gly Ser Phe Asn Gly Tyr Ala Gly Asn

65 70 75 80

Phe Asp Ala Ala Thr Ile Glu Glu Ile Lys Asn Ser Pro Glu Val Ala

85 90 95

Ala Val Glu Leu Asp Gln Lys Trp Thr Leu Tyr Ala Leu Thr Thr Gln

100 105 110

Ser Asn Val Pro His Gly Leu Ala Thr Ile Ser His Arg Gln Ser Gly

115 120 125

Ala Ser Asn Tyr Ile Tyr Asp Ser Thr Ala Gly Gln Gly Ala Tyr Ala

130 135 140

Tyr Val Val Asp Ser Gly Val Asn Ile Gly His Val Glu Phe Glu Gly

145 150 155 160

Arg Ala Thr Arg Gly Tyr Asn Ala Ala Gly Gly Glu Asp Val Asp Thr

165 170 175

Leu Gly His Gly Thr His Val Ser Gly Thr Ile Ala Ser Lys Ser Tyr

180 185 190

Gly Val Ala Lys Lys Ala Ser Ile Val Ser Val Lys Val Phe Ser Gly

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Arg Thr Ala Asp Thr Ser Val Ile Leu Asp Gly Tyr Asn Trp Ala Val

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Asn Asp Ile Val Ala Lys Arg Arg Gln Thr Arg Ser Val Ile Asn Leu

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Ser Leu Gly Gly Pro Ala Ser Thr Ala Phe Asp Ser Ala Val Ala Ser

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Ala Tyr Asn Gln Gly Val Leu Thr Val Val Ala Ala Gly Asn Glu Asn

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Gln Asp Ser Arg Asn Val Ser Pro Ala Arg Ala Pro Gln Ala Ile Thr

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Val Ala Ala Val Gly Ala Thr Trp Ala Arg Trp Val Trp Asn Ala Gln

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Asp Val Leu Ser Thr Trp Ile Gly Ser Thr Thr Ala Thr Asn Thr Ile

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<213> Cordyceps militaris (cordyces militaris)

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atggtcggct tcaaaagctt cgccgctctc ttcctcgcgg cccttggcac cgctgcccct 60

gctccttctg gcggcaagta cattgtcacg ctcaaggacg gtgccactgc cagcaaggtt 120

gagtctcact tgcagtgggt aaacgatgtc cacgctcgca gcgtcggtcg tcgcgatctc 180

aacctcaacg gtgtcgaaaa gacctacggc attggtagct tcaacggcta cgctggcaac 240

tttgacgctg ccaccattga ggagatcaag aacagccccg aggttgcggc cgtcgagctt 300

gaccagaagt ggactctgta tgccctgacc acgcagtcca acgtccccca cggactggcc 360

accatctcgc accgtcagtc cggtgccagc aactacatct acgacagcac cgcaggccag 420

ggcgcctacg cctacgtcgt cgactcaggt gtcaacattg gacacgtcga gttcgagggc 480

cgtgctaccc gcggctacaa cgcggccggc ggcgaggacg tcgacaccct cggccacggc 540

actcacgtct ctggcaccat tgcttccaag agctacggcg tggccaagaa ggccagcatc 600

gtctccgtca aggttttcag cggccgcacc gccgacacct ccgtcatcct cgatggctac 660

aactgggccg tcaacgacat cgtcgccaag cgccgccaga ctcgctccgt cattaacctg 720

tccctcggtg gtcccgcctc gaccgctttc gacagcgcag ttgccagtgc ctacaaccag 780

ggtgttctga ccgtcgtcgc cgccggcaac gagaaccagg acagcagaaa cgtctccccc 840

gcccgcgccc cccaggccat caccgtcgcc gctgtcggcg ccacctgggc tcgctgggtc 900

tggaacgcgc agcagggctc caactacggc actcccgtcg acatctacgc tcccggtgag 960

gatgttctgt ccacctggat tggctcgacc actgccacca acaccattac tggcacctcc 1020

atggcctctc cccacattgc tggtctggcc atttaccttg ccgttctcga gaacctcaac 1080

acccctgccg ctgtcaccaa ccgcatcaag gcgcttggca ccgcgaacaa ggtgactggc 1140

aacgttggca gcactgtcaa cttgctctcg tacaacggta accagtaa 1188

Claims

1. An application of Cordyceps militaris fibrinolytic enzyme in preparing thrombolytic medicine;

the amino acid sequence of the cordyceps militaris fibrinolytic enzyme is shown in SEQ ID NO. 1.