KR101884394B1 - Recombinant fibrinolytic protease from Vibrio furnissii and uses thereof - Google Patents

Abstract

The present invention relates to a recombinant thrombolytic enzyme derived from Vibrio furnissii, a composition for thrombolysis comprising the recombinant thrombolytic enzyme as an active ingredient, a method for decomposing thrombus by treating the composition with an object containing thrombus, a method for producing a recombinant thrombolytic enzyme using a recombinant microorganism transformed with a recombinant vector comprising a gene encoding the recombinant thrombolytic enzyme, the recombinant thrombolytic enzyme produced by the method, a health food composition and a pharmaceutical composition for preventing or ameliorating thrombosis comprising the recombinant thrombolytic enzyme as an active ingredient. The recombinant thrombolytic enzyme of the present invention may be effectively used for the treatment and prevention of thrombosis or similar diseases.

Description

Recombinant fibrinolytic protease from Vibrio frissini and uses thereof < RTI ID = 0.0 >

The present invention relates to a recombinant thrombolytic enzyme derived from Vibrio vulnificus and to its use, and more particularly to a recombinant thrombolytic enzyme derived from Vibrio furnissii comprising the amino acid sequence of SEQ ID NO: 1, A composition for thrombolysis comprising a degrading enzyme as an active ingredient, a method for decomposing thrombi by treating the composition with an object containing thrombus, a recombinant vector containing the gene encoding the recombinant thrombolyticase, A method for producing a recombinant thrombolytic enzyme using microorganisms, a recombinant thrombolytic enzyme produced by the method, a health functional food composition for preventing or improving thrombosis comprising the recombinant thrombolytic enzyme as an active ingredient, Prevention or prevention of thrombosis with enzyme as active ingredient For relates to a pharmaceutical composition.

Proteolytic enzymes that dissolve thrombosis can be classified into two types depending on the mechanism of action. One class is a proteolytic enzyme that acts directly on the thrombus to dissolve it, and the other class is a proteolytic enzyme that activates plasminogen to produce plasmin. Examples of the proteolytic enzymes that dissolve the fibrin clot which is the cause of the thrombus include papain, trypsin, brinase, plasmin, etc., and plasminogen Activated enzymes include tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), and streptokinase.

Currently, there are tPA and uPA in the clinical use, but uPA has the advantage of being able to be administered orally, but there are many side effects such as systemic hemorrhage due to low selectivity to thrombus, tPA is highly selective for thrombus, There is a drawback that it is short and expensive. Therefore, research on developing high-fibrinolytic enzymes specific for thrombolysis is still an important field with few side effects from various biological resources.

Currently, various studies have been conducted to minimize the side effects, to mass-produce thrombolytic enzymes, and to develop thrombolytic enzymes capable of oral administration, but the industrialization thereof is extremely limited.

Korean Patent Publication No. 2002-0079719 discloses a novel Bacillus mechatellium jbi-42 and a novel thrombolytic enzyme produced therefrom. Korean Patent No. 1217713 discloses a novel thrombolytic enzyme derived from thymus mushroom-derived thrombolytic enzyme And a method for producing the fermentation product of the present invention, and a method for producing the fermentation product, and a use thereof. However, the recombinant thrombolytic enzyme derived from Vibrio vulnificus of the present invention and its use are not disclosed.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and the present inventors have found that a recombinant thrombolytic enzyme derived from vibrio rhubarb may selectively cleave A ?, B? Or? Chains of fibrinogen and fibrin polymer formed in fibrin and human plasma And the fibrin clusters are efficiently decomposed by direct fibrin dissolution, thus completing the present invention.

In order to solve the above problems, the present invention provides a recombinant thrombolytic enzyme derived from Vibrio furnissii comprising the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a thrombolytic composition comprising the recombinant thrombolytic enzyme as an active ingredient.

In addition, the present invention provides a method for decomposing thrombus by treating the above composition with an object containing thrombus.

The present invention also provides a method for producing a recombinant thrombolytic enzyme using a recombinant microorganism transformed with a recombinant vector comprising a gene encoding the recombinant thrombolytic enzyme.

The present invention also provides a recombinant thrombolytic enzyme produced by the above method.

The present invention also provides a health functional food composition for preventing or ameliorating thrombosis comprising the recombinant thrombolytic enzyme as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating thrombosis comprising the recombinant thrombolytic enzyme as an active ingredient.

The recombinant thrombolytic enzyme derived from Vibrio vulnificus of the present invention is a novel thrombolytic enzyme that degrades fibrin and fibrinogen, and can be effectively used for the treatment and prevention of thrombosis or similar diseases.

FIG. 1 shows a gene cloning step for the production of a recombinant protein of Vibrio furnissii- derived thrombolytic enzyme. The amplified gene region of 1,827 bp in size was amplified by PCR, and Eco RI of FLAG-ATS vector And a plasmid construction procedure for producing a recombinant thrombolytic enzyme by inserting a gene into the plasmid.
FIG. 2 is a graph illustrating the purification and separation process of recombinant thrombolytic enzyme, wherein A and B are ion exchange chromatography using a Hitrap Q column and a Source 15Q 4.6 / 100 PE column, and C is a size using Sepedex G-75 column Excretion chromatography, and D is the result of separation of each of the separation step-wise proteins on an SDS-PAGE gel. ●, protein concentration (absorbance); ○, proteolytic activity.
3 is an analysis of the fibrinogen cleavage pattern by the recombinant thrombolytic enzyme (rvFMP) of the present invention.
FIG. 4 is a graph showing the relative turbidity measured by measuring the fibrin polymer degrading ability by the recombinant thrombolytic enzyme of the present invention.
FIG. 5 shows the results of comparing and analyzing the fibrin plate resolution of plasmin and the recombinant thrombolytic enzyme of the present invention.
FIG. 6 shows the results of analyzing the relative turbidity of a fibrin polymer formed in plasma of a normal person.
FIG. 7 shows the anti-thrombotic effect of rvFMP in the carotid artery thrombosis model by FeCl ₃ , where A is a blood vessel treated with saline, B is a blood vessel causing thrombosis with FeCl ₃ , C is a blood vessel treated with urokinase pretreatment FeCl ₃ , and D is a photograph showing blood vessels that caused thrombosis with FeCl ₃ after pretreatment with rvFMP.

In order to accomplish the object of the present invention, the present invention provides a recombinant thrombolytic enzyme derived from Vibrio furnissii comprising the amino acid sequence of SEQ ID NO: 1.

The term "thrombus" refers to a core caused by local coagulation of blood components in a blood vessel or heart. In general, thrombus consists of platelets, fibrin, red blood cells and white blood cells. Thrombosis is largely divided into arterial and venous blood. The proportion of its components is not the same for all blood types, and platelets or other blood cells are mainly present in the fibrin network. Thrombosis is solved by the fibrinolytic enzyme plasmin.

The term "thrombolytic enzyme" in the present invention means an enzyme that acts directly on fibrin to degrade fibrin. It can be used interchangeably with the expression of thrombolytic enzyme, fibrinolytic enzyme or proteolytic enzyme, . ≪ / RTI >

The range of the recombinant thrombolytic enzyme according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 1 and a functional equivalent of the protein. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably at least 70% or more, Refers to a protein having a homology of at least 95% and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 1.

Preferably, the recombinant thrombolytic enzyme of the present invention is a thrombolytic enzyme derived from Vibrio furnissii . The recombinant thrombolytic enzyme has metalloprotease characteristics, a molecular weight of 35 kDa, and an optimum pH of enzyme activity of 7.0 , And the optimum activation temperature is 50 캜. In addition, the thrombolytic enzyme of the present invention selectively cleaves A?, B? Or? Chain of fibrinogen, efficiently decomposes fibrin polymer formed in fibrin and human plasma, And the fibrin clot is efficiently decomposed by the fibrin clot.

In addition, the present invention provides a thrombolytic composition comprising the recombinant thrombolytic enzyme as an active ingredient.

The recombinant thrombolytic enzyme of the present invention selectively cleaves the A?, B? Or? Chain of fibrinogen and efficiently decomposes the fibrin polymer formed in fibrin and human plasma and efficiently decomposes the fibrin clot by direct fibrinolysis And can effectively decompose thrombus.

In addition, the present invention provides a method for decomposing thrombus by treating the above composition with an object containing thrombus.

The term "individual" as used in the present invention means all animals such as human, monkey, dog, goat, pig or rat which can decompose thrombus by treating the composition of the present invention. For example, it means.

As used herein, the term "treatment" means providing a composition of the invention to a subject in any suitable manner.

The present invention also provides a method for producing a recombinant thrombolytic enzyme using a recombinant microorganism transformed with a recombinant vector comprising a gene encoding the recombinant thrombolytic enzyme.

Specifically, the method of the present invention comprises: (a) culturing a recombinant microorganism transformed with a recombinant vector comprising a gene encoding a recombinant thrombolytic enzyme of the present invention; And (b) recovering the recombinant thrombolytic enzyme from the culture or the culture supernatant of step (a).

The gene encoding the recombinant thrombolytic enzyme according to the present invention may include the nucleotide sequence shown in SEQ ID NO: 2. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 2 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In the present invention, the term "vector" means a nucleic acid molecule capable of carrying other nucleic acid to which it is linked. One type of vector, "plasmid" refers to a circular double-stranded DNA loop in which additional DNA fragments can be ligated.

The vector of the present invention can direct expression of a gene encoding a target protein operatively linked, which is referred to as an "expression vector. &Quot; Generally, in the use of recombinant DNA technology, the expression vector is in the form of a plasmid.

When the E. coli is used as a host, the expression vector may be a pET28a vector. When yeast is used as a host, the expression vector may be YEp13, YCp50, pRS, pYEX-based vectors, and the like. As the promoter, for example, GAL promoter, AOD promoter and the like can be used.

In addition, the expression vector may include a fragment for suppressing expression, having various functions for suppressing, amplifying, or inducing expression of a target gene, a marker for selecting a transformant, a gene resistant to antibiotics, A gene encoding a signal for the purpose of secretion of a secreted protein, and a customized fusion factor suitable for a hung-off protein.

In the present invention, the term "transformation" means that DNA is introduced into a host cell so that DNA can be replicated as an extrachromosomal factor or by chromosomal integration.

Transformation methods in the present invention can be carried out by transforming the expression vector of the present invention by methods known in the art, including, but not limited to, transient transfection, microinjection, transduction, But are not limited to, phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, , A spiropalat method, or a lithium acetate method, and transformed into a host cell.

The host cell used for the transformation according to the present invention may be any host cell well known in the art. However, the present invention is not limited to the host cell having excellent introduction efficiency of the gene encoding the recombinant thrombolytic enzyme of the present invention, Efficient hosts may be used, including, for example, bacteria, fungi, yeast, plant or animal (e.g., mammalian or insect) cells.

The medium for culturing the transformant of the present invention and the culture conditions can be appropriately selected depending on the host cell. The nutrient medium preferably contains an inorganic nitrogen source or an organic nitrogen source which is necessary for growth of the host cell. Examples of the carbon source include glucose, dextran, soluble starch, sucrose, and methanol. Examples of the inorganic or organic substance include ammonium salts, nitrates, amino acids, corn steep liquer, peptone, casein, bovine extract, soybean bag, and potato extract. (Nutrients such as sodium chloride, calcium chloride, sodium dihydrogenphosphate, inorganic salts such as magnesium chloride, vitamins, and antibiotics (tetracycline, neomycin, ampicillin, and kanamycin). During the culture, the conditions such as the temperature, the pH of the medium and the incubation time can be appropriately adjusted so as to be suitable for cell growth and mass production of the protein.

The step of recovering the recombinant thrombolytic enzyme can be carried out by a conventional biochemical separation technique or by separating and purifying the thrombolytic enzyme in an immuno-friendly manner using an antibody against one part or another part of the protein. Another method may be purification by affinity tagging with a protein sequence and using antibodies or other materials with a suitably high affinity for these tags. Typical biochemical separation techniques include, but are not limited to, treatment with protein precipitants (salting-out), centrifugation, ultrasound disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, And various chromatographies such as chromatography. Typically, proteins having high purity can be separated by using them in combination.

The present invention also provides a recombinant thrombolytic enzyme produced by the above method.

The recombinant thrombolytic enzyme of the present invention selectively cleaves the A?, B? Or? Chain of fibrinogen and efficiently decomposes the fibrin polymer formed in fibrin and human plasma and efficiently decomposes the fibrin clot by direct fibrinolysis Have.

The present invention also provides a health functional food composition for preventing or ameliorating thrombosis comprising the recombinant thrombolytic enzyme as an active ingredient.

The term "thrombosis" refers to a disease caused by a thrombus, which is also referred to as a thromboembolism. The causes of thrombosis are three of the following: slow blood flow, excessive coagulation, and vascular injury. These three causes are directly or indirectly caused by thrombosis. Symptoms of thrombosis include a wide variety of symptoms, depending on the location of the organs in which the thrombosis has occurred and the type of blood vessels that are developed. In the case of arterial thrombosis, ischemic symptoms that may occur when the blood is not properly supplied and peripheral blood flow is insufficient In the case of venous thrombosis, congestion or congestion may occur due to the blood reaching the periphery but not returning to the heart.

There is no particular limitation on the kind of the food. Examples of the food to which the thrombolytic enzyme can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, soups, Alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

In addition, the present invention provides a pharmaceutical composition for preventing or treating thrombosis comprising the recombinant thrombolytic enzyme as an active ingredient.

The term "treatment ", as used herein, refers to dissolution of thrombus by thrombolytic enzyme of the present invention in thrombosis or similar diseases. Examples of diseases to which the pharmaceutical composition of the present invention can be applied include brain diseases such as cerebral thrombosis, cerebral embolism, pulmonary diseases such as obstructive pulmonary disease, pulmonary infarction, deep vein thrombosis, gait disorder, blood flow obstructive anemia, Peripheral neurological diseases such as neuralgia and hyperlipemia, renal diseases such as renal hypertension, kidney disease, renal failure, angina pectoris, ischemic heart disease, and cardiac diseases such as myocardial infarction.

The composition provided herein may comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutically acceptable carrier for the composition to be formulated into a liquid solution include sterilized and suitable for the living body such as saline, sterilized water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The pharmaceutical composition of the present invention may be various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical compositions of the present invention are administered by a suitable route of administration according to the formulation form. The method of administration is not particularly limited, and it can be administered by contents, external application, and injection. Injections can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and the like. The dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. For example, in the case of an adult, 0.00001 to 0.1 mg / kg per day may be administered. The administration can be carried out once a day or divided into several times. The dosage may be appropriately changed depending on factors such as the method of administration and the age, health, weight, nature and degree of symptoms of the patient, kind of simultaneous treatment, frequency of treatment, and desired effect.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1. Cloning of proteolytic enzyme gene derived from Vibrio germ

The entire genomic DNA was isolated from the strain Vibrio furnissii KCCM41679, and the gene was amplified by a polymerase chain reaction based on the metalloprotease gene sequence derived from the already known Vibrio vulnificus ATCC35016. The primer sequences used were as follows. EcoRI restriction sites (underlined) were inserted into forward and reverse primers for cloning into expression vectors.

Forward primer: 5'-CAAGCTTCTCGA GAATTC ATGAAAACATTACAAC-3 '(SEQ ID NO: 3)

Reverse primer: 5'-TGCAGGTACCCGG GAATTC TTAGTCCAGACGCAG-3 '(SEQ ID NO: 4)

The PCR reaction conditions for gene amplification were 1 minute denaturation at 95 ° C, binding at 55 ° C for 40 seconds, and extension at 72 ° C for 2 minutes and 30 seconds. The size of the amplified gene product was 1,827 bp. After digestion with Eco RI, the product was cloned into pFLAG-ATS vector and transformed into E. coli DH5α to obtain a transformant. The obtained recombinant plasmid was named pFLAG-ATS-vFMP (Fig. 1).

Example 2. Purification of recombinant thrombolytic enzyme

E. coli strain DH5? Was used for expression of recombinant thrombolytic enzyme (hereafter rvFMP), and a single clone containing the recombinant plasmid pFLAG-ATS-vFMP was inoculated into 50 ml of LB medium containing 100 μg / ml of ampicillin antibiotic And incubated overnight at 37 ° C. Then, the inoculated culture medium of 10ml of fresh LB medium containing antibiotics of 500ml and was cultured at A _600, until it reaches 0.8. After incubation, 0.2 mM IPTG (isoprophyl-β-D-thiogalactopyranoside) was treated at 37 ° C. for 12 hours to induce protein expression, and cells were collected using a centrifuge. The collected cells were dissolved in 100 ml of elution buffer (30 mM Tris-HCl (pH 8.0), 20% sucrose, 1 mM EDTA, 0.3 mg / ml lysozyme, 1 mM PMSF) and reacted at 4 ° C for 30 minutes. Cells Was centrifuged at 13,000 rpm for 20 minutes to collect only the supernatant. The supernatant was concentrated using 70% ammonium sulfate and then subjected to two types of ion exchange chromatography (Hitrap Q column and Source 15 Q 4.6 / 100 PE column ) And size exclusion chromatography (Superdex G-75 column). It was confirmed that the purified protein was loaded on SDS-polyacrylamide gel to have a size of 35 kDa and 32 kDa (FIG. 2).

Example 3 Measurement of Fibrinogen Resolution

The fibrinogen degrading ability of the recombinant thrombolytic enzyme (rvFMP) of the present invention was measured. 30 μg of fibrinogen was added to 0.5 μg of rvFMP and reacted at 37 ° C. for 0, 10, 30, 60, 180, 300 and 600 seconds, and terminated with 1,10-phenanthroline. The reactions were then confirmed on a 12% SDS-polyacrylamide gel.

As a result, it was confirmed that rvFMP completely decomposed A? And B? Of fibrinogen within 10 seconds, and γ was more resistant to rvFMP but completely decomposed after 20 minutes (FIG. 3).

Example 4. Measurement of thrombolysis ability

In order to measure the thrombotic resolution of rvFMP, 10 μl of thrombin (17.7 U) was added to a 96-well plate of fibrinogen and reacted at 25 ° C for 1 hour to form a fibrin polymer. Then, 2, 4 and 6 μg rvFMP and 2 의 of plasmin were added, respectively, and the absorbance A ₃₅₀ was measured at 37 캜 for 1 hour. The turbidity of the fibrin polymer formed by adding thrombin to fibrinogen was taken as 100%, and the relative turbidity of the solution containing rvFMP or plasmin was analyzed. This is to evaluate the ability of the rvFMP of the present invention to degrade the fibrin polymer by reducing the turbidity of the solution.

As a result, it was confirmed that the rvFMP of the present invention had a turbidity lowering ability more pronounced than that of plasmin at all treatment concentrations, which means that the fibrinolytic effect of rvFMP is relatively larger than that of plasmin (Fig. 4).

Example 5. Measurement of fibrin resolution

The fibrin resolution of rvFMP was measured using the fibrin plate method. 2 ml of 1% fibrinogen and 2 ml of 1% agarose were mixed and 70 ml of 17.7 U of plasmin was added to prepare a fibrin plate. Thereafter, a hole having a diameter of 5 mm was drilled, and 2 μg of plasmin, 2 μg of rvFMP, and Tris-HCl (pH 7.5) were placed in the hole and activated at 37 ° C. for 5 hours. Finally, the size of the transparent ring was 0.7 cm for plasmin and 1.1 cm for rvFMP, and it was confirmed that rvFMP had better fibrin resolution 2.5 times than plasmin (Fig. 5).

Example 6 Measurement of thrombolytic activity using human plasma

In order to measure the thrombolytic ability of rvFMP in human plasma, 10 μl of 17.7 U of thrombin was added to 90 μg of human plasma in a 96-well plate and reacted at 25 ° C. for 1 hour to form a fibrin polymer. Then, 2, 4 and 6 by the addition of plasmin of the ㎍ rvFMP 2㎍ and for 2 hours at 37 ℃ was measured for absorbance a _350. The turbidity of the fibrin polymer formed by adding thrombin to plasma of a normal person was determined as 100%, and the relative turbidity was analyzed. Since the decrease in the turbidity of the solution is due to the decomposition of fibrin in the solution, the thrombolytic ability of the rvFMP of the present invention can be measured by observing the turbidity change.

As a result, when 4 and 6 μg of the rvFMP of the present invention were added, a marked decrease in turbidity was confirmed as compared with that of plasmin, which means that the fibrinolytic effect of rvFMP in human plasma was relatively larger than that of plasmin 6).

Example 7. Ferric chloride (FeCl ₃ Analysis of Antithrombotic Effect in Animal Model of Carotid Thrombosis

The vascular injury model induced by FeCl ₃ is widely used to study thrombus formation in vivo. To confirm the antithrombotic / anticoagulant effect of rvFMP of the present invention, an animal model of carotid thrombosis by FeCl ₃ was used.

All animal experiments were carried out according to the experimental method approved by Chosun University Experimental Animal Management Committee (approval number: CIACUC2016-A0027). In the present invention, Sprague-Dawley (SD) rats (hereinafter referred to as SD-rats) were used. The SD-Rette was kept under controlled conditions (22 ± 2 ° C, 12-hour day / night cycle) after being subjected to a purifying period in a stainless steel cage and the food and water were free-diet. 0.9 mg / kg of rvFMP was mixed with physiological saline buffer solution and injected into the tail vein of SD-rat for 5 days. After exposure to 3% isoflurane using an anesthesia mask, the animals were anesthetized and anesthesia was maintained throughout the surgical procedure by mixing 70% nitrous oxide, 1.5% isoflurane and 30% oxygen. SD-rats were treated with urokinase (4,000 IU / kg; 8 mg / kg) or rvFMP (0.9 mg / kg) 10 minutes before thrombolysis with FeCl ₃ reagent, Control animals received 0.9% saline. The right carotid artery was exposed and the skin and muscles near the jaw were cut and the vagus nerve was not injured. The formation of thrombus was induced by covering two blood filter paper (6 mm x 6 mm) impregnated with 4% FeCl ₃ solution in a blood vessel. Ten minutes later, the filter paper was removed, the carotid artery containing the thrombus was exposed, and the blood vessel portion (length 20 mm) was cut. The cut blood vessels were fixed with 4% paraformaldehyde at 4 ° C for one day, and blood vessels were cut under a microscope to confirm the antithrombotic effect by rvFMP in order to confirm the cross section of the blood vessels.

As a result, it was confirmed that no blood clot was induced in the blood of the 0.9% saline-treated blood vessel that did not induce thrombus (FIG. 7A), and blood clot was formed due to coagulation of the blood vessel in the FeCl ₃ treatment (Fig. 7B), the urokinase pretreatment group (Fig. 7C) used as a positive control for antithrombotic effect and the rvFMP pretreatment group of the present invention (Fig. 7D) In particular, it was confirmed that the anti-thrombotic effect was more excellent in the animal model in which rvFMP was administered than the urokinase-treated rats.

<110> Industry-Academic Cooperation Foundation, Chosun University <120> Recombinant fibrinolytic protease from Vibrio furnissii and uses          the <130> PN17032 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 608 <212> PRT <213> Vibrio furnissii <400> 1 Met Lys Thr Leu Gln Arg Gln Val Lys Ala Tyr Ser Gln Ser Val Arg   1 5 10 15 Leu Trp Leu Ser Arg Phe Arg Gln Arg Asn Gly Ser Pro Ser Lys Thr              20 25 30 Val Val Ser Phe Ser Lys His Lys Trp His Lys Lys Ala Ala Ser Ser          35 40 45 Arg Leu Pro Met Ala Ile Arg Pro Ser Lys Pro Phe Asn Cys Pro Thr      50 55 60 Glu Lys Leu Lys Cys Val Ile Ser Ser Cys Ile Thr Ala Phe Arg Tyr  65 70 75 80 Ser Thr Arg Arg Trp Phe Arg Pro Asn Arg Val Lys Gly Ser Pro Lys                  85 90 95 Cys Arg Ala Gly Trp His Arg Ala Leu Lys Pro Met Leu Pro Pro Leu             100 105 110 Ser Arg Arg Ser Ser Arg Ser Ser Pro Ser Ser Lys Gln Gln Thr Ile         115 120 125 Leu Ala Pro Pro Thr Arg His Leu Arg Gly Arg Ile Cys Arg Trp Lys     130 135 140 Ile Asn Arg Arg Tyr Leu Trp Cys Ala Trp Met Thr Ser Ser Arg His 145 150 155 160 Asn Trp Cys Ile Trp Leu Thr Ser Leu Trp Arg Pro Ile Arg Leu Arg                 165 170 175 Val Phe Leu Leu His Asp Ala Asn Ser Gly Asp Val Val Lys Gln Trp             180 185 190 Asp Gly Leu Ala His Ala Glu Ala Thr Gly Thr Gly Pro Gly Gly Asn         195 200 205 Gln Lys Thr Gly Met Tyr Gln Tyr Gly Thr Asp Tyr Pro Gly Phe Ala     210 215 220 Val Ser Lys Thr Gly Ser Thr Cys Thr Met Leu Ser Ser Ala Val Lys 225 230 235 240 Thr Val Asp Leu Lys Asn Lys Thr Ser Gly Thr Thr Ala Tyr Ser Tyr                 245 250 255 Asp Cys Asn Asn Ser Ser Asn Tyr Asn Asp Tyr Lys Ala Val Asn Gly             260 265 270 Ala Tyr Ser Pro Leu Asn Asp Ala His Tyr Phe Gly Lys Val Val Phe         275 280 285 Asp Met Tyr Asn Asp Trp Leu Asn Thr Ser Pro Leu Thr Phe Gln Leu     290 295 300 Thr Met Arg Val Tyr Gly Ser Asn Tyr Glu Asn Ala Phe Trp Asp 305 310 315 320 Gly Ser Ala Met Thr Phe Gly Asp Gly Tyr Ser Thr Phe Tyr Pro Leu                 325 330 335 Val Asp Ile Asn Val Ser Ala His Glu Val Ser His Gly Phe Thr Glu             340 345 350 Gln Asn Ser Gly Leu Val Tyr Glu Gly Met Ser Gly Gly Ile Asn Glu         355 360 365 Ala Tyr Ser Asp Ile Ala Gly Glu Ala Ala Glu Tyr Tyr Met Arg Gly     370 375 380 Ser Val Asp Trp Val Val Gly Ser Asp Ile Phe Lys Ser Ser Gly Gly 385 390 395 400 Leu Arg Tyr Phe Asp Thr Pro Ser Lys Asp Gly Ser Ser Ile Asp His                 405 410 415 Ala Ser Gln Tyr Tyr Ser Gly Ile Asp Val His His His Ser Ser Gly Val             420 425 430 Phe Asn Arg Ala Phe Tyr Leu Leu Ser Asn Lys Gln Gly Trp Asp Val         435 440 445 Arg Lys Gly Phe Glu Val Phe Ala Val Ala Asn Gln Leu Tyr Trp Thr     450 455 460 Pro Asn Ser Thr Phe Asp Glu Gly Ala Cys Gly Val Val Lys Ala Ala 465 470 475 480 Gln Asp Leu Gly Tyr Asn Val Asn Asp Val Thr Ala Ala Phe Thr Thr                 485 490 495 Val Gly Val Asn Ser Ser Cys Ser Val Asp Ser Gly Asn Glu Leu Val             500 505 510 Lys Gly Gln Pro Val Thr Gly Leu Ser Gly Ser Ala Gly Ser Glu Ser         515 520 525 Phe Tyr Thr Phe Thr Val Asn Ser Ala Thr Thr Ala Thr Val Ser Ile     530 535 540 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser. 545 550 555 560 Pro Thr Thr Ser Ser Trp Asp Cys Arg Pro Tyr Arg Ser Gly Asn Asn                 565 570 575 Glu Gln Cys Ser Ser Ser Ala Val Ala Gly Thr Thr Tyr His Val Met             580 585 590 Leu Lys Gly Tyr Ser Ala Tyr Leu Pro Gly Val Thr Leu Arg Leu Asp         595 600 605 <210> 2 <211> 1827 <212> DNA <213> Vibrio furnissii <400> 2 atgaaaacat tacaacgtca agttaaagct tactcgcagt cggtacggtt atggctttcc 60 cggtttcggc agcgcaatgg gtcgccgtcg aagacagtgg tcagtttcag caaacataag 120 tggcacaaaa aggcagcatc gtcacgcctg ccaatggcta tcaggccatc aaaaccattc 180 aactgcccaa cggaaaagtt aaagtgcgtt atcagcagtt gtatcacggc gttccggtat 240 tcaacacggc ggtggtttcg accgaatcga gtaaagggat caccaaagtg cagggcagga 300 tggcacaggg cattgaagcc gatgttgcca ccgttaagcc gacgctcgat gagaagcagg 360 ccatcgccaa agcagcagac aattttagcg ccgccaacgc gtcatttacg gggcaggatc 420 tgccgatgga aaatcaatcg gcggtattta tggtgcgctt ggatgaccag cagcaggcac 480 aactggtgta tctggttaac ttctttgtgg cgtccgatac gcctgcgcgt ctttctactt 540 catgatgcca acagtggcga cgtggtgaaa cagtgggatg gtttggcgca cgcggaagcg 600 acgggcactg gccctggcgg taaccagaaa acaggcatgt atcaatacgg caccgattat 660 ccgggatttg cggtgagtaa aaccggttca acctgtacca tgctgagttc tgcggtcaaa 720 accgtagacc taaagaacaa aacatcaggc acgacagcct acagctacga ctgtaacaac 780 agcagtaact acaacgatta caaagcggtg aatggtgcct attcgccgct caatgacgcc 840 cactacttcg gtaaagtggt gttcgacatg tacaacgatt ggttgaatac ctcgccgctg 900 acgtttcagc taaccatgcg tgtgcattac ggcagcaatt atgagaacgc gttctgggat 960 ggctctgcca tgacgtttgg tgacggctat tcaacctttt atccgctggt ggacatcaac 1020 gtgagtgcgc acgaagtcag ccatggcttt actgagcaaa actcaggtct ggtatatgaa 1080 ggtatgtcag gcggtatcaa cgaagcctac tcggatatcg cgggcgaagc ggcggaatac 1140 tacatgcgcg gttcggtaga ctgggtggtc ggcagcgaca tctttaagtc gtccggcggc 1200 ctgcgctact tcgatacgcc gtcgaaagat ggcagctcga ttgatcacgc ttctcagtac 1260 tacagcggca tcgacgtgca ccattcaagc ggtgtgttca accgtgcgtt ctacttgctt 1320 tcgaacaaac aaggttggga tgtgcgcaaa ggttttgaag tgtttgccgt ggcaaaccaa 1380 ctctactgga cgccaaacag cacctttgat gaaggcgcgt gtggtgtggt gaaagctgcg 1440 caagatttgg gttacaacgt caatgatgtg actgcggcct ttaccacggt tggcgtgaac 1500 tcgtcatgtt ctgtggattc tggcaatgaa cttgttaaag gccaacccgt gactggtctt 1560 tctggttctg cgggttctga atcgttctac acctttacgg tgaacagcgc gacaaccgcg 1620 acggtctcga tcagttctgg ttcgggtgat gtggatctgt acgtgaaagc gggcagtaaa 1680 ccgaccacca gttcttggga ttgccgtcca tatcgttcgg gcaataacga gcagtgttcg 1740 atctcggctg ttgcaggcac cacgtatcac gtgatgctga aaggctacag cgcctatttg 1800 cccggtgtga cgctgcgtct ggactaa 1827 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caagcttctc gagaattcat gaaaacatta caac 34 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgcaggtacc cgggaattct tagtccagac gcag 34

Claims (9)

A recombinant thrombolytic enzyme derived from Vibrio furnissii consisting of the amino acid sequence of SEQ ID NO: 1. The recombinant thrombolytic enzyme according to claim 1, wherein the thrombolytic enzyme selectively degrades the A?, B? Or? Chain of fibrinogen. A composition for thrombolysis comprising the recombinant thrombolytic enzyme of claim 1 or 2 as an active ingredient. A method for decomposing thrombus by treating the composition of claim 3 except for a human containing thrombus. (a) culturing a recombinant microorganism transformed with a recombinant vector comprising a gene encoding the recombinant thrombolytic enzyme of claim 1; And
(b) recovering the recombinant thrombolytic enzyme from the culture or the culture supernatant of the step (a). [Claim 6] The method according to claim 5, wherein the gene encoding the recombinant thrombolytic enzyme comprises the nucleotide sequence of SEQ ID NO: 2. A recombinant thrombolytic enzyme produced by the method of claim 5. A health functional food composition for preventing or improving thrombosis comprising the recombinant thrombolytic enzyme of claim 1 or 7 as an active ingredient. A pharmaceutical composition for preventing or treating thrombosis comprising the recombinant thrombolytic enzyme of claim 1 or 7 as an active ingredient.

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