CN1482139A - Mucroslysin protein and gene thereof - Google Patents

Abstract

The present invention provides one new kind of mucroslysin protein and gene, carrier for producing the protein and host cell. The mucroslysin protein of the present invention has fast thrombolysis effect without the side effect of hemorrhage.

Description

Mucroslysin protein and gene thereof

Technical field

The present invention system is about a kind of Mucroslysin (mucroslysin) protein and gene thereof, and the carrier and the host cell of this protein of manufacturing.

Background technology

Thrombosis is a kind of process that forms clot in blood vessel, can cause tissue injury, as processing in time, even can cause death.The formation of thrombus is relevant with the agglutination reaction of thrombocyte.Interaction between the endothelial cell surface of thrombocyte and injured blood vessel and other thrombocyte is to cause the main factor of thrombus.Thrombus is in case formation may cause vasculature part or whole obturations, cause many serious cardiovascular complications, comprise stenocardia, Acute Myocardial Infarction, cerebral vessels embolism (apoplexy), pregnant woman's pulmonary infarction, deep venous thrombosis and arterial thrombus etc., wherein myocardial infarction and apoplexy are to cause one of main causes of death.

Now have the medicine of many elimination clots, as urokinase (urokinase), streptokinase (streptokinase), acetylsalicylic acid (aspirin) and recombinant tissue plasminogen activator (recombinant tissue plasminogen activator, rt-PA), two pyramine alcohol (dipyridamole) and heparin.These medicines also can't directly dissolve clot, and are that Profibrinolysin produces endogenous plasmin (plasmin) in order to eliminate the clot that decomposition has produced in patient's body by activating.Yet said medicine all has the potential side effect that causes continuous bleeding.The shortcoming of these medicines is the interior Profibrinolysin that exhausts for the thrombi of resisting constant generation in the blood circulation of thrombus patient's body, and the Profibrinolysin that this moment, the manufacturing of patient's liver organization made new advances can't in time replenish, make the dynamic mechanism overbalance that forms thrombi, more therefore patient's thrombus district continues to enlarge, worsen.Medical personnel then are to use the medicine of more heavy dose of this type, want to utilize the formation and the continuation accumulation that are used for turning back or slowing down thrombi of its only surplus inhibition platelet aggregation, the result not only is helpless to thrombus dissolving and dwindles the thrombus district, the side effect that produces haemophilia on the contrary.For example, rt-PA is the optimal selection for the treatment of acute ischemic stroke and Acute Myocardial Infarction (AMI) at present, but the hemorrhage side effect that the effect of rt-PA is slow and tool is serious uses in 3 hours that its treatment only limits to that apoplexy begins, and can't treat the patient (Huang of tool non-fusibility thrombus (lytic-resistant thrombi), T.C., Jordan, R.E., Hantgan, R.R. reach Alevriadou, B.R. (2001) Thromb.Res.102,411-425).Rt-PA is not direct thrombus, and is to activate plasmin (plasmin) and activate the path with the cracking thrombus by a whole process (de novo) tissue plasmin.When sufferer is suffered from occluding thrombus, the common variation of this mechanism of action.The therapeutical agent that other uses clinically, as urokinase and streptokinase, then tool hangs down pipeline recanalization rate (recanalization rate) and side effect (as general dissolved state and hemorrhage complication) (Mueller, H.S., Roberts, R., Teichman, S.L. reach Sobel, B.E. (1989) Med.Clin.North Am.73, shortcoming such as 387-407).Therefore, the thrombolytic agent that presses for effective thrombus at present and have no side effect.

Has the multiple proteolytic enzyme that influences coagulation of blood in the venom of known poisonous snake.For example, three not fragrant (triflavin) and three lattice bright (trigramin) enzyme from the snake venom separation has resist coagulation activity (Cercek, B., Lew, A.S., Hod, H., Yano, J., Lewis, B., Reddy, K.N. and Ganz, W. (1987) Thromb.Res.47,417-426).Fibrinogen lytic enzyme and fibrinolytic enzyme present the resist coagulation activity, thus have potentiality as thrombolytic agent (Trikha, M., Schmitmeier, S. and Markland, F.S. (1994) Toxicon 32,1521-1531).Many these fermentoids that have been separated are for containing the metalloprotease of zinc; Except that the resist coagulation activity, also have a hemorrhagic activity.Therefore, this fermentoid is not suitable for clinical application.The at present existing non-hemorrhagic metalloprotease of minority separates from snake venom, as cellulase (fibrolase), H2-proteolytic enzyme (H2-proteinase), draw shellfish enzyme (lebetase) and fibrinogenase (vipera lebetina fibrinogenase, VIF) (Sanchez, E.F., Bush, L.R., Swenson, S. reach Markland, F.S. (1997) Thromb.Res.87,289-302; Takeya, H., Arakawa, M., Miyata, T., Iwanaga, S., and Omori-Satoh, T. (1989) J.Biochem. (Tokyo) 106,151-157; Trummal, K., Vija, H., Subbi, J. and Siigur, J. (2000) Biochim.Biophys.Acta 1476,331-336; And Gasmi, A., Chabchoub, A., Guermazi, S., Karoui, H., Elayeb, M. and Dellagi, K. (1997) Thromb.Res.86,233-242.).Yet these protein are difficult to separate in the hemorrhagic isozyme of venom, and can't use in a large number clinically.In addition, these protein need at least 90 minutes just can be with thrombolysis.The existing at present venom protein that utilizes the method manufacturing of genetic engineering, as MT-C, MT-d-I and MT-d-II etc., all can in experiment in vitro, show the activity (Jeon of decomposes collagen (collagenolytic) and decomposition gelatin (gelatinolytic), O.H. reach Kim, D.S. (1999a) Biochem.Mol.Biol.Int.47,417-425; And Jeon, O.H, and Kim, D.S. (1999b) Eur.J.Biochem.263,526-533).

Summary of the invention

The present invention relates to a kind of new Mucroslysin (mucroslysin) protein, comprise the aminoacid sequence shown in SEQ ID NO:1, and the equivalent of tool thrombus dissolving function.

The present invention comprises the nucleotide sequence of Mucroslysin protein aminoacid sequence shown in the coding tool SEQ ID NO:1 also about a kind of nucleic acid molecule.

The present invention is also about a kind of host cell that comprises the expression vector of nucleotide sequence of the present invention and comprise carrier of the present invention.

The present invention comprises and will include the host cell of nucleic acid molecule of the present invention again about the method for a kind of manufacturing protein of the present invention, cultivates under the condition of tolerable by the protein expression of this nucleic acid molecule encoding; Reach and in this host cell nutrient solution, reclaim this protein.

The present invention comprises the present invention's Mucroslysin protein again about a kind of pharmaceutical composition.

Description of drawings

Fig. 1 is the present invention's recombinant DNA sequence, reaches the aminoacid sequence according to the cDNA sequence derivation of Mucroslysin.Top line is the nucleotide sequence of the cDNA of 2139bp; Bottom line is the aminoacid sequence according to the derivation of cDNA sequence.The sequence number of Nucleotide and amino acid is marked in the right side.Amino acid system is with single alphabetical marked.The signal peptide that straight line under the aminoacid sequence expresses possibility (signal peptide).Kozak sequence aaaATGA and polyadenylation signal AATAAA are all with box indicating.In the proenzyme zone, the conserved sequence system in the total zone of zine ion calmodulin binding domain CaM and RGD represents in the mode of stroke bottom line.Part shown in the arrow is GSP5b and GST3b primer.

Fig. 2 is the tissue specificity transcription analysis of Mucroslysin.The Northern blotting is that Mucroslysin cDNA with [32P] mark is as probe.The used separation of each row is 10 micrograms from total RNA of trimeresurus mucrosquamatus Cantor different tissues in this experiment.1: poison gland; 2: brain; 3: lung; 4: testis; 5: liver; 6: the heart.Figure bottom system is with the 18S ribosome-RNA(rRNA) of [32P] mark identical trace figure as probe.

Fig. 3 is tricine SDS-PAGE and the immunoassay trace figure of generation from the reorganization Mucroslysin of the cDNA of Mucroslysin.(A) with the Mucroslysin protein distribution plan of the 10%tricineSDS-PAGE analysis of tool Coomassie indigo plant; And (B) purifying from the Western blotting analysis chart of the reorganization Mucroslysin of His-binding resin.The Western blotting is to dilute 5000 times antiserum(antisera) analysis.Arrow is depicted as the Mucroslysin protein of performance.1: molecular weight protein marker (9-115kDa); 2:pET21a (I) is the vehicle Control of inducing with IPTG; 3:pMucroslysin; And 4:pMucroslysin (I) is the reorganization Mucroslysin protein of His-bind purifying, induces (2 hours) with IPTG respectively or do not induce with IPTG.

Fig. 4 is for folding the fibrinogenolysis activity and the time relation of the Mucroslysin protein of back again.The analysis of proteolytic activity system utilizes 10 microgram bovine fibrinogen and unfolded protein co-cultivation again to inspect.Incubation time is shown in the figure top, and cracking pattern system analyzes with 10%SDS-PAGE.The A α of Fibrinogen-, B β-and γ-chain be shown in the right side.Molecular weight protein marker is shown in left side (20-182kDa).

Fig. 5 is the thrombolysis analysis of the interior Mucroslysin of body to artificial thrombus.Artificial thrombus lies in tail postcava through anesthetized rat and utilizes bovine fibrinogen and thrombus to induce to form.Thrombolysis system uses vasography record 2 hours.(A) the demonstration thrombus is induced the angiogram of back.When beginning (0 o'clock), artificial thrombus total blockage artery blood flow.Locate interface shown in the arrow into thrombus and rear side blood.(B) angiogram behind 1.0 milligrams of (1.0 milligrams/kg body weight) Mucroslysin proteins of injection per kilogram of body weight.Arrow is represented the zone of siphunculus again.

Embodiment

The invention provides a kind of Mucroslysin (mucroslysin) protein and gene thereof, and make the carrier and the host cell of this protein.The present invention clones and shows non-hemorrhagic thrombus dissolving snake venom protein from trimeresurus mucrosquamatus Cantor (Trimeresurusmucrosquamatus), and reproduces the biological function activity of this protein.The snake venom protein of this genetic engineering is called Mucroslysin, and (in 15 minutes) present effective thrombus dissolving effect and do not have hemorrhage side effect at short notice. Protein

The present invention system comprises the aminoacid sequence shown in SEQID NO:1 (as Fig. 1) about a kind of Mucroslysin (mucroslysin) protein, and the equivalent of tool thrombus dissolving function.As used herein, words and phrases " equivalent of thrombus dissolving function " are one to have the protein identical with the thrombus dissolving function of Mucroslysin protein of the present invention.According to the present invention, Mucroslysin protein of the present invention has the former activity of solution fibrin.

According to the present invention, the clone provides the 52kDa protein of thrombus activity, this protein and string wonderful suffering (trimucin) tool high homology, and be the snake of separation from same breed.Protein of the present invention is containing metal protein, and it comprises a proenzyme forefoot area (zymogenprodomain), proteolytic enzyme zone and a disinterin zone.Signal peptide is made up of 18 conserved amino acids and the proenzyme sequence is made up of 171 conserved amino acids.One high conservative sequence PKMCGVT system is positioned at the terminal near the proenzyme zone.The proteolytic enzyme zone comprises 203 amino-acid residues, and comprising 7 cysteine residues, and wherein 6 cysteine residues relate to intrachain disufide bond.Similar with other snake venom metalloprotease, the activated positions conservative amino acid sequence HEXXHXXGXXH of Mucroslysin is found in the proteolytic enzyme zone.Yet the metalloprotease zone of Mucroslysin is the N-glycosylation position not.This lack the N-glycosylation position may with non-hemorrhagic characteristic relevant (Nikai, T., Taniguchi, the K. of snake venom metalloproteinase, Komori, Y., Masuda, K., Fox, J.W. and Sugihara, H. (2000) Arch.Biochem.Biophys.378,6-15).After the proteolytic enzyme zone is the zone of one 16 amino acid, and it is connected to proteinic second zone, promptly proteinic disintegrin zone.

According to the present invention, the preparation of recombinant protein ties up under the condition of this protein expression of tolerable, and cultivate the host cell of the nucleic acid molecule that comprises code book invention protein and reclaim this protein, and optionally folding again this protein.

According to the present invention,,, will lose its biological activity as bacterial expression system at the protein of some expression system expression.Therefore, the process that recover the unfolding (unfolding) of the proteinic biological activity protein of the present invention and folding again (refolding) is essential.22 cysteine residues are arranged in the sequence of protein of the present invention, can form 11 intramolecularly cystine linkages at most.It is successfully folding again that these cystine linkages can make this protein be difficult to.Yet the present invention can successfully folding again Mucroslysin protein.According to the present invention, this folds in the buffering system that ties up to the amino acid (for example, gsh) that comprises metal ion (for example, zinc and calcium ion) and carries out. Nucleic acid

The invention provides a kind of isolated nucleic acid molecule, comprise the nucleotide sequence (being the gene order of Mucroslysin protein) of Mucroslysin protein aminoacid sequence shown in the coding tool SEQ ID NO:1 (see figure 1).The words and phrases that this paper used " nucleic acid molecule " mean DNA or RNA congener and derivative, fragment and the homologue that comprises that dna molecular (as cDNA or genomic dna), RNA molecule (as mRNA), use nucleosides congener are produced.Nucleic acid molecule can be strand or two strands, but preferable with double-stranded DNA.The words and phrases that this paper used " isolating nucleic acid " molecule means other nucleic acid person who separates from being stored in natural origin.

According to the present invention, nucleic acid can only comprise the part fragment of coding Mucroslysin biological activity.The words and phrases that this paper used " fragment " mean to encode still has a nucleotide sequence part of Mucroslysin protein fragment of biological activity.

In the preferred embodiment, isolated nucleic acid molecule of the present invention has nucleotide sequence (see figure 1) or its degenerate sequence (wobble hypothesis, Wobble hypothesis) of SEQ ID NO:2 one of according to the present invention.In another preferred embodiment, isolated nucleic acid molecule of the present invention comprises the nucleotide sequence shown in SEQ ID NO:2.As those skilled in the art approval person,, can make the nucleic acid of many code book invention Mucroslysin proteins based on the degeneracy (wobble hypothesis) of gene-code.Therefore, at one by the specific amino acids sequence of evaluation, those skilled in the art can be under the situation of the aminoacid sequence that does not change Mucroslysin protein, and mat is simply modified one or more password and made various different nucleic acid.

According to the present invention, the nucleic acid of coding Mucroslysin can the use standard hybridization and clone technology separate.Especially, nucleic acid of the present invention can the use standard be cloned and triage techniques, separates from the cDNA storehouse of trimeresurus mucrosquamatus Cantor venom.The amplification of nucleic acid of the present invention (amplification) can be according to Standard PC R amplification technique, uses cDNA, mRNA or genomic dna to get as template and suitable Oligonucleolide primers.Can be cloned into suitable carrier and the mat dna sequence analysis is identified through the nucleic acid of amplification.Especially, the cDNA library of trimeresurus mucrosquamatus Cantor venom system constructs in a suitable expression, as λ gt11.Mucroslysin cDNA is mat screening recombinant clone strain and separating, and the nucleotide sequence of gained is shown in SEQ ID NO:2, and size is 2139bp, has had the nucleotide position of starting from 96 and has ended at the open reading frame of the termination codon (TAA) of position 1541. Expression vector and host system

The present invention also provides an expression vector, comprises the Mucroslysin nucleotide sequence of a coding shown in SEQ ID NO:1.In a present invention preferred embodiment, expression vector comprises the nucleotide sequence shown in SEQ ID NO:2.The words and phrases that this paper used " expression vector " are directly to express the gene nucleic acid molecule that is connected on it.Preferable carrier can duplicate and express connection nucleic acid person thereon voluntarily for they.General speech, the expression vector that can be used for recombinant DNA technology is generally " carrier " form, is generally the DNA of ring-type two strands, does not merge to karyomit(e) when it is carrier format.

Be to express the Mucroslysin of tool biological activity, the encode nucleotide sequence of Mucroslysin protein or its function equivalent of the present invention can insert in the suitable expression vector, and this carrier need contain the necessary assemblies such as transcribing and translate that inserts encoding sequence.According to the present invention, those skilled in the art can utilize the method for knowing to construct to contain the coding Mucroslysin and suitably transcribe and translate the expression vector of control unit.These methods comprise the extracorporeal recombinant DNA technology, synthetic technology and vivo gene recombinant technology etc.

Another purpose system of the present invention provides the host cell that comprises expression vector, this carrier to comprise the nucleotide sequence of the Mucroslysin of encoding.The words and phrases that this paper used " host cell " are can be through the host cell of carrier (as plasmid) infection.Be applicable to that the present invention's host comprises usually and be generally used in the host of this area.According to the present invention, many host systems can be used for comprising and expressing the sequence of coding Mucroslysin.These host systems include but not limited to microorganism (as the bacterium with recombinant plasmid or expression vector conversion), yeast (as the yeast with yeast expressed carrier conversion) or zooblast system. Pharmaceutical composition

The present invention also comprises a kind of pharmaceutical composition, comprises the present invention's recombinant protein.Except that protein of the present invention, this pharmaceutical composition can contain suitable pharmaceutically acceptable supporting agent.

The present invention's pharmaceutical composition can be known in the art mode (for example mat common method) make, this common method comprises mixings, dissolving, granulation, makes ingot, levigate, emulsification, encapsulate, bag is sunken and/or step such as lyophilize.

But the dispensing of any approach of the present invention's pharmaceutical composition mat, that these approach include, but is not limited to is oral, in the intravenously, intramuscular, intra-arterial, marrow, in the sheath, Intraventricular, wear in skin, subcutaneous, the peritoneal cavity, in the nose or enteron aisle throw with. Practicality

According to the present invention, the present invention's Mucroslysin protein is preferably in 15 minutes at short notice, can present the thrombus effect and not have the hemorrhagic side effect.In other words, Mucroslysin only needs the short period of time (about 15 minutes) can remove thrombus, and it presents unique characteristic on the treatment thrombus.The present invention's Mucroslysin protein has the former activity of solution fibrin, and direct thrombus is so provide very fast dissolution rate.Therefore, protein of the present invention can be used for treating cardiovascular complication, comprises stenocardia, Acute Myocardial Infarction, cerebral vessels embolism (apoplexy), pregnant woman's pulmonary infarction, deep venous thrombosis and arterial thrombus.The special system of protein of the present invention is useful on treatment apoplexy, Acute Myocardial Infarction, pregnant woman's pulmonary infarction and deep venous thrombosis.

Embodiment Embodiment 1 The nucleotide sequence analysis of the cDNA of MucroslysinAntibody Preparation

The crude venom system of 35 microlitres takes from trimeresurus mucrosquamatus Cantor, and does not at room temperature activate 1 hour with 10% formalin of equal volume amounts.Do not activate venom again with PBS damping fluid (137 millimolar concentration NaCl, 2.68 millimolar concentration KCl, 10 millimolar concentration Na ₂HPO ₄And 1.76 millimolar concentration KH ₂PO ₄, pH7.4) be diluted to 0.5 milliliter volume, and then mix fully with 0.5 milliliter of Fu Shi (Freund) Freund's complete adjuvant.This mixture (1.0 milliliters) then is injected in the rabbit body with intracutaneous dosing mode repeatedly.Supplementary immunization reaction 3 times again in during 2 to 3 weeks.CDNA library construction and screening

According to the described method of people such as Sambrook (Maniatis, T., Fristsch, E.F., and Sambrook, J. (1989) in Molecular Cloning:A Laboratory Manual, editor: Ford, N., Nolan, C., and Ferguson, M., Cold Spring Habor LaboratoryPress, New York, 8.3-8.80 page or leaf) uses 10 milligrams the poly (A) that separates from ripe trimeresurus mucrosquamatus Cantor venom ⁺RNA prepares double-stranded cDNA.Venom cDNA storehouse is implemented in λ gt11 cloning system (Promega Co.), and this cDNA library comprises about 1.0 * 10 ⁵Individual bacteriolyze spot is with the anti-three not polyclonal antiserum screenings of fragrant (triflavin).Hybridization was carried out 16 hours in the mixture of the salmon sperm dna behind a-protein, secondary antibody and the ultrasonic oscillation of tool [125I] mark that comprises 10 * Denhardt solution, 6 * SSC, 50 Dimethyl formamides and 100 mg/ml under 37 ℃.Use the purifying composition that separates from the T.flavoviridis venom, three not fragrant (triflavin) antigens, synthetic anti-three not fragrant (triflavin) antiserum(antisera)s.The 5 '-rapid amplifying (5 '-RACE) of cDNA terminal

From synthetic two specific antisenses (anti-sense) primer of 5 ' terminal sequence of the cDNA of the part Mucroslysin in snake venom gland cDNA library, it is respectively the GST5b primer according to separation: 5 '-TAT TTG AAG ACT GCA TGG GC-3 ' (SEQ ID NO:3) and nido (nested) GST3b primer: 5 '-GGT ACG TCT CCC CTT GAA GC-3 ' (SEQ ID NO:4) (see figure 1).Mucroslysin 5 '-end cDNA synthetic use 5 '-RACE system test kit (U.S.A. is according to producer's flow process for Life Technologies, NY) carries out.First complementary strand uses GST5b primer and ThermoScript II to synthesize, and product prolongs at 3 ' end with few d (C) and terminal enzyme (DNA).The support primer that 5 ' end regions of full-length cDNA then uses nido GST3b primer and is complementary to widow (C) increases through polymerase chain reaction (PCR).Carry out 39 circulations altogether, each circulation be 60 seconds, 51 ℃ of 94 ℃ of thermally denatures reactions annealing reaction 40 seconds, and 72 ℃ carry out 5 minutes (Perkin Elmer of primer extension with the UlTma polysaccharase, CA, U.S.A.), use 1605 Air Thermo-Cycler (Idaho Technology, ID, U.S.A.).The DNA sequencing

The cDNA of Mucroslysin is subcloned on the M13mp18/19 phage, and use the described dideoxy chain termination of previous Sanger (dideoxy chain termination method) order-checking (Sanger, F., Nicklen S. and Coulson, A.R. (1977) Proc.Natl.Acad.Sci.USA.74,5463-5467).The sequence analysis software bag that relatively uses GCG company to provide of nucleotide sequence and institute's deduced amino acid carry out (Genetics Computer Group, Inc., WI, U.S.A.). The evaluation of the nucleotide sequence of Mucroslysin cDNA and analysis

The cDNA library construction of trimeresurus mucrosquamatus Cantor venom is in expression vector λ gt11.The cDNA of Mucroslysin is with anti-three not fragrant (triflavin) antiserum(antisera) mat screenings 1.0 * 10 ⁵Individual recombinant clone and separating.Protein-A antibody screening method of using [125I] mark is to obtain 20 positive immune clones that comprise the cDNA of Mucroslysin.The magnitude range that the DNA inset is pointed out in the analysis of 20 positive colonies is from 700 to 1,400 base pairs (promptly 0.7～1.4Kb).1.4Kb the clone is through screening and be used to clone the cDNA of total length.As a result, 5 of mat cDNA end '-rapid amplifying method (RACE) obtains a 0.67Kb DNA except that the 1.4Kb sequence.The aminoacid sequence of nucleotide sequence (SEQID NO:2) and derivation (SEQ ID NO:1) as shown in Figure 1.The cDNA of this 2139bp size has the open reading frame of the termination codon TAA that starts from Nucleotide 96 together and finish from the position 1541.(in-frame) methionine(Met) password system is found and is positioned at 96 positions and is contained in the common sequence of rotaring intertranslating start, AAAATGA in first frame.Coding region has 481 amino acid of 1475 base pairs and codified tool 52.1KDa molecular weight.5 '-do not translate regional rising to start from Nucleotide 1 to 95.3 '-not translate the zone and comprised long extension of 598 Nucleotide that starts from Nucleotide 1542 and extend to Nucleotide 2139, it has polyadenylation signal, and AATAAA is located at the 20bp upstream (Fig. 1) of position 2114 poly-(A)+tails.The tissue specificity of embodiment 2 Mucroslysin mRNA is translated

The organ of excision trimeresurus mucrosquamatus Cantor is existed side by side, and soon it is frozen in the liquid nitrogen.Total RNA extraction is to use to be described in Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, the method for 5294-5299 from poison gland, brain, lung, testis, liver and the heart of trimeresurus mucrosquamatus Cantor.The cDNA probe use rediprimer dna marker system (Amersham, IL, U.S.A.) with [α-32P] dCTP mark (Amersham, IL, U.S.A.).The hybridization of Northern blotting was carried out 16 hours as probe with the Mucroslysin cDNA through mark under 42 ℃.

The analysis of Northern blotting shows the poison gland specificity hybridization product (Fig. 2) of a 2.1Kb.The analysis of Northern blotting points out that Mucroslysin gene organization specificity only finds expression in poison gland, with and the cDNA of corresponding length. Embodiment 3 The preparation of Mucroslysin proteinThe structure of expression plasmid

The structure of expression plasmid is that mat connects the coding region of Mucroslysin cDNA to pET21a carrier (Novagen Inc., WI, BamHI U.S.A.) and EcoRI position.Then will comprise the plasmid of 1.47Kb inset, and be called pMucroslysin, transfection is carried out protein expression to E.coli BL21 (DE3) cell.The expression of fused protein and purifying

BL21 (DE3) cell that comprises pMucroslysin grows to late logarithmic phase (A600=0.3～0.4) in the Luria-Bertani nutrient solution and (isopropyl-β-D-thiogalactoside IPTG) induces 2 hours with the isopropyl-of 1 millimolar concentration.At 8M urea buffer solution (8M urea, 0.1M NaH ₂PO ₄, 10mM Tris-HCl pH8.0) and with the ultrasonic wave effect, cell can form agglomerate.The lysate that gained is comprised the Mucroslysin protein of recombinating was cultivated 2 hours with His-binding affinity resin under 4 ℃.This resin then cleans 2 times with the 8M urea buffer solution.Recombinant protein is to contain the same buffer wash-out of 400mM imidazoles (imidazole).The productive rate of protein purification is 10 mg/litre inoculums.Purified fused protein is analyzed (Fig. 3 A) with 10%tricine SDS-PAGE.The Western blotting is analyzed

According to the method for Burnette carry out the Western blotting (Burnette, W.N. (1981) Anal.Biochem.112,195-203).Film is at first resisted-processing of trimeresurus mucrosquamatus Cantor poison gland (1: 5000) antiserum(antisera) with rabbit, then react with the peroxidase-conjugated secondary antibody of goat anti-rabbit igg.Use 3,3 '-diaminobenzidine (3,3 '-diaminobenzidine) (DAB) and hydrogen peroxide detect protein.Western trace figure system is shown in Fig. 3 B.Via the Mucroslysin of His-combination-resin purification is that clonal antibody is confirmed more than the anti-trimeresurus mucrosquamatus Cantor venom of mat, and finds to have the molecular weight of 52kDa.Be expressed in the folding again and evaluation of the recombinant protein of E.coli

The Mucroslysin protein of purified back is carrying out folding under 4 ℃ in the buffered with amino acid liquid that contains metal ion (folding again damping fluid) again.Again the functional selection of unfolded protein under 37 ℃ in containing 1mM CaCl ₂And among the 10 microgram Fibrinogens, carried out respectively 0,3,6,18,24,30 and 36 hour.Fibrinogen is 100/1 (w/w) to the protein ratio of unfolded protein again, and with 100 ℃ down heating came termination reaction in 5 minutes, and the gained sample is utilized sds gel electrophoresis analysis.

In this Fibrinogen was analyzed, the A α chain of Fibrinogen can be by complete digestion back 18 hours of cultivation.Yet the digestion of B β chain began after 18 hours and can be by complete digestion after 36 hours.Mucroslysin also presents the ability of the γ chain of cracking Fibrinogen.The gel bottom is in 30kDa place aggegation one extra band, and it corresponds to the B β chain and the γ chain (Fig. 4) of the Fibrinogen of disappearance. Embodiment 4 Thrombolysis is analyzedThe thrombolysis analysis of angiogram

The former lytic activity of the body inner fibrin of recombinant protein system tests to induce in the artificial thrombus of the tail postcava of Sprague-Dawley (SD) mouse, and with vasography analysis thrombolysis situation, analysis time was above 2 hours.At first conduit is inserted right chain vein through anesthesia SD mouse to carry out dosing and blood sampling.After artificial thrombus tied up to abdominal, mat was injected the bovine fibrinogen solution (5%) of 15 microlitres and 5 microlitre thrombus (100u/ml) and is induced tail postcava in separation.Thrombus was induced back 1 hour, and the reorganization Mucroslysin protein of 1.0 milligrams/kilogram of dosage is injected in rat through the thigh bone ductus venosus.Before getting angiogram, through the intravascular contrast media (angiograffin) of tube injection 0.5 ml volumes) (Angiovist 370, Berlex Labs).Respectively when 0 minute, 15 minutes, 30 minutes of experiment beginning back and 120 minutes, absorb altogether four angiograms (Willis, T.W., Tu, A.T. and Miller, C.W. (1989) Thromb.Res.53,19-29).

The intravenously dispensing of the reorganization Mucroslysin that dosage is 1.0 milligrams/kilogram showed the dispensing Mucroslysin after 15 minutes, and the thrombus of original occluding vascular begins dissolving and dissolving (Fig. 5) fully in 2 hours.Histology experiment through the animal of reorganization Mucroslysin protein processing

10 Sprague-Dawley mouse are through the Mucroslysin protein of 10 milligrams/kg body weight of intravenous injection dosage.After 24 hours, the tissue slice of taking from kidney, liver, the heart and lung is with 10% formalin fixed and be embedded in the paraffin.Section is with phenodin (hematoxylin) and Yihong (eosin) dyeing.Each tissue carries out necrosis and hemorrhage detection.The tissue detection demonstration of kidney, liver, the heart and lung tissue there is no necrosis and bleeding.

Nucleic acid and the protein of the foregoing description system in order to further specify the present invention is not the restriction as the present invention.

Claims (15)

1. a Mucroslysin (mucroslysin) protein comprises the aminoacid sequence shown in SEQ ID NO:1, and the equivalent of tool thrombus dissolving function.

2. according to the protein of claim 2, the activity that its tool solution fibrin is former.

3. isolated nucleic acid molecule, it comprises the nucleotide sequence of Mucroslysin protein aminoacid sequence shown in the coding tool SEQ ID NO:1.

4. according to the nucleic acid molecule of claim 3, wherein this molecule comprises nucleotide sequence or its degenerate sequence shown in SEQ ID NO:2.

5. an expression vector comprises the nucleotide sequence as claim 3.

6. an expression vector comprises the nucleotide sequence as claim 4.

7. a host cell comprises as the carrier according to claim 5.

8. a host cell comprises as the carrier according to claim 6.

9. the method for protein of manufacturing such as claim 1, it comprises the following step:

A) will include host cell, under the condition of tolerable, cultivate by the protein expression of this nucleic acid molecule encoding as the nucleic acid molecule of claim 3; And

B) reclaim this protein in this host cell nutrient solution certainly.

10. according to the method for claim 9, other is included in the step that folds this protein in the buffered with amino acid system that contains metal ion again.

11. a pharmaceutical composition comprises the protein as claim 1.

12., have the former activity of solution fibrin according to the pharmaceutical composition of claim 11.

13., be used to remove thrombus according to the pharmaceutical composition of claim 11.

14. according to the pharmaceutical composition of claim 11, be used for the treatment of and be selected from following cardiovascular complication: stenocardia, Acute Myocardial Infarction, cerebral vessels embolism (apoplexy), pregnant woman's pulmonary infarction, deep venous thrombosis and arterial thrombus.

15., be used for the treatment of apoplexy, Acute Myocardial Infarction, pregnant woman's pulmonary infarction and deep venous thrombosis according to the pharmaceutical composition of claim 11.

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