CN100355884C - Novel earthworm fibrinolytic enzyme, its encoding sequence and uses - Google Patents
Novel earthworm fibrinolytic enzyme, its encoding sequence and uses Download PDFInfo
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- CN100355884C CN100355884C CNB2005100232419A CN200510023241A CN100355884C CN 100355884 C CN100355884 C CN 100355884C CN B2005100232419 A CNB2005100232419 A CN B2005100232419A CN 200510023241 A CN200510023241 A CN 200510023241A CN 100355884 C CN100355884 C CN 100355884C
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Abstract
The present invention provides novel earthworm fibrinolysin named EFE-3D with a changed structure of genetic engineering, and a polynucleotide sequence for encoding the fibrinolysin, which belongs to the fields of biotechnology and medicine. The fibrinolysin modified by genetic engineering can be suitable for being expressed in yeast (Pichia pastoris), and consequently, earthworm fibrinolysin EFE-3D is generated by a recombination technology. The present invention also discloses a method of the earthworm fibrinolysin for treating multiple diseases such as thromboembolic disease, etc. The present invention also provides a medical composition containing the earthworm fibrinolysin.
Description
Technical field
The invention belongs to biotechnology and medical field, be specifically related to new earthworm fibrinolysin, called after EFE-3D, and the polynucleotide sequence of this plasmin of encoding, the invention still further relates to and utilize gene recombination technology to prepare the method for this plasmin, and this plasmin also relates to the pharmaceutical composition that contains this plasmin in the purposes of aspects such as treatment thrombotic disease.
Background technology
Earthworm fibrinolysin is the enzyme that the class extracted from earthworm can hydrolysis fibrinogen (former).It is present in the digestive tube of earthworm, molecular weight from 15,000 to 60,000 dalton.Earthworm fibrinolysin is not single a kind of enzyme, but has the general name of the multiple protein lytic enzyme of identical function.
Result of study shows that earthworm fibrinolysin has dual-use function, except energy direct hydrolysis scleroproein, can also activate profibr(in)olysin and become plasminogen, thereby have the effect of indirect hydrolysis of fibrin.The result of external pharmacological evaluation points out that earthworm fibrinolysin can also stimulate vascular endothelial cell to discharge tPA, thereby strengthen intravital fibrinolytic effect except directly dissolving the clot.Reach external thrombus formation model experiment and artificial thrombolysis experimental result in the body and point out that all earthworm fibrinolysin has tangible anti-bolt and thrombolytic effect.
Based on the such specific character of earthworm fibrinolysin, it has been made oral capsule both at home and abroad and be used for treating clinically thrombotic disease.For example, the commodity imperial heart by name, Lumbrukinase, Bo Luoke, general grace reach medicine such as thrombolysis capsule again and all make as raw material with earthworm fibrinolysin.
In view of plasmin has great application prospect at aspects such as treatment thrombotic diseases, so this area presses for the new plasmin of exploitation.But the content of natural earthworm fibrinolysin in earthworm is extremely low, is about 1,/20 ten thousand of earthworm body weight, and this has just brought very big technology barrier for extracting this enzyme, has also expended a large amount of natural resourcess.Therefore, the present invention carries out genetic engineering modified to natural earthworm fibrinolysin, makes it can be adapted at expression among the yeast Pichia Pastoris, and is suitable for genetically engineered production.
Summary of the invention
The purpose of this invention is to provide a kind of new earthworm fibrinolysin that changes structure through genetically engineered.
Another object of the present invention provides the polynucleotide sequence of the above-mentioned plasmin of coding.
Another object of the present invention provides the method for the above-mentioned plasmin of production and the purposes of above-mentioned plasmin and encoding sequence.
In a first aspect of the present invention, provide a kind of new genetically engineered to change the earthworm fibrinolysin protein of structure, called after EFE-3D, this protein come to change the structure product on the comfortable natural earthworm fibrinolysin genes encoding basis, and it comprises the protein with the aminoacid sequence shown in the SEQ ID NO:2; The significant difference of it and natural earthworm fibrinolysin gene is: the new fibrinolytic enzyme gene sequence through genetic engineering modified is a yeast preference type gene, can be incorporated in the yeast chromosomal and stablely goes down to posterity and duplicate; The new partial amino-acid that in genetic engineering modified fibrinolytic enzyme gene sequence, has substituted in the natural earthworm fibrinolysin gene, and this substituting improved the expression amount of plasmin in genetically engineered is produced significantly.
In a second aspect of the present invention, the polynucleotide that provide a kind of genetically engineered to change structure, these polynucleotide comprise and have a kind of nucleotide sequence that is selected from as next group:
(a) coding has the proteinic nucleotide sequence of aminoacid sequence shown in the SEQID NO:2;
(b) be complementary to the nucleotide sequence of above (a).
These polynucleotide are the nucleotide sequences with 16-738 position among the SEQ ID NO:1.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell (Piehia Pastoris) that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, a kind of method of utilizing gene recombination technology to prepare earthworm fibrinolysin as claimed in claim 1 is provided, this method comprises: (a) under the condition that is fit to expression EFE-3D, cultivate above-mentioned by transformed host cells; (b) from culture, isolate protein with plasmin activity.
In a fifth aspect of the present invention, above-mentioned earthworm fibrinolysin EFE-3D is provided or its polynucleotide of encoding in the purposes of treatment aspect the thrombotic disease.A kind of pharmaceutical composition also is provided, and it contains the earthworm fibrinolysin EFE-3D of safe and effective amount or encodes its polynucleotide and pharmaceutically acceptable carrier.
The present invention at first analyzes from multiple earthworm known array, kept wherein total conserved dna sequence, adopt the method for amino acid replacement simultaneously, substituted the aminoacid sequence that is not suitable in yeast, expressing in the natural plasmin, adopt the conserved dna sequence in the alternative natural plasmin of yeast preference type codon simultaneously, naming it is plasmin EFE-3D.Then the encoding sequence of plasmin EFE-3D is inserted suitable carrier and changes proper host cell over to, express and separated plasmin EFE-3D.And identified by the Western engram analysis.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The proteinic coding region sequence of encoding mature can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the plasmin EFE-3D protein of reorganization.In general following steps are arranged:
(1). with the proteinic polynucleotide of coding plasmin EFE-3D of the present invention (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). in suitable medium, cultivate host cell;
(3). separation, protein purification from substratum or cell.
Host cell is a yeast Pichia Pastoris cell.
Earthworm fibrinolysin EFE-3D of the present invention is of use in many ways.These purposes include, but is not limited to: direct disease as the pharmacological agent thrombotic disease.The protein molecule that can suppress or stimulate the plasmin function that can be used for seeking therapeutic value with the reorganization plasmin EFE-3D screening protein library of expressing.
Utilize plasmin EFE-3D of the present invention,, can filter out with plasmin interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Plasmin EFE-3D of the present invention and antibody, inhibitor, agonist or antagonist etc. when using (administration) in treatment, can provide different effects.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.
Protein of the present invention can be directly used in disease treatment, for example, is used for the treatment of thrombus aspect.When using plasmin EFE-3D of the present invention, also can use the other treatment agent simultaneously, as antitumor drug etc.
The present invention also provides a kind of pharmaceutical composition, and it contains plasmin EFE-3D protein of the present invention and the pharmaceutically acceptable carrier or the vehicle of safe and effective amount.This class carrier comprises (but being not limited to): physiological saline, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, protein of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that plasmin EFE-3D with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 microgram/skies, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 microgram/skies-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The method that whether has plasmin EFE-3D in a kind of test sample is to utilize the specific antibody of plasmin EFE-3D to detect, and it comprises: sample is contacted with plasmin EFE-3D specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample plasmin EFE-3D.
The invention has the advantages that: the plasmin that contains kind more than 10 in the earthworm.Separation and purification a kind of composition wherein has certain degree of difficulty to single purity on technology from earthworm.The present invention is to multiple earthworm fibrinolysin gene, analyze its gene conserved sequence, reject and be difficult in the gene structure of expressing smoothly in the yeast in its primary structure, and successfully express, and this expression product secretes in nutrient solution, more helps separation and purification.These achievements in research not only are that the development research gene engineering product has been created good condition from now on, also provide necessary chemical structure data for developing the plasmin injection from now on.In addition, plasmin EFE-3D of the present invention provides new treatment approach for diseases such as treatment thrombotic diseases, thereby has great application prospect.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 has shown the pcr amplification band of plasmin EFE-3D, and swimming lane 1 is the DNA standard; Swimming lane 2,3 is pcr amplification product (750bp).
Fig. 2 has shown the pcr amplification band of MFL gene and plasmin EFE-3D.Swimming lane 1 is the DNA standard; Swimming lane 2,3 is pcr amplification product MFL gene and plasmin EFE-3D fusion gene (1000bp)
Annotate: Fig. 1, the DNA standard molecular weight is from top to bottom in 2: 2000,1000,750,500,250 and 100bp.
Fig. 3 has shown the building process of expressing the recombination yeast Pichia Pastoris of plasmin EFE-3D.
Fig. 4 has shown the SDS-PAGE collection of illustrative plates and the western collection of illustrative plates of plasmin EFE-3D expression product.
Wherein: 1: a plurality of plasmin components all are positive with antibodies in the natural earthworm; 2: the yeast that does not contain the EFE-3D gene; 3,4,5: the yeast that contains the EFE-3D gene.
Fig. 5 has shown plasmin EFE-3D and natural earthworm fibrinolysin specific activity.Fibrinolytic circle maximum in the Lower Half is natural earthworm fibrinolysin 5 microgram amounts, and other are the yeast that contains the EFE-3D gene, different strains.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The natural earthworm fibrinolysin of separation and purification from earthworm
It is some to get fresh and alive earthworm (William strengthens the tail earthworm), through mechanical disintegration, is cooled to 4-10 degree centigrade rapidly, 10000rpm, and 4 ℃ of frozen centrifugations, and collect supernatant liquor.Add 50% ammonium sulfate, stirred 10000rpm frozen centrifugation, collecting precipitation 10 hours.Precipitation is dissolved in an amount of distilled water, through ion exchange column, and affinity column, multistep separation and purification such as molecular sieve column obtain the refining sample of earthworm fibrinolysin.Sample is through the HPLC method, or the SDS electrophoretic analysis, presents single band.
The preparation of the anti-earthworm fibrinolysin antibody of rabbit
Get body weight and be one of male new zealand rabbit about 2 kilograms.Add Freund's complete adjuvant with 1mg earthworm fibrinolysin sample and grind to form emulsion state, at the rabbit neck part multi-point injection.After raising two weeks, add Freund's incomplete adjuvant with 1mg earthworm fibrinolysin sample and grind to form emulsion state, again at the rabbit neck part multi-point injection.After one month, add Freund's incomplete adjuvant with same method booster immunization with 1.5mg earthworm fibrinolysin sample.Behind the two weeks, add Freund's incomplete adjuvant booster immunization once more with 1mg earthworm fibrinolysin sample again.After raising two weeks, carotid artery is got blood, 4 ℃ of standing over night, 2, centrifugal 3 minutes of 000rpm.Upper serum is the anti-earthworm fibrinolysin antibody of rabbit.
The gene design of plasmin EFE-3D
According to a plurality of known earthworm fibrinolysin gene orders, it is as follows to redesign new EFE-3D fibrinolytic enzyme gene sequence:
cgcctcgaga?aaagaatcct?tggtggtact?aaggctcgtg?ttggtgagat?cccatggcaa 60
cttagtcaac?aacgtggtgg?tagtcacagt?tgtggtgcta?gtcttcttcg?tccaggtagt 120
gctcttagtg?ctgctcactg?tgttgacggt?gctccaccag?ctgacgttcg?tatcgttgct 180
ggtcttcacc?ttcgtagtga?cgagagtact?gctgttgcta?gtcttgctga?gagtttcctt 240
atccacccaa?gttacaacgt?tggtgagggt?actttcccaa?acgacatcgc?tatcatctac 300
cttcttacta?acatcaacag?tgctccagtt?gagaacatcg?acttcgctct?tcttccacca 360
gacaacgttg?agcaattcgt?tggtttcact?tgtgttctta?gtggttgggg?tcgtactagt 420
gctagtaacg?ttcttccaga?cgctcttcaa?aaggttagta?tcgacgttat?cactactgct 480
gagtgtgaca?gtcgtatggc?tgctgttgct?ggtgctgact?gtactgacgc?tcacatcgct 540
gttttcgacc?cagctcttca?aaagggtagt?tgtaacggtg?acagtggtgg?tccaatgaac 600
tgtccactta?gtggtgagtt?cgttgttgct?ggtgttacta?gttggggtat?cagtggtggt 660
ggtgcttgtc?ttccagagta?cccaagtgtt?tacactcgta?ctggtttcta?ccgtcaatgg 720
atccttgaca?acatccgtta?gaattctc 748
Plasmin EFE-3D gene is synthetic
Plasmin EFE-3D gene order according to design among the embodiment 3 is divided into 32 dna fragmentations, 16 fragments of positive-sense strand wherein, and 16 fragments of antisense strand are through the synthetic splicing of artificial gene; And the employing pcr amplification, obtain a single-minded band of the DNA about 750bp.See shown in Figure 1.
Plasmin EFE-3D gene is connected with yeast MFL gene Fusion
Yeast α-MFL (yeast saccharomyces cerevisiae secretory gene) is the pPIC9K carrier (plasmid) that provides from Invitrogen company of GENE SOURCES (270bp), behind pcr amplification, Xho I restriction endonuclease by inside is connected with plasmin EFE-3D gene, again through PCR reaction, obtain one about 1000bp MFL and the fusion gene of EFE-3D.See shown in Figure 2.α-MFL gene is the leading peptide gene, and the EFE-3D gene after can helping to express is secreted in the nutrient solution.
Embodiment 6
The expression of plasmin EFE-3D and evaluation
The system constructing reconstitution cell that adopts Invitrogen company to provide.Wherein the plasmid construction collection of illustrative plates is seen Fig. 3.
(1) expresses
Plasmid transforms in bacterial strain Pichia Pastoris cell.Obtain to contain the recombinant cell strain of EFE-3D foreign gene through PCR screening, when treating that cell grows into certain density, adopt methyl alcohol to induce.Centrifugal collection supernatant liquor.The concrete operations step, the method that provides according to Invitrogen company.
(2) identify
After SDS-PAGE finishes, take off running gel 100V electricity under the condition of ice bath and change 2h to nylon membrane.Film is put into hybridization bag, add 5mL confining liquid (phosphate buffered saline buffer of 5% skim-milk), on oscillating platform, shake 1h..With confining liquid with 1: 1000 the dilution first antibody (the anti-earthworm fibrinolysin antibody of the rabbit that makes among the embodiment 2).Open hybridization bag, outwell confining liquid, add the good first antibody of dilution, shake 1h..Wash nylon membrane 3 times with phosphate buffered saline buffer, each 15min.Film that washing is good and confining liquid dilute good goat-anti rabbit alkaline phosphatase (dilution in 1: 3000) hybridization 1h..Film alkaline phosphatase damping fluid (100mmol/L Tris-HCl, pH9.5,5mmol/L MgCl
2, 100mmol/LNaCl) after the washing, with BCIP/NBT (5-bromo-4-chloro-3-one indoles phosphoric acid/nitroblue tetrazolium(NBT)) colour developing.
Results of hybridization shows, combines the EFE-3D protein of expression and earthworm fibrinolysin antibody specificity.See Fig. 4.
Embodiment 7
The determination of activity of plasmin EFE-3D
Press the described fibrin plate methods of people such as Deogny (Clinica ChimicaActa, 1975,60,85), calculate the activity of plasmin according to the area size of solusphere.The natural earthworm fibrinolysin of the control sample that is adopted for extracting among the embodiment 1, the result of vitality test is respectively: natural earthworm fibrinolysin is 21,000mm
2/ mg protein, plasmin EFE-3D are 20,000mm
2/ mg protein.See Fig. 5.
Both vigor are suitable.The point sample amount is calculated during according to the mensuration activity, and reference substance contains the natural earthworm fibrinolysin of 5 micrograms, and the mensuration group is 25 microlitre fermented liquids.Calculate, EFE-3D protein expression amount is 0.2 grams per liter.When adopting 50 liters of fermentations, just can obtain 10 gram EFE-3D protein, and adopt the natural earthworm fibrinolysin of same preparation 10 grams of method of natural extract to need 2 tons of earthworms.As seen, the method addressed of the present invention and the new gene of employing demonstrate tangible technical progress and technical superiority aborning.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
SEQUENCE?LISTING
＜110〉East China University of Science, Guoyuan Bio-Technology Co Ltd, Shanghai
＜120〉new earthworm fibrinolysin and encoding sequence and purposes
＜130〉specification sheets, claims
<160>2
<170>PatentIn?version?3.1
<210>1
<211>748
<212>DNA
＜213〉artificial sequence
<400>1
cgcctcgaga?aaagaatcct?tggtggtact?aaggctcgtg?ttggtgagat?cccatggcaa 60
cttagtcaac?aacgtggtgg?tagtcacagt?tgtggtgcta?gtcttcttcg?tccaggtagt 120
gctcttagtg?ctgctcactg?tgttgacggt?gctccaccag?ctgacgttcg?tatcgttgct 180
ggtcttcacc?ttcgtagtga?cgagagtact?gctgttgcta?gtcttgctga?gagtttcctt 240
atccacccaa?gttacaacgt?tggtgagggt?actttcccaa?acgacatcgc?tatcatctac 300
cttcttacta?acatcaacag?tgctccagtt?gagaacatcg?acttcgctct?tcttccacca 360
gacaacgttg?agcaattcgt?tggtttcact?tgtgttctta?gtggttgggg?tcgtactagt 420
gctagtaacg?ttcttccaga?cgctcttcaa?aaggttagta?tcgacgttat?cactactgct 480
gagtgtgaca?gtcgtatggc?tgctgttgct?ggtgctgact?gtactgacgc?tcacatcgct 540
gttttcgacc?cagctcttca?aaagggtagt?tgtaacggtg?acagtggtgg?tccaatgaac 600
tgtccactta?gtggtgagtt?cgttgttgct?ggtgttacta?gttggggtat?cagtggtggt 660
ggtgcttgtc?ttccagagta?cccaagtgtt?tacactcgta?ctggtttcta?ccgtcaatgg 720
atccttgaca?acatccgtta?gaattctc 748
<210>2
<211>241
<212>PRT
＜213〉artificial sequence
<400>2
Ile?Leu?Gly?Gly?Thr?Lys?Ala?Arg?Val?Gly?Glu?Ile?Pro?Trp?Gln?Leu
1 5 10 15
Ser?Gln?Gln?Arg?Gly?Gly?Ser?His?Ser?Cys?Gly?Ala?Ser?Leu?Leu?Arg
20 25 30
Pro?Gly?Ser?Ala?Leu?Ser?Ala?Ala?His?Cys?Val?Asp?Gly?Ala?Pro?Pro
35 40 45
Ala?Asp?Val?Arg?Ile?Val?Ala?Gly?Leu?His?Leu?Arg?Ser?Asp?Glu?Ser
50 55 60
Thr?Ala?Val?Ala?Ser?Leu?Ala?Glu?Ser?Phe?Leu?Ile?His?Pro?Ser?Tyr
65 70 75 80
Asn?Val?Gly?Glu?Gly?Thr?Phe?Pro?Asn?Asp?Ile?Ala?Ile?lle?Tyr?Leu
85 90 95
Leu?Thr?Asn?Ile?Asn?Ser?Ala?Pro?Val?Glu?Asn?Ile?Asp?Phe?Ala?Leu
100 105 110
Leu?Pro?Pro?Asp?Asn?Val?Glu?Gln?Phe?Val?Gly?Phe?Thr?Cys?Val?Leu
115 120 125
Ser?Gly?Trp?Gly?Arg?Thr?Ser?Ala?Ser?Asn?Val?Leu?Pro?Asp?Ala?Leu
130 135 140
Gln?Lys?Val?Ser?Ile?Asp?Val?Ile?Thr?Thr?Ala?Glu?Cys?Asp?Ser?Arg
145 150 155 160
Met?Ala?Ala?Val?Ala?Gly?Ala?Asp?Cys?Thr?Asp?Ala?His?Ile?Ala?Val
165 170 175
Phe?Asp?Pro?Ala?Leu?Gln?Lys?Gly?Ser?Cys?Asn?Gly?Asp?Ser?Gly?Gly
180 185 190
Pro?Met?Asn?Cys?Pro?Leu?Ser?Gly?Glu?Phe?Val?Val?Ala?Gly?Val?Thr
195 200 205
Ser?Trp?Gly?Ile?Ser?Gly?Gly?Gly?Ala?Cys?Leu?Pro?Glu?Tyr?Pro?Ser
210 215 220
Val?Tyr?Thr?Arg?Thr?Gly?Phe?Tyr?Arg?Gln?Trp?Ile?Leu?Asp?Asn?Ile
225 230 235 240
Arg
Claims (9)
1. a genetically engineered changes the earthworm fibrinolysin of structure, it is characterized in that it is the protein with the aminoacid sequence shown in the SEQ ID NO:2.
2. a genetically engineered changes the polynucleotide of structure, it is characterized in that, it comprises the proteinic nucleotide sequence that coding has aminoacid sequence shown in the SEQ ID NO:2.
3. polynucleotide as claimed in claim 2 is characterized in that these polynucleotide are the nucleotide sequences with 16-738 position among the SEQ ID NO:1.
4. carrier is characterized in that it contains polynucleotide as claimed in claim 2.
5. a genetically engineered host cell is characterized in that, it contains the described polynucleotide of claim 2, and behind methanol induction the described earthworm fibrinolysin of secreting, expressing claim 1.
6. host cell as claimed in claim 5 is characterized in that it is a yeast Pichia Pastoris cell.
7. a method of utilizing gene recombination technology to prepare earthworm fibrinolysin as claimed in claim 1 is characterized in that, this method comprises:
(a) use the described polynucleotide of claim 2, or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(b) being fit to express under the condition of plasmin, cultivate host cell;
(c) from culture, isolate protein with plasmin protein active.
8. the application of the polynucleotide of the earthworm fibrinolysin of claim 1 or claim 2 in preparation treatment thrombotic disease medicine.
9. pharmaceutical composition is characterized in that it contains the earthworm fibrinolysin of claim 1 of safe and effective amount or the polynucleotide and the pharmaceutically acceptable carrier of claim 2.
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| CN113234708A (en) * | 2021-05-10 | 2021-08-10 | 北京中医药大学 | Fibrinolysis active protein, preparation method and application thereof, pharmaceutical composition and nucleic acid for coding protein |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1361280A (en) * | 2000-12-29 | 2002-07-31 | 中国科学院上海生物化学研究所 | New plasmin and its coding sequence and use |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1361280A (en) * | 2000-12-29 | 2002-07-31 | 中国科学院上海生物化学研究所 | New plasmin and its coding sequence and use |
Non-Patent Citations (3)
| Title |
|---|
| 编码蚯蚓纤溶酶组A基因的cDNA克隆与表达 刘俊峰等.科学通报，第47卷第22期 2002 * |
| 蚯蚓纤溶酶在大肠杆菌中的克隆与表达 赵晓瑜等.河北大学学报(自然科学版)，第22卷第4期 2002 * |
| 蚯蚓纤溶酶基因的cDNA克隆及其序列分析 胡燕等.武汉大学学报(理学版)，第50卷第2期 2004 * |
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