CN1108817C - Thrombolytic medicine - Google Patents

Abstract

The present invention relates to a streptokinase preparation. A gene engineering technology is adopted, proteinase genes which can strongly decompose fibrin are extracted from particular bacillus subtilis through PCR gene magnification and transferred to obtain gene engineering bacteria, and the gene engineering bacteria are cultivated, fermented, separated and purified to obtain the streptokinase preparation. Tests show that the streptokinase preparation has the capability of dissolving fibrin (zymogen), particularly cross-linked fibrin, inhibiting thrombosis effectively and dissolving thrombus matrixes to dredge blood flow. As oral liquid, the streptokinase preparation can prevent and cure cardiovascular and cerebrovascular diseases. The present invention has the advantages of extensive raw material sources, economical raw materials, mature technology, simple and convenient extraction and low cost.

Description

Thrombolytics

The present invention is a thrombolytics, belongs to technical field of bioengineering.

Thrombotic disease such as myocardial infarction, cardiovascular and cerebrovascular diseases such as lung infraction, coronary artery infraction, cerebral infarction apoplexy and arteriovenous vascular infraction have become the No.1 killer of the healthy and life security of serious harm people.People are in capturing the process of this disease, and the Fibrinogen of understanding thrombotic one of the main reasons gradually and being in the blood of human body forms cancellated fibrin under thrombin action.Therefore, people wish to find can dissolve these fibrinous thrombolytics, makes cancellated fibrinolysis, reaches the mobile effect of unblocked blood flowing.Mainly contain streptokinase (being called for short SK) that from the Streptococcus hemolyticus culture fluid, separates acquisition and the urokinase (UK) that from human urine, extracts purification as present commercial thrombolytics, and the human histiotype fibrinolysin activator (being called for short tPA) that adopts gene engineering method to produce, because these thrombolytics are fibrinolysin activator in the human body, so the direct fibrin in the thrombus.Simultaneously, these thrombolytics are intravenous form in the use, can not be oral, can only can not be used for the prevention of thrombotic disease as treatment acute thrombus disease.In its preparation,, be difficult to extensively promote the use of because complicated process of preparation costs an arm and a leg.

Japan scholar Sumi H etc. once reported and be separated to a kind of fibrinous protease that can decompose from japanese traditional fermented food " natto ", this is called nattokinase (being called for short NK), and to its gene structure, physicochemical properties etc. have been done comparatively detailed research, but commercialization not yet so far, (Sumi.H, et al.Experientia, 43 (10) 1110-1111,1987; Fujita.M, et al.Biol Pharm Bull 18 (10), 1387～1391,1995).

In Chinese patent (application number a 97106442.3) literary composition, mention that orientation is separated to large quantities of antibacterials from soil, Radix Glycyrrhizae, and therefrom screen a strain and can produce justacrine in a large number and have a strong fibrin degradation hydrolytic enzyme bacterial strain to external, but the enzymatic productivity of this bacterial strain is still lower, need further to improve, and need gene structure to enzyme, the key property of enzyme, pharmacological effect is further studied.

Based on the present situation of present thrombolytics development, the present invention attempts to develop a kind of thrombolytics, and it is extensive that it has raw material sources, and preparation easily, purification is convenient, and price is more cheap, and high specificity, curative effect are more remarkable, side effect is little, be suitable for oral, a kind of thrombolytics with treatment and the two effects of prevention.

Technical characterictic of the present invention is to use engineered method, and on specific carrier and change over to through in the bacillus subtilis receptor of transforming, acquisition can correctly efficiently express the genetic engineering bacterium of streptokinase with isolated streptokinase gene clone.Adopt the microbial fermentation engineering method then, through the bacterial classification inoculation cultivation and fermentation, steps such as separation and purification make work simplification, output raising, quality meet the thrombolytics of oral formulations requirement.

The used bacterial strain of the present invention is that directed screening obtains from Radix Glycyrrhizae, and called after MBL-1, it is characterized in that in solid or fluid medium, can produce a kind of strong fibrinolytic proteolytic enzyme of secretion by cultivating, by " the outstanding third constellations of uncle Bacteria Identification handbook method, to the strain morphology feature, physio-biochemical characteristics etc. are tested, carry out analysis-by-synthesis relatively according to result of the test, with the MBL-1 dientification of bacteria is bacillus subtilis (Bacillus Subtilis) MBL-1, the fibrin hydrolytic enzyme that it produced be called BACILLUS-KINASE BK (Bacillus-Kinase, BK).

Thrombolytics of the present invention-streptokinase gene comes from the MBL-1 bacterial strain, according to Nakaomura T.et al. report (Biosci Biotec Biochem 56 (11): 1869-1871,1992), design a pair of primer, with conventional method purification MBL-1 chromosomal DNA, (PCR) amplifies dna fragmentation with the gene amplification technology, cuts PCR product and pUB simultaneously with restriction endonuclease ₁₈Plasmid DNA, the required dna fragmentation of agarose gel electrophoresis Separation and Recovery is used T ₄Dna ligase carries out coupled reaction, obtain recombinant DNA molecules, the transformation receptor bacterium obtains transformant, contain the transformant (monoclonal that has transparent circle) of streptokinase gene with the selectivity plate screening that contains 1% defatted milk powder and kanamycin (5 μ g/ml), obtain genetic engineering bacterium through identifying.The insertion streptokinase genetic fragment of genetic engineering bacterium is carried out sequence analysis, finished by the ABI automatic sequencer, the structural gene sequence of gained streptokinase as shown in Figure 1.

The structural gene of streptokinase of the present invention is made of 825 nucleotide, from the nucleotide sequence deduced amino acid as shown in Figure 1.Study on Physico-chemical to streptokinase shows: this enzyme is an alkaline serine protease, and molecular weight is 28000 (SD S-PAGE electrophoresis method), further mass spectral analysis, and accurately determining molecular weight is 27735.37 ± 6.99; Its isoelectric point, IP is 8.6.

Thrombolytics of the present invention-streptokinase gene structure also comprises its variant.Design a pair of primer as stated above,, amplify dna fragmentation, cut PCR product and pUB simultaneously with restriction endonuclease Bam HI and EcoRI with the strict gene amplification technology of controlling reaction condition with conventional method separation and purification MBL-1 strain chromosome DNA ₁₈Plasmid DNA, the required dna fragmentation of agarose gel electrophoresis Separation and Recovery is used T ₄Dna ligase carries out coupled reaction, obtain recombinant DNA molecules, transform the bacillus subtilis recipient cell, obtain transformant, with the selectivity flat board that contains 1% defatted milk powder and kanamycin (5 μ g/ml), screening contains the transformant (monoclonal that has transparent circle) of streptokinase gene, obtains genetic engineering bacterium through identifying.The streptokinase genetic fragment of genetic engineering bacterium is carried out sequence analysis, finishes by the ABI automatic sequencer, the structural gene sequence of gained streptokinase compared to Figure 1, the 61st bit base is C, the 21st codon is CAC, its corresponding aminoacid is H.The 581st bit base is T, and the 194th codon is TTT, and its corresponding aminoacid is F.The coded enzyme of this gene structure variant has strong solution fibrin and thrombus ability.

Streptokinase preparation of the present invention, by Qi Chen's method (herbal pharmacology research methodology, Beijing people's health publishing house, front page in 1994) experimental result of measuring thrombolytics dissolving human fibrin and clot shows: this thrombolytics has the ability of very strong dissolving human fibrin and human blood sludged blood; Thrombolytics is made oral formulations show that this thrombolytics can obviously shorten the blood plasma euglobulin lysis time for experiment white mice and rat oral test result, improve plasmin activity in the body, improve the secretory volume of tPA; Adopt thrombolytics of the present invention that mice is carried out oral test, its result shows obvious reduction whole blood viscosity, suppresses the formation of thrombosis; Give 48-65 year crowd oral 3 days by the thrombolytics of the embodiment of the invention 1, the 2 methods preparations enteric solubility capsule of packing into, show that after tested the human plasma euglobulin lysis time shortens 33～50% from former person.These results show obviously induction of vascular endothelial emiocytosis tPA of thrombolytics of the present invention, improve the fibrinolytic of human body, prevent thrombosis and thrombolytic usefulness thereby play.

Thrombolytics of the present invention comes from microbial fermentation product, but the indeterminate growth fermentation overcomes raw material sources difficulty as UK, and the bacterium of streptokinase is a bacillus subtilis, and gene outcome is the external secretion type, can simplify purifying process like this, and safety simultaneously is better.Thrombolytics of the present invention is oral effectively, can overcome existing thrombolytics and mostly be intravenous drip, plays prevention and treats dual curative effect.

Embodiment 1 eluriates selected high quality soybean clean, puts into 2-10% (W/V) concentration amounts and fills the buffer container that contains inorganic compounding salt, through 1.1-1.2kg/cm ²The pressure steaming and decocting under high pressure prepares the Semen sojae atricolor leachate, make fermentation medium, behind autoclaving, insert a certain amount of genetic engineering bacterium that contains fibrinolytic enzyme gene, placed under the 25-40 ℃ of condition aerobic culture 20-48 hour, 3000-8000 rev/min 4 ℃ of centrifugal 10-30 minutes, abandon precipitate supernatant, add the analytical pure solid ammonium sulfate, be the saturation (or fractional precipitation) of 30-85%, 4 ℃ of quiescent settings spend the night, 3000-5000 rev/min 4 ℃ centrifugal 20 minutes, abandon the supernatant taking precipitate, it is dissolved in contains 2-10mM CaCl ₂The buffer of 0.01M Tris-HCl PH 7.0-7.5 in, the bag filter of packing into same buffer 4C dialysis 12-36 hour, concentrates dialysis solution, lyophilization makes the streptokinase goods.Can be directly used in the preparation oral formulations.

Embodiment 2 prepares the streptokinase semifinished product by embodiment 1 method, and it is dissolved in the 5mMPH6.0 phosphate buffer, and last sample is at the CMSepharose Fast Flow post of crossing with same buffer balance in advance.Carry out linear gradient elution with the same buffer that contains 0～1M Nacl behind the last sample, collect the plasmin activity peak.With the solid ammonium sulfate precipitation fibrinolysin of 50-80% saturation, go up Sephadet G-75 post again and carry out gel filtration, mobile phase is the phosphate buffer that contains the 0.1M PH 7.5 of 0.05M Nacl, collect active peak, the ammonium sulfate precipitation dialysis desalting, lyophilization gets the streptokinase highly finished product.

Embodiment 3 is dissolved in the phosphate buffer of 0.03M PH7.8 by the streptokinases of embodiment 1 or 2 preparations and becomes finite concentration, get respectively 10ul add three kinds on the fibrin plate of different disposal (with reference to Astrup T.et al.Biochem.Biophs, 40:346-351,1952).Promptly contain the human fibrin flat board of plasminogen and reach the cattle fiber egg flat boards that do not contain plasminogen through 80 ℃ of human fibrin flat boards of handling to make the plasminogen inactivation in 2 hours, and compare with the UK sample, 37 ℃ are incubated 5 hours, with the size of transparent circle around the accurate measuring samples of the slide calliper rule hole.The result shows: containing not the heating on the human fibrin flat board of plasminogen, the BK streptokinase can both produce the similar transparent circle of size with UK; Around heated plate (plasminogen inactivation) is gone up the UK sample, do not have transparent circle and occur, around the BK sample transparent circle is arranged still, show that the BK streptokinase has direct plasmin activity; And do not containing on the former cattle fibrin plate of fibrinolytic, be added with bright aquatic foods of transparent circle around the sample well of BK streptokinase and plasminogen simultaneously greater than the transparent circle that only adds around the BK streptokinase sample well, do not have any transparent circle around only adding the plasminogen sample well, show that the BK streptokinase has the ability that plasminogen activation becomes fibrinolysin.

Embodiment 4 presses embodiment 1, the preparation the BK streptokinase be dissolved in the normal saline, make certain density streptokinase solution, press 5400u/kg/ days oral to Kunming kind white mice, for three days on end, get tPA in hematometry blood plasma plasmin activity and the blood plasma (operating) by the detectable description.The result shows that medicine group blood plasma fibrinolytic increases by 30% than matched group, and tPA content increases by 51%.

Embodiment 5 is dissolved in the normal saline by the BK streptokinase of embodiment 2 preparations, make the not streptokinase solution of concentration, suppress thrombosis and thrombolytic effect (concrete grammar is undertaken by thrombosis detector description) in vitro tests BK streptokinase with micro computer thrombosis detector.The results are shown in Table 1,2.

Table 1, the BK streptokinase suppresses the thrombosis effect

Group	Enzyme concentration (u)	Thrombosis length (mm)	Wet weight of thrombus (mg)	Thrombosis dry weight (mg)
					Matched group	0	17.0	88.2	21.9
Administration group (low concentration)	480	4.0	18.8	2.5
					Administration group (high concentration)	960	0	0	0

Table 2, the effect of BK streptokinase thrombus

Group	Enzyme concentration (u)	Thrombosis length (mm)	Wet weight of thrombus (mg)	Thrombosis dry weight (mg)
					Matched group	0	30.0	216.2	54.8
Administration group (low concentration)	480	24.0	168.0	43.1
					Administration group (high concentration)	960	3.0	19.5	3.1

Table 1,2 presentation of results BK streptokinases brightly suppress thrombosis that thrombosis and dissolving formed and along with the increase of enzyme concentration, thrombosis length, weight in wet base, dry weight all reduce thereupon, have a certain amount of effect relationship in that external energy is bright.

Claims (3)

1. thrombolytics, it is characterized in that: be strong fibrinolytic proteolytic enzyme, promptly be called BACILLUS-KINASE BK (Bacillus-Kinase, BK) alkaline serine protease, molecular weight is 27735.37 ± 6.99, isoelectric point, IP is 8.6, and its structural gene is made of 825 nucleotide, and this nucleotide is pressed following series arrangement: GCGCAATCTG TTCCTTATGG CATTTCTCAA ATTAAAGCGC CGGCTCTTCA CTCTCAAGGC

A Q S V P Y G I S Q I K A P A L H S Q G TACACAGGCT CTAACGTAAA AGTAGCTGTT ATCGACAGCG GAATTGACTC TTCTCATCCT

Y T G S N V K V A V I D S G I D S S H P GACTTAAACG TCAGAGGCGG AGCAAGCTTC GTTCCTTCTG AAACAAACCC ATACCAGGAC

D L N V R G G A S F V P S E T N P Y Q D GGCAGTTCTC ACGGTACGCA TGTCGCCGGT ACGATTGCCG CTCTTAATAA CTCAATCGGT

G S S H G T H V A G T I A A L N N S I G GTTCTGGGCG TAGCGCCAAG CGCATCATTA TATGCAGTAA AAGTGCTTGA TTCAACAGGA

V L G V A P S A S L Y A V K V L D S T G AGCGGCCAAT ATAGCTGGAT TATTAACGGC ATTGAGTGGG CCATTTCCAA CAATATGGAT

S G Q Y S W I I N G I E W A I S N N M D GTTATCAACA TGAGCCTTGG CGGACCTACT GGTTCTACAG CGCTGAAAAC AGTAGTTGAT

V I N M S L G G P T G S T A L K T V V D AAAGCGGTTT CCAGCGGTAT CGTCGTTGCT GCCGCAGCCG GAAACGAAGG TTCATCCGGA

K A V S S G I V V A A A A G N E G S S G AGCACAAGCA CAGTCGGCTA CCCTGCAAAA TATCCTTCTA CTATTGCAGT AGGTGCGGTA

S T S T V G Y P A K Y P S T I A V G A V AACAGCAGCA ACCAAAGAGC TTCATTCTCC AGCGTAGGTT CTGAGCTTGA TGTAATGGCT

N S S N Q R A S F S S V G S E L D V M A CCTGGCGTGT CCATCCAAAG CACACTTCCT GGAGGCACTT ACGGCGCTTA TAACGGAACG

P G V S I Q S T L P G G T Y G A Y N G T TCCATGGCGA CTCCTCACGT TGCCGGAGCA GCAGCGCTAA TTCTTTCTAA GCACCCGACT

S M A T P H V A G A A A L I L S K H P T TGGACAAACG CGCAAGTCCG TGATCGTTTA GAAAGCACTG CAACATATCT TGGAAACTCT

W T N A Q V R D R L E S T A T Y L G N S TTCTACTATG GAAAAGGGTT AATCAACGTA CAAGCAGCTG CACAA

F Y Y G K G L I N V Q A A A Q

2. the preparation of a thrombolytics, operating procedure comprises:

First step bacterium source

Used bacterial strain is that directed screening obtains from Radix Glycyrrhizae, and called after MBL-1, at solid or fluid medium, can produce a kind of strong fibrinolytic proteolytic enzyme of secretion by cultivating, by " uncle Jie Shi Bacteria Identification handbook method, to the strain morphology feature, physio-biochemical characteristics etc. are tested, carry out analysis-by-synthesis relatively according to result of the test, with the MBL-1 dientification of bacteria is bacillus subtilis (Bacillus Subtilis) MBL-1, the proteolytic enzyme that it produced be called BACILLUS-KINASE BK (Bacillus-Kinase, BK);

Second step obtained gene

According to Nakaomura T.et al. report, design a pair of primer, with conventional method separation and purification MBL-1 strain chromosome DNA, amplify dna fragmentation with the gene amplification technology, cut PCB product and pUB simultaneously with restriction endonuclease Bam HI and EcoRI ₁₈Plasmid DNA, the required dna fragmentation of agarose gel electrophoresis Separation and Recovery;

The 3rd step obtained genetic engineering bacterium T ₄Dna ligase carries out coupled reaction, obtain recombinant DNA molecules, transform the bacillus subtilis recipient cell, obtain transformant, with the selectivity flat board that contains 1% defatted milk powder and kanamycin (5 μ g/ml), screening contains the transformant (monoclonal that has transparent circle) of streptokinase gene, obtains genetic engineering bacterium through identifying; It is characterized in that: the insertion streptokinase genetic fragment of this genetic engineering bacterium has the described nucleotide collating sequence of claim 1,

Make thrombolytics with the 4th step

Selected high quality soybean is eluriated totally, put into the buffer container that contains inorganic compounding salt with 2-10% (W/V) concentration amounts, through 1.1-1.2kg/cm ²The pressure steaming and decocting under high pressure prepares the Semen sojae atricolor leachate, make fermentation medium, behind autoclaving, insert a certain amount of genetic engineering bacterium that contains fibrinolytic enzyme gene, placed under the 25-40 ℃ of condition aerobic culture 20-48 hour, 3000-8000 rev/min 4 ℃ of centrifugal 10-30 minutes, abandon precipitate supernatant, add the analytical pure solid ammonium sulfate, be the saturation (or fractional precipitation) of 30-85%, 4 ℃ of quiescent settings spend the night, 3000-5000 rev/min 4 ℃ centrifugal 20 minutes, abandon the supernatant taking precipitate, it is dissolved in contains 2-10mMCaCl ₂The buffer of 0.01M Tris-HCl pH 7.0-7.5 in, the bag filter of packing into 4 ℃ of dialysis of same buffer 12-36 hour, concentrates dialysis solution, lyophilization makes the streptokinase goods, i.e. the thrombolytics semifinished product.

3. the preparation of thrombolytics according to claim 2 is characterized in that: be the continuation of the preparation of claim 2,

The 5th step purification thrombolytics semifinished product

The thrombolytics semifinished product that the 4th step of claim 2 is made is dissolved in 5mM pH 6.0 phosphate buffers, last sample is at the CM Sepharose FastFlow post of crossing with same buffer balance in advance, carry out linear gradient elution with the same buffer that contains 0-1M NaCl behind the last sample, collect the plasmin activity peak, solid ammonium sulfate precipitation fibrinolysin with the 50-80% saturation, go up Sephadet G-75 post again and carry out gel filtration, mobile phase is the phosphate buffer that contains the 0.1MpH 7.5 of 0.05M NaCl, collect active peak, the ammonium sulfate precipitation dialysis desalting, cool drying gets streptokinase highly finished product, i.e. thrombolytics highly finished product.