WO1999004017A1 - Staphylokinase recombinante et sa souche manipulee pour une expression elevee - Google Patents

Staphylokinase recombinante et sa souche manipulee pour une expression elevee Download PDF

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Publication number
WO1999004017A1
WO1999004017A1 PCT/CN1998/000129 CN9800129W WO9904017A1 WO 1999004017 A1 WO1999004017 A1 WO 1999004017A1 CN 9800129 W CN9800129 W CN 9800129W WO 9904017 A1 WO9904017 A1 WO 9904017A1
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Prior art keywords
staphylokinase
sak
recombinant
dna
sequence
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PCT/CN1998/000129
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English (en)
Chinese (zh)
Inventor
Qijiu Zhang
Guicai Zhang
Guangfeng Xu
Guiwu Qu
Liliang Bie
Wanxue Xu
Yunshan Wu
Original Assignee
Bai Hansan
Qijiu Zhang
Guicai Zhang
Guangfeng Xu
Guiwu Qu
Liliang Bie
Wanxue Xu
Yunshan Wu
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Application filed by Bai Hansan, Qijiu Zhang, Guicai Zhang, Guangfeng Xu, Guiwu Qu, Liliang Bie, Wanxue Xu, Yunshan Wu filed Critical Bai Hansan
Priority to AU84297/98A priority Critical patent/AU8429798A/en
Publication of WO1999004017A1 publication Critical patent/WO1999004017A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a new recombinant staphylokinase (r-Sak) and a high-expression engineering strain thereof.
  • the expression product has the action of plasminogen activator and has high hydrolytic activity on human fibrin.
  • Staphylokinase is a thrombolytic drug. It is a non-enzymatic protein produced by Staphylococcus aureus. It is similar to streptokinase (SK) and forms in combination with plasminogen. The complex is converted into a staphylokinase-plasmin complex, which further catalyzes plasminogen into plasmin, thereby dehydrolyzing fibrin on the thrombus, thereby achieving the purpose of thrombolysis.
  • Sak-activated plasminogen is highly specific for fibrin. It preferentially binds to plasminogen on the blood test and hydrolyzes fibrin in it.
  • CN1109508 specification A discloses that a staphylokinase gene was obtained from S. aureus phage DNA and cloned. There are documents: Collen, D. et al. (1992) Fibrinolysis, 6: 226-231. The paper disclosed that the staphylokinase gene was cloned from the chromosomal DNA of S. aureus. Both of these gene fragments were cloned using large DNA fragments. The gene fragment is large, and the number of copies assembled into the vector plasmid is low, which makes it difficult to obtain a high expression effect. The expression level of chromosomal DNA of this S.
  • aureus bacteria is generally 10-15%, as disclosed in the article "High Yield Production and Purification of Recombinant Staphylokinase for the Treatment of Thrombosis Drugs", Volume 12, 1994.2 Transfection of E. coli TG1 cells produces staphylokinase in the cell, which accounts for 10-15 3 ⁇ 4 of the total protein in the cell. "This is the currently recognized level of staphylokinase expression in publicly available reports. Object of the invention
  • One of the objectives of the present invention is to provide a recombinant staphylokinase with strong selectivity, good safety, high heat resistance and good thrombolytic effect;
  • Another object of the present invention is to provide an engineered strain that can highly express the Recombinant staphylokinase
  • Another object of the present invention is to provide a safe and effective pharmaceutical composition for the treatment of cardiovascular and cerebral thrombotic diseases.
  • a recombinant staphylokinase has an amino acid sequence of sequence 1.
  • the recombinant staphylokinase of the present invention has high heat resistance and biological activity, and is obtained by a method of genetic recombination.
  • an engineered strain is constructed using a genetic engineering method, capable of efficiently expressing the recombinant staphylokinase of the present invention, and using the engineered strain of the present invention to produce recombinant staphylokinase, the yield of which can reach more than 40% of the total cell protein. .
  • a pharmaceutical composition for treating a thrombotic disease contains the recombinant staphylokinase of the present invention as an active ingredient.
  • the pharmaceutical composition of the present invention shows high safety and effectiveness.
  • Figure la-lc Comparison of the nucleotide sequences of staphylokinase genes and their encoded amino acid sequences from different sources.
  • Figure 2 Scanning electron microscope image of Staphylococcus aureus SL1.063.
  • Figure 3 Colony characteristics of S. aureus SL1.063 strain on mannitol high-salt agar plate.
  • Figure 4 Colony characteristics of Staphylococcus aureus SL 1.063 strain on yolk high-salt agar plate.
  • Figure 5 Hemolysis of Staphylococcus aureus SL 1.063 strain on blood agar flat fox.
  • Figure 6 Staphylococcus aureus SL 1.063 strain thermostable nuclease test.
  • Figure 7a Sak in Staphylococcus aureus SL 1.063 strain, detected by human fibrin standard.
  • Figure 7b Sak in Staphylococcus aureus SL 1.063 strain, detection results with human fibrin heat level.
  • Figure 8 Assay of DNA fragments inserted into recombinant plasmids
  • Figure 9a Flow chart of the construction of recombinant plasmid pUK408 high-efficiency expression plasmid.
  • Figure 9b Flow chart of the construction of recombinant plasmid pHK408 high expression plasmid.
  • Figure 10 SDS-PAGE (12%) electrophoresis analysis of the highly expressed product of engineered bacteria TG2 (pHK408).
  • Figure 11 Thermal stability of the recombinant staphylokinase of the present invention.
  • E. coli strains suitable for use in the present invention and their genotypes are as follows: Genotype
  • an isolated and purified staphylokinase-producing golden staphylococcus aureus strain SL1.063 the amino acid sequence of the glucokinase produced by this strain is sequence 1.
  • This strain has been deposited in the General Microbiology Center of the China Microbial Species Collection Management Committee under the accession number CGMCC No.0353. This strain has the following characteristics:
  • Morphological characteristics Gram staining is positive, the bacteria are spherical, piled up into grape-like, no spores, and the size of the bacteria is 0.8-0.9 ⁇ .
  • thermostable nuclease and plasma coagulase With the ability to produce lecithin, thermostable nuclease and plasma coagulase.
  • a recombinant staphylokinase is produced by a genetic engineering method, the amino acid sequence of the recombinant staphylokinase is sequence 1, and the nucleotide of the gene encoding the recombinant staphylokinase is The sequence is sequence 2.
  • nucleotide sequence of the r-Sak gene and the 136 amino acid sequence encoded by the r-Sak gene of the present invention are different from r-Sak scabs reported at home and abroad, as shown in Table 1 and FIG. 1.
  • Table 1 Differences in the nucleotide sequence of staphylokinase genes and their encoded amino acid sequences from different sources Gene Gene Source 34th 36th 43th 2nd 133rd Nucleotide Data Source
  • the r-Sak gene and Sak star of the present invention are both from S. aureus chromosomal DNA.
  • the strains are SL 1.063 and 23 S. aureus strains.
  • Inventions are glycine and arginine, and Sak star is Serine and histidine also show differences in the nucleotide sequence of the corresponding DNA.
  • the r-Sak gene of the present invention differs from the lysogenic phage-derived gene in the nucleotide sequence and amino acid sequence.
  • the r-Sak gene of the present invention is compared with Sak SY.
  • the amino acid at position 36 is glycine, while Sak SY is arginine.
  • the nucleotide sequence of the gene of the present invention is G at positions 105 and 106 of the gene.
  • the T and 399 are T, and the nucleotides at Sak SY are A. It is not difficult to see that the difference between the r-Sak gene of the present invention and these two Sak genes from different strains on the nucleotide sequence and the amino acid sequence encoded by them is significant.
  • the r-Sak of the present invention not only has an amino acid sequence different from the amino acid sequence of known staphylokinase, but also has higher heat resistance and therefore higher biological stability.
  • Sak42D was treated at 100 ° C for 1.5 h, the enzyme activity was almost completely lost.
  • the residual enzyme activity was only 10%.
  • Sak star was treated at 100 ° C for 1.5 h, and the residual enzyme activity was close to zero.
  • the residual enzyme activity was about 60%.
  • Sak star was more heat resistant than Sak42D.
  • FIG. 11 after the Sak63 of the present invention is treated at 100 ° C for 1.5 h, its residual enzyme activity is 55%; 70. C 2. Oh treatment, the alcohol activity is 70%.
  • the r-Sak of the present invention has higher heat resistance.
  • the staphylokinase-encoding gene of the present invention can be recombined into a vector.
  • the recombined vector contains the gene encoding the recombinant staphylokinase of the present invention, and the nucleotide sequence of the gene is the sequence 2.
  • the carrier used in the present invention may be a commonly used carrier. For example, you can choose pUC19, pBV220 and so on.
  • a host containing the recombinant vector of the present invention By transferring the recombinant vector of the present invention into a host cell, a host containing the recombinant vector of the present invention can be obtained for high expression of the recombinant staphylokinase of the present invention.
  • the host suitable for the present invention may be commonly used E. coli, for example: C600, MC1061, JM83, JM109, DH5a, TGI, TG2, etc., among which DH5a, TGI, TG2 is preferred
  • a method for constructing an engineered strain producing recombinant staphylokinase includes screening staphylokinase-producing Staphylococcus aureus. Natural Staphylococcus aureus is collected from nature and isolated and purified. DNA polymerase chain reaction (PCR amplification) was performed using the S. aureus chromosomal DNA as a template. It is preferable to use S. aureus chromosomal DNA with high staphylokinase expression as a template. In PCR amplification, the primers used are: Primer I
  • a plasmid (vector) is selected, and a small fragment of the complete staphylokinase gene DNA obtained after the amplification step is introduced into the plasmid to achieve recombination of the plasmid.
  • the gene DNA fragment and the vector plasmid were digested with EcoRI and BamHI, respectively; and the above-digested plasmid and small staphylokinase gene DNA fragments were ligated with T 4 DNA ligase to obtain a recombinant plasmid.
  • the recombined plasmid can be directly transferred into host cells to complete the construction of engineered bacteria.
  • the staphylokinase gene DNA fragment in the recombinant plasmid can also be excised and used to recombine another plasmid, and the post-recombinant plasmid can be transferred into a host cell to construct an engineering bacterium.
  • the choice of plasmids and hosts has been described above.
  • Preferred plasmids are pUC19, pBV220; preferred hosts are E. coli DH5a, TGI and TG2.
  • a highly expressed engineering bacterium TG2 (pHK408) efficiently expresses the recombinant staphylokinase of the present invention.
  • This strain has been deposited in the General Microbial Center of the China Microbial Species Collection Management Committee under the accession number CGMCC No. 0333.
  • the engineered strain is a high expression system, and the recombinant staphylokinase of the present invention is produced by using the engineered strain with a yield of 40% or more.
  • the expression level was increased by nearly 1.7 times or more.
  • the recombinant staphylokinase of the present invention can be used to treat myocardial infarction and thrombotic diseases, for example, it can be used to treat diseases such as myocardial infarction, deep venous thrombosis, pulmonary embolism, acute or subacute peripheral arterial thrombosis and chronic arterial embolism, and central retinal artery or vein embolism. Therefore, according to the present invention, a pharmaceutical composition includes an effective dose of the recombinant staphylokinase of the present invention and a pharmaceutical excipient.
  • Pharmaceutical excipients can be commonly used excipients, such as: human albumin, hydrocolloid protein, glutamic acid, glycine, and inorganic salts.
  • R-Sak was administered intravenously at two doses of 6.9xl0 4 AU / kg and 13.8xl0 4 AU / kg, and there was no significant change in blood pressure, heart rhythm and electrocardiogram wave of anesthetized mini-pigs;
  • Miniature Pigs Biochemical Analysis of Femoral Artery Red Blood Cell Thrombosis, Coronary Thrombosis, Fibrinolytic System after Blood Flow Coronary thrombosis intravenous 4.6xl0 4 AU / kg and 9.2xl0 4 AU / kg.
  • Rats Carotid arterial thrombosis, 1.8xl0 4 , 6xl 0 4 , 1.8xl0 5 AU / kg administered intravenously.
  • Rabbit Pulmonary embolism, 3xl0 3 , lxlO 4 , 3xl0 4 AU / kg intravenously.
  • Recombinant staphylokinase is an indirect mode of plasminogen activator.
  • in vivo and in vitro models were used to evaluate the thrombolytic activity and fibrin specificity of recombinant staphylokinase. experiment result shows:
  • RA method RA method
  • HPLC separation and radioactivity detection method HPLC-RA method
  • rats were intravenous (iv) r-Sak 3 x 10 4, 9 x 10 4, and 3 x 10 5 AU / kg dose pharmacokinetic three learning trials to method RA serum total radioactivity to
  • HPLC-RA method was used to determine the concentration and concentration-time data of the serum prototype drug, and the pharmacokinetic parameters were calculated. It was proved that the total radioactive elimination was faster after r-Sak iv administration, and t 1/2 was about 134min. Fast, t 1/2 is around 56min.
  • AUC Area Under the Curve
  • Rats were dosed with iv 9 X 10 4 AU / kg by RA and HPLC-RA to determine the drug content in different tissues. It was found that at the same time, the kidney drug content was the highest and the brain concentration was the lowest.
  • Rats were dosed with iv 9 10 4 AU / kg, and the total radioactivity in urine and feces was measured by RA method. Within 24 hours, 81.54% of the radioactivity was recovered from urine and 2.05% was recovered from feces. The total excretion of urine and feces reached 83.59% of the administered dose, indicating that excretion is fast and complete.
  • Staphylococcus aureus In order to isolate Staphylococcus aureus, more than 90 samples were collected from hospital surgery, ophthalmology, and ENT, etc., and were diluted or streaked on the egg yolk sodium chloride agar medium, cultured at 36 ° C. After 24-48 hours of cultivation, pick golden or lemon-colored colonies with pearly white precipitates in the surrounding medium, transfer them into the common nutrient medium, and culture at 36 ° C overnight. Gram staining, microscopy, and G + bacteria selection The body is spherical and accumulates into grape-like, spore-free strains for further purification on egg yolk medium. The purified strains were respectively connected to a high-salt mannitol agar medium and cultured at 36 ° C for 24-48 hours. Bright yellow circles appeared around the colonies. After isolation, purification and identification, a total of 167 S. aureus strains were obtained. Their morphological and cultural characteristics are as follows:
  • Gram staining is positive, the bacterial cells are spherical, piled into grape-like, no spores, and the size of the bacterial cells is usually 0.8-0.9 ⁇ m. See Figure 2.
  • thermostable nuclease In order to determine whether a thermostable nuclease is produced, the test strain is cultured in an ordinary nutrient culture solution at 37 ° C for 18-24 hours, the bacterial solution is boiled for 10 minutes, and the toluline blue DNA agar plate is opened at 37 ° C. C kept warm for 3-5 hours, and the positive ones produced pink circles, as shown in Figure 6.
  • 1, 4 are physiological saline
  • 2, 3, 5, 6, 7, are fermentation supernatants of SL1.063 strain.
  • Plasma coagulase test Rabbit plasma: physiological saline (1: 1) was mixed with an equal volume of each common culture medium. Control: An equal volume of normal saline. Incubate at 37 ° C overnight. Plasma of the positive control and test strains coagulated, and the control tube plasma flowed freely.
  • the 063 enzyme is By activating plasminogen to generate plasmin to hydrolyze fibrin, heat it at 85 ° C for 40 minutes to inactivate all plasminogen. Therefore, the enzyme produced by SL1.063 heats the fiber like urokinase t-PA No more transparent circles on flat surfaces.
  • the organic nitrogen sources are tryptone (Beijing Haidian), soybean mash (Shanghai Yunzhou), whey protein hydrolysate (Japan), Maokou Juyue, (polypeptone, Japanese version).
  • Fermentation conditions 37 shake culture (180rpm) 16 hours.
  • Viability determination Fibrin-agar level method, fixed centrifugation, take 10 ⁇ l of fermentation supernatant, 37 ° C, 16 hours. The results are as follows:
  • Animal serums include: rabbit, chicken, sheep, bovine and other animal serums, which have comparable effects.
  • Fermentation cycle 37 shake culture for 12-16 hours is appropriate.
  • a single colony of the strain was inoculated in 25 ml of LB liquid medium, cultured with shaking at 37 ° C overnight, centrifuged at 4000 rpm for 8 minutes to collect the bacterial cells, and the cells were suspended in 2.5 ml of GTE solution (50 mM Glucose-50 mM Tris-HCl-1 O mM EDTA, PH8 .0).
  • GTE solution 50 mM Glucose-50 mM Tris-HCl-1 O mM EDTA, PH8 .0.
  • Add lysozyme at a final concentration of 10 mg / ml, incubate at 37 ° C for 60 minutes, add 300 ⁇ 1 of 10% SDS, RNase A (10 mg / ml) and RNase TiPOOOU / ml) each at 40 ⁇ 1, incubate at 37 ° C for 60 minutes, etc.
  • phenol-chloroform-isoamyl alcohol (12: 12: 1) was extracted three times.
  • TE solution (20mM Tris-HCl (H8.0) -lmM EDTA) was back-extracted twice. After the aqueous phases were combined, 10 volumes of 3 M NaCl and 2 volumes of 95% ethanol were added. The DNA was washed twice with 70% ethanol and absolute ethanol, and the ethanol was removed. DNA was dissolved in a proper amount of TE solution at a concentration of about 0.2 g ⁇ l. On SDS-PAGE, no RNA was present.
  • DNA fragment The DNA was dissolved in 20 ⁇ l TE (PQ8.0) solution. 2 enzyme digestion:
  • pUC19 plasmid was extracted according to the method of Sambrook et al. The pUC19 plasmid was digested with EcoRI and Bami ⁇ as described above, and passed through Genelute Agarose Spin.
  • Recombinant plasmid DNA was extracted by alkaline SDS-lysed cell PEG NaCl method, and was checked by 7% Agarose gel electrophoresis. It was basically free of RNA and could be used for DNA sequence determination. The DNA sequence was determined using the T 7 polymerase sequencing system (Promega's product results are as previously described).
  • the high-efficiency expression plasmid pHK408 was constructed according to the procedure of FIG. 9b.
  • the plasmid DNA was prepared according to the method of Sambrook et al. A single colony of DH5a (pUK408) plasmid was taken and placed in LB-AP liquid medium, and cultured at 37 ° C with shaking overnight. Colonic rod cells were lysed with alkaline SDS, and RNA was precipitated with PEG-NaCl to obtain plasmid DNA. After SDS-PAGE examination, it was basically free of RNA. Digest pUK408 DNA with EcoRI and BamH. 1.5% Agarose electrophoretic separation 'Cut out small fragments of DNA, and follow the Genelute Agarose Spin Columns method to obtain purified DNA.
  • the pBV220 vector plasmid was isolated and purified as described above. Digest with EcoRI and BamHi. ddH 2 0 36 ⁇ 1
  • Plasmid DNA (0.5 ⁇ ⁇ ⁇ 1) 5 ⁇ 1
  • the digested reaction solution was run by 0.7% Agarose electrophoresis, and large DNA fragments were cut under a long-wavelength UV lamp. Purified by Genelute Agarose Spin Columns. The above gene fragment was ligated with pBV220 plasmid DNA, reacted at 16 ° C for 12 hours, and then transferred into E. coli TG2 receptor cells. Recombinant plasmid DNA was extracted, digested with EcoRI and BamHi, and 1.2% Agarose gel electrophoresis was performed to confirm the insertion of the DNA gene fragment.
  • the TG2 (pHK408) strain containing the recombinant plasmid was put into LB liquid medium, cultured at 37 ° C with shaking overnight, the supernatant was centrifuged, and the supernatant was spotted on a human fibrin plate at 37 ° C for 16 hours. Circle to confirm that the strain can express staphylokinase.
  • the pH 408 plasmid was transferred into different E. coli strains, such as JM109, DH5a, TGI or TG2 with higher expression levels.
  • Engineering bacteria TG2 (pHK408), deposit number CGMCC NO.0333, inoculated in 50ml PY medium (1000ml, 16g tryptone, 5g yeast powder, 5g NaCl, PH7.2) at 30 ° C with shaking for 6 hours, immediately raise temperature Continue shaking culture at 42 ° C for 5-6 hours.
  • the bacterial cells were suspended in 5 ml of TSE solution (20 mM Tns-HCl-50 mM NaCl-10iTiM EDTA pH8.0), sonicated with a JY92-II ultrasonic cell pulverizer, and the supernatant was collected by centrifugation and analyzed by SDS-PAGE electrophoresis.
  • Recombinant staphylokinase was prepared by fermentation with engineered bacteria TG2 (pHK408).
  • the crude enzyme solution was purified in three steps by SP-Sepharose FF column ( ⁇ 45 x 200mm) and Phenyl- Sepharose FF column ( ⁇ 45 x 200i) and Sephacryl S-300 ((j) 26 x 800). Concentrated and sterilized to obtain 970mg of recombinant staphylokinase.
  • the obtained recombinant staphylokinase samples were detected by SDS-PAGE and HPLC with a purity of 96% and 100%, respectively.
  • the specific activity of the fibrin plate was 6.5 X 10 4 AU / mg protein (equivalent to 1 ⁇ 1.2 X 10 5 HU / mg protein).
  • Mass spectrometry and SDS-PAGE determined that the MW was 15.4kD.
  • the N-terminal amino acid measurement result is the same as the theoretical value (N-SSSSFDKGKYKKGDDA). .
  • Sak S-29 is adenylate at positions 105, 128, and 399, thymidine at position 275, and guanylate at position 398;

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Abstract

L'invention concerne un Staphylococcus aureus SL 1,063 produisant la staphylokinase (Sak), une nouvelle staphylokinase recombinante et leur souche manipulée pour une expression élevée. On a obtenu un gène codant pour Sak par amplification ACP avec un ADN chromosomique de Staphylococcus aureus produisant la Sak en tant que matrice. La séquence nucléotidique du gène et les acides aminés codés par ces séquences était différente de celles du gène Sak qui ont été reportées. Le fragment d'ADN recombinant a été obtenu par amplification au moyen d'une amorce ayant une séquence nucléotidique particulière, purifiée, identifiée, coupée avec des enzymes, ligaturée et transformée en une cellule hôte permettant de construire une souche manipulée d'expression élevée de Sak. Le produit issu de l'expression sert d'activateur du plasminogène et présente une action hydrolytique très forte sur la fibrine humaine. On peut guérir les thrombo-embolies, telles que les infarctus du myocarde, les thromboses cérébrale et pulmonaire, ainsi que d'autres maladies analogues.
PCT/CN1998/000129 1997-07-19 1998-07-17 Staphylokinase recombinante et sa souche manipulee pour une expression elevee WO1999004017A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU84297/98A AU8429798A (en) 1997-07-19 1998-07-17 Recombinant staphylokinase and their high expression engineered strain

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CN97105988.8 1997-07-19
CN97105988A CN1059926C (zh) 1997-07-19 1997-07-19 葡激酶基因及其高表达工程菌株

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WO1999004017A1 true WO1999004017A1 (fr) 1999-01-28

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1064406C (zh) * 1998-05-11 2001-04-11 中国人民解放军军事医学科学院微生物流行病研究所 利用基因工程技术生产葡激酶的方法
CN101240284B (zh) * 2007-09-20 2012-05-30 三峡大学 一种重组葡激酶及其高效分泌表达的方法
CN114480353B (zh) * 2022-01-17 2023-06-23 景泽生物医药(合肥)股份有限公司 一种制备重组人奥克纤溶酶的方法

Citations (4)

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JPH0398595A (ja) * 1989-09-11 1991-04-24 Taisho Pharmaceut Co Ltd ペプチド類の遺伝子工学的製造法
WO1993013209A1 (fr) * 1991-12-30 1993-07-08 Medac Gesellschaft für klinische Spezialpräparate mbH Staphilokinases exemptes de peptides-signal d'expression
CN1096325A (zh) * 1994-04-04 1994-12-14 上海医科大学 一种重组葡激酶的制备方法
CN1109508A (zh) * 1995-01-23 1995-10-04 成都世新药物技术开发中心 重组葡激酶的全dna序列

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0398595A (ja) * 1989-09-11 1991-04-24 Taisho Pharmaceut Co Ltd ペプチド類の遺伝子工学的製造法
WO1993013209A1 (fr) * 1991-12-30 1993-07-08 Medac Gesellschaft für klinische Spezialpräparate mbH Staphilokinases exemptes de peptides-signal d'expression
CN1096325A (zh) * 1994-04-04 1994-12-14 上海医科大学 一种重组葡激酶的制备方法
CN1109508A (zh) * 1995-01-23 1995-10-04 成都世新药物技术开发中心 重组葡激酶的全dna序列

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FIBRINOLYSIS, 6(4), 1992, COLLEN D. et al., "Primary Structure and Gene Structure of Staphylokinase", p. 226-231. *
GENBANK, M57455, 24 Apr. 1996. *
GENBANK, U77328, 12 Nov. 1997. *
MOL. GEN. GENET., 210(3), (1987), BEHNKE D., "Cloning and Expression in Escherichia Coli, Bacillus Subtilis and Streptococcus Sanguis of a Gene for Staphylokinase-a Bacterial Plasminogen Activator", p. 528-534. *
NUCLEIC ACIDS RES., 11(22), (1983), SAKO T. and TSUCHIA N., "Nucleotide Sequence of the Staphylokinase Gene from Staphylococcus Aureus", p. 7679-7693. *

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CN1180106A (zh) 1998-04-29
AU8429798A (en) 1999-02-10

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