WO1999004017A1 - Recombinant staphylokinase and their high expression engineered strain - Google Patents

Recombinant staphylokinase and their high expression engineered strain 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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French (fr)
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/en

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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

The invention disclosed is a staphylokinase (Sak)-producing Staphylococcus aureus SL1.063, a novel recombinant staphylokinase and their high expression engineered strain. A gene coding for Sak was obtained by PCR amplification with chromosomal DNA of Sak-producing Staphylococcus aureus as a template. The nucleotide sequence of the gene and the amino acids they encoded was different from those of the Sak gene which had been reported. And the recombinant DNA fragment was obtained by amplification using a primer which had a particular nucleotide sequence, purified, identified, cut with enzymes, ligated and transformed into a host cell to construct a Sak-high expressing engineered strain, the expression product acted as plasminogen activator and had very high hydrolytic activity to human fibrin. It cold treats thrombembolia disease such as myocardial infarction, cerebral, pulmonary thrombus and the like.

Description

重组葡激酶及其高表达工程菌株 背景技术  Recombinant staphylokinase and its highly expressed engineering strain
本发明涉及一种新的重组葡激酶 （r-Sak )及其高表达工程菌株， 该表达产物具有纤溶酶原激活剂作用， 对人体纤维蛋白具有很高的水 解活性。  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.
葡激酶 (Staphylokinase, Sak)是一种溶栓药物， 是由某种金葡菌 (Staphylococcus aureus )产生的非酶蛋白， 它像链激酶 (Streptokinase, SK)—样， 与纤溶酶原结合形成复合物， 并转化为葡激酶一纤溶酶复 合物， 进而催化纤溶酶原成为纤溶酶， 以此去水解血栓上的纤维蛋白， 从而达到溶栓目的。 Sak激活纤溶酶原对血纤維蛋白具有高度的专一 性， 它优先选择与血检上的纤溶酶原结合， 并水解其中的血纤维蛋 白。 与此同时， 几乎不伤害維持正常血液循环所必须的纤维蛋白原、 因子 V和因子珊等血浆蛋白（Collen D et al. Blood, 1994, 74:387; Okada K et al. Am. J. Hematol. 1996, 53: 151)。  Staphylokinase (Sak) 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. At the same time, it hardly harms the plasma proteins such as fibrinogen, factor V, and factor Shan necessary for maintaining normal blood circulation (Collen D et al. Blood, 1994, 74: 387; Okada K et al. Am. J. Hematol 1996, 53: 151).
葡激酶的基因 已被多 家实验室克隆 。 有公开专利文献 The staphylokinase gene has been cloned in several laboratories. There are published patent documents
CN1109508 A号说明书中，披露从 S.aureus噬菌体 DNA获得了葡激酶 基因并克隆化。 有文献： Collen, D.et al. (1992)Fibrinolysis, 6:226-231 文中披露从 S.aureus 菌染色体 DNA克隆了葡激酶基因。 这两种基因 片段都是采用酶切大片段 DNA进行克隆的。 其基因片段较大， 组装 到载体质粒的拷贝数较低， 难以得到较高的表达效果。 这种 S.aureus 菌染色体 DNA的表达水平一般在 10 - 15 %，如《生物工程》 1994.2 第 12 卷 "治疗血栓用药重组葡激酶的高产率生产和纯化" 一文中披 露： "用发酵罐培养转染大肠杆菌 TG1细胞， 细胞内产生葡激酶， 占 细胞总蛋白的 10 - 15 ¾ 。 " 这是目前公开资料报导中， 葡激酶表达 的公认水平。 发明目的 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 ¾ of the total protein in the cell. "This is the currently recognized level of staphylokinase expression in publicly available reports. Object of the invention
本发明的目的之一是提供一种具有选择性强、 安全性好、 耐热性 高和溶栓效果好的重组葡激酶；  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;
本发明的另一个目的是提供一种工程菌株， 可以高表达本发明的 重組葡激酶； Another object of the present invention is to provide an engineered strain that can highly express the Recombinant staphylokinase
本发明的又一个目的是提供一种安全、 有效的药物组合物， 用于 心脑血栓类疾病的治疗。  Another object of the present invention is to provide a safe and effective pharmaceutical composition for the treatment of cardiovascular and cerebral thrombotic diseases.
本发明的其它目的， 将在对本发明的描述中阐明。 发明简述  Other objects of the present invention will be clarified in the description of the present invention. Brief description of the invention
根据本发明， 一种重组葡激酶， 其氨基酸序列为序列 1 。 本发明 的重组葡激酶具有较高的耐热性和生物活性， 通过基因重组的方法获 得。  According to the present invention, 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.
根据本发明， 一种工程菌株， 采用基因工程的方法抅建， 能够高 效表达本发明的重组葡激酶， 用本发明的工程菌株生产重组葡激酶， 其产率可达细胞总蛋白的 40 %以上。  According to the present invention, 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. .
根据本发明， 一种治疗血栓疾病的药物组合物， 含有本发明的重 组葡激酶作为活性组分。本发明的药物组合物表现出较高的安全性和 有效性。 附图简要说明  According to the present invention, 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. Brief description of the drawings
图 la-lc: 不同来源葡激酶基因的核苷酸序列及其编码的氨基酸序 列的比较。  Figure la-lc: Comparison of the nucleotide sequences of staphylokinase genes and their encoded amino acid sequences from different sources.
图 2: 金葡菌 SL1.063扫描电镜图。  Figure 2: Scanning electron microscope image of Staphylococcus aureus SL1.063.
图 3: 金葡菌 SL1.063 菌株在甘露醇高盐琼脂平 ^上的菌落特 征。  Figure 3: Colony characteristics of S. aureus SL1.063 strain on mannitol high-salt agar plate.
图 4: 金葡菌 SL 1.063菌株在卵黄高盐琼脂平 ^上菌落特征。  Figure 4: Colony characteristics of Staphylococcus aureus SL 1.063 strain on yolk high-salt agar plate.
图 5: 金葡菌 SL 1.063菌株在血琼脂平狐上溶血现象。  Figure 5: Hemolysis of Staphylococcus aureus SL 1.063 strain on blood agar flat fox.
图 6: 金葡菌 SL 1.063菌株耐热核酸酶试验。  Figure 6: Staphylococcus aureus SL 1.063 strain thermostable nuclease test.
样品说明：  Sample description:
1. 4. 为生理盐水  1. for saline
2. 6. 为 SL 1.063 菌株发酵上清液  2. 6. Fermentation supernatant for SL 1.063 strain
3. 5. 7. 为 SL 1.063 菌株发酵上清液  3. 5. 7. Fermentation supernatant for SL 1.063 strain
图 7a ： 金葡菌 SL 1.063菌株中的 Sak , 以人纤维蛋白标准平 检测结果。 图 7b ： 金葡菌 SL 1.063菌株中的 Sak， 以人纤维蛋白加热平 检测结果。 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.
样品说明：  Sample description:
1 . t-PA  1 .t-PA
2. 尿激酶  Urokinase
3. 本发明的葡激酶 （ Sak 63 )  3. Staphylokinase (Sak 63) of the present invention
4. 蚓激酶 （纤溶酶）  4. Earthworm kinase (plasmin)
5. Sak 63粗酶液  5. Sak 63 crude enzyme solution
图 8: 重组质粒插入 DNA片段的检定  Figure 8: Assay of DNA fragments inserted into recombinant plasmids
说明：  Explanation:
Ml pBR322 DNA/MSPI DNA小片段标准分子量。  Standard molecular weight of Ml pBR322 DNA / MSPI DNA fragments.
M2 SPPI DNA/EcoRI DNA大片段标准分子量。  Standard molecular weight of M2 SPPI DNA / EcoRI DNA large fragments.
1 . pHK408 DNA/EcoRI + BamH 1双酶切。  1. pHK408 DNA / EcoRI + BamH 1 double digestion.
2. pBV220 DNA/EcoRI + BamHl双酶切。  2. pBV220 DNA / EcoRI + BamHl double digestion.
3. pUC 19 DNA/EcoRI + BamHl双酶切。  3. pUC 19 DNA / EcoRI + BamHl double digestion.
4. pUK408 DNA/EcoRI + BamHl双酶切。  4. pUK408 DNA / EcoRI + BamHl double digestion.
图 9a ： 重组质粒 pUK408高效表达质粒的抅建流程图。  Figure 9a: Flow chart of the construction of recombinant plasmid pUK408 high-efficiency expression plasmid.
图 9b: 重组质粒 pHK408高表达质粒的抅建流程图。  Figure 9b: Flow chart of the construction of recombinant plasmid pHK408 high expression plasmid.
图 10: 工程菌 TG2(pHK408)高效表达产物的 SDS-PAGE ( 12%) 电泳分析图。  Figure 10: SDS-PAGE (12%) electrophoresis analysis of the highly expressed product of engineered bacteria TG2 (pHK408).
样品说明：  Sample description:
1 . 9. 细胞色素 C(MW 13370)  1. 9. Cytochrome C (MW 13370)
2. l O.RNase A(MW 15000)  2. l O.RNase A (MW 15000)
3. 未诱导 DH5a(pHK408)全细胞裂解液  3. Uninduced DH5a (pHK408) whole cell lysate
4. 未诱导 DH5 (ρΗΚ408)细胞超声破碎上清液  4. Ultrasonically disrupted DH5 (ρΗΚ408) cells
5. 未诱导 DH5 a(pHK408)细胞超声破碎沉淀  5. Ultrasonication of DH5 a (pHK408) cells without precipitation
6. 诱导的 DH5a(pHK408)全细胞裂解液  6. Induced DH5a (pHK408) whole cell lysate
7. 诱导的 DH5 a(pHK408)细胞超声破碎上清液  7. Ultrasonically disrupted supernatant of induced DH5a (pHK408) cells
8. 诱导的 DH5 a(pHK408)细胞超声破碎沉淀  8. Ultrasonication of DH5 a (pHK408) cells induced by precipitation
图 1 1 : 本发明的重組葡激酶对热的稳定性。 适用于本发明的大肠杆菌菌株的举例及其基因型如下： 基因型 Figure 11: Thermal stability of the recombinant staphylokinase of the present invention. Examples of E. coli strains suitable for use in the present invention and their genotypes are as follows: Genotype
F [el4"(McrA-)or el4+(McrA+)] thr-1 F [el4 "(McrA-) or el4 + (McrA + )] thr-1
leuB6 thi-1 lacYl supE44 rfbDl fhuA21  leuB6 thi-1 lacYl supE44 rfbDl fhuA21
F araD139 A(ara-leu)7679 galE15  F araD139 A (ara-leu) 7679 galE15
galkl6 A(lac)X74 rpsL(Stf) hsdR2  galkl6 A (lac) X74 rpsL (Stf) hsdR2
(ΐγ τα ) mcrA mcrBl  (ΐγ τα) mcrA mcrBl
F ara A(lac-proAB) rpsL{ tf)  F ara A (lac-proAB) rpsL (tf)
^0dlacA(lacZ)M15] thi  ^ 0dlacA (lacZ) M15] thi
F'traD36 laclq A(lacZ)M15 proA+B+/ F'traD36 lacl q A (lacZ) M15 proA + B + /
el4"(McrA") Aflac-proAB) thi gyrA96  el4 "(McrA") Aflac-proAB) thi gyrA96
(NaY)endAl hsdRl 7(νΰ k +) relAl supE44 (NaY) endAl hsdRl 7 (νΰ k + ) relAl supE44
DH5 F 'lendAl hsdRl 7(rk' mk +) supE44 thi-1 DH5 F 'lendAl hsdRl 7 (r k ' m k + ) supE44 thi-1
relAl gyrA(NaY) relAl A(laclZYA-argF)  relAl gyrA (NaY) relAl A (laclZYA-argF)
U169 deoR{^0dlacA(lacZ)M15)  U169 deoR (^ 0dlacA (lacZ) M15)
TGI F 'traD36 laclqA(lacZ)M15 proA+B+/s pEA TGI F 'traD36 lacl q A (lacZ) M15 proA + B + / s pEA
(hsdM-mcrB)5(rk'mk'McrB') thi Aflac-proAB) TG2 supEhsdA5thiA(lac-proAB) Δ (srl-recA)  (hsdM-mcrB) 5 (rk'mk'McrB ') thi Aflac-proAB) TG2 supEhsdA5thiA (lac-proAB) Δ (srl-recA)
306:: TnlO(tef)F'[traD36 proAB+ laclq lacZAM15] 发明详述 306 :: TnlO (tef) F '[traD36 proAB + lacl q lacZAM15] Detailed description of the invention
为了寻找具有优越特性的葡激酶， 本发明人进行了广泛和细致的 研究， 发现了一种金葡菌株， 能产理想的葡激酶。 因而， 根据本发明， 一种分离和纯化的、 产葡激酶的金葡菌株 SL1 .063 , 该菌株所产葡激 酶的氨基酸序列为序列 1 。该菌株已保藏在中国微生物菌种保藏管理 委员会普通微生物中心， 保藏号为 CGMCC No.0353 。 该菌株具有下 列特征：  In order to find staphylokinase with superior characteristics, the present inventors conducted extensive and detailed research and found a gold staphylococcus strain that can produce ideal staphylokinase. Therefore, according to the present invention, 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:
形态特征： 革兰氏染色为阳性， 菌体为球形， 堆积成葡萄状， 无 芽孢， 菌体大小为 0.8-0.9μΐΏ 。  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 μΐΏ.
培养特征：  Training characteristics:
(1)在甘露醇高盐琼脂培养基上 36 °C培养 24-48 小吋， 菌落为金 黄色， 图形凸起， 外围呈微黄色环。  (1) Cultivate 24-48 hours at 36 ° C on mannitol high-salt agar medium. The colonies are golden yellow, the figure is convex, and the periphery is a yellowish ring.
(2)在卵黄高盐琼脂培养基上， 36 °C培养 24-48小吋， 菌落为金黄 色或柠檬色， 其周围呈白色月暈状油膜。 (3)在血琼脂培养基上划线， 36 °C培养 18-24小吋， 菌落呈现透明 的溶血环。 (2) On a yolk high-salt agar medium, culture for 24-48 hours at 36 ° C. The colonies are golden yellow or lemon-colored, and there is a white halo-like oil film around them. (3) Streak on blood agar medium, and culture for 18-24 hours at 36 ° C. The colonies show a transparent hemolytic ring.
生化特征：  Biochemical characteristics:
具有产卵磷脂酶、 产耐热核酸酶和血浆凝固酶等能力。  With the ability to produce lecithin, thermostable nuclease and plasma coagulase.
根据本发明， 以 SL1.063 的染色体 DNA为模板， 通过基因工程 的方法，生产一种重组葡激酶，该重組葡激酶的氨基酸序列为序列 1 ， 而编码该重组葡激酶的基因的核苷酸序列为序列 2 。  According to the present invention, using chromosomal DNA of SL1.063 as a template, 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.
本发明 r-Sak基因的核苷酸序列及其编码的 136个氨基酸序列与 国内外已报道的 r-Sak结抅相比， 均有所不同， 如表 1和图 1所示。 表 1: 不同来源葡激酶基因的核苷酸序列及其編码的氨基酸序列的差别 基因 基因来源 第 34位 第 36位 第 43位 第 2位 第 133位 核苷酸不同处 资料来源 The 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
Sak 63 金葡 ¾ 甘氨酸 甘氨酸 精氨酸 ¾氨酸 异亮氨酸 本发明 Sak 63 Gold glucose ¾ Glycine Glycine Arginine ¾ Isoleucine The present invention
SL 1.063 ¾  SL 1.063 ¾
色体 DNA  Chromosomal DNA
Sak star 金葡 23 丝氨酸 甘氨酸 組氦酸 氨酸 异亮氛酸 100A(G)105A(G) Fibrinolysis, 染色体 128A(G) 399A(T) 1992, 6: 226 DNA  Sak star Gold glucose 23 Serine Glycine Histidine Histidine Isoleucine 100A (G) 105A (G) Fibrinolysis, Chromosome 128A (G) 399A (T) 1992, 6: 226 DNA
Sak 42D D42噬 a 甘氨酸 精氣酸 精氨酸 鉻氨酸 异亮氨 ¾ 105A(G) 106A(G) MMG, 1987, 体 DNA 399A(T) 210:528 Sak 42D D42 phagolysine glycine arginine arginine chromine isoleucine ¾ 105A (G) 106A (G) MMG, 1987, somatic DNA 399A (T) 210: 528
Sak s(jic 5φ ¾ 8体 甘氨酸 甘氨酸 组氨酸 鉻氨酸 异亮氨鲛 33G(A) 105A(G) NAR, 1983, Sak s (jic 5φ ¾ 8-body glycine glycine histidine chromine isoleucine G 33G (A) 105A (G) NAR, 1983,
DNA 128A(G) 399A(T) 1 1 :7679 DNA 128A (G) 399A (T) 1 1: 7679
Sak s-29 金葡菌噬 甘氨酸 甘氛酸 组氛酸 苯丙氨酸 精氨酸 105A(G) 128A(G) CN1 109508A 菌体 DNA 275T(A) 398G(T) Sak s-29 Staphylococcus aureus Glycine Glyoxalic acid Histanoic acid Phenylalanine Arginine 105A (G) 128A (G) CN1 109508A Bacterial DNA 275T (A) 398G (T)
399A(T)  399A (T)
Sak SY 金葡 ¾大 甘氨酸 精氨酸 精 ll酸 ^氨酸 异亮氨酸 105A(G) 106A(G) CN 1096325A 分子 DNA 346A(T) 399A(T)  Sak SY Gold glucose ¾ Big glycine Arginine Arginine ll Alanine Isoleucine 105A (G) 106A (G) CN 1096325A Molecular DNA 346A (T) 399A (T)
注： 下方划线的氛基酸表明与本发明不同， 括号内的核苷酸为本发明的核苷酸。  Note: The underlined amino acid indicates that it is different from the present invention, and the nucleotides in parentheses are the nucleotides of the present invention.
从 r-Sak基因来源分析， 本发明的 r-Sak基因与 Sak star均来自金 葡菌染色体 DNA, 其菌株分別为 SL 1.063和 23金葡菌株， 酶的氨基酸 序列第 34位和 43位， 本发明分别为甘氨酸和精氨酸， 而 Sak star则是 丝氨酸和组氨酸， 在相应的 DNA 的核苷酸序列上也表现出差异。 本 发明 r-Sak基因与溶原性噬菌体来源的基因在核苷酸序列和氨基酸序 列上， 亦有所不同。 本发明 r-Sak基因与 Sak SY比较， 第 36位氨基 酸， 本发明为甘氨酸， 而 Sak SY则是精氨酸， 基因核苷酸序列本发 明基因第 105位和 106位均为 G ， 346位和 399位则为 T，而 Sak SY 这四个位点上的核苷酸均为 A 。 不难看出， 本发明 r-Sak基因与这两 个来自不同菌株的 Sak基因在核苷酸序列上及其编码的氨基酸序列的 差异是显著的。 According to the analysis of the r-Sak gene 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.
本发明 r-Sak不仅氨基酸序列不同于已知葡激酶的氨基酸序列， 而且还具有较高的耐热性，因而有较高的生物稳定性。 Sak42D经 100 °C 1.5h处理， 酶活性几乎全部丧失； 70 °C 2.0h处理， 酶的残留活性 仅有 10 % 。 Sak star经 100 °C 1.5h处理， 残存酶活性接近于零； 70 °C 2.0h处理， 残存酶活性约 60 %， 显然， Sak star比 Sak42D耐热性 强。 正如图 11所揭示的那样， 本发明 Sak63经 100 °C 1.5h处理， 其 残存酶活性为 55 %； 70 。C 2. Oh处理， 醇活性为 70 % 。本发明 r-Sak 与已经报道的上述两个葡激酶相比， 具有更高的耐热性。  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. After Sak42D was treated at 100 ° C for 1.5 h, the enzyme activity was almost completely lost. At 70 ° C for 2.0 h, 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. At 70 ° C for 2.0 h, the residual enzyme activity was about 60%. Obviously, Sak star was more heat resistant than Sak42D. As disclosed in 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%. Compared with the above two staphylokinases, the r-Sak of the present invention has higher heat resistance.
根据本发明， 可以将本发明的编码葡激酶的基因重组入一种载 体。 重组后的载体含有編码本发明重組葡激酶的基因， 该基因的核苷 酸序列为序列 2 。 用于本发明的载体可以采用常用的载体。 例如， 可 以选用 pUC19 ， pBV220等等。  According to the present invention, 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.
将本发明的重组载体转入一种宿主细胞， 可以得到含有本发明重 组载体的宿主，用于高表达本发明的重组葡激酶。适于本发明的宿主， 可以为常用的大肠杆菌，例如： C600 、 MC1061 、 JM83 、 JM109 、 DH5a 、 TGI 、 TG2等等， 其中优选 DH5a 、 TGI 、 TG2  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
根据本发明， 一种抅建产重组葡激酶的工程菌株的方法， 包括筛 选产葡激酶的金葡菌。 从自然界中采集天然金葡菌并分离、 纯化。 以 该金葡菌染色体 DNA为模板进行 DNA聚合酶链式反应 (PCR扩增）。 优选对葡激酶表达高的金葡菌染色体 DNA作模板。 在 PCR扩增中， 采用的引物为： 引物 I According to the present invention, 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
5'-CGCGAATTCATGTCAAGTTCATTCGACAAAGG-3', 链长： 5'-CGCGAATTCATGTCAAGTTCATTCGACAAAGG-3 ', chain length:
32 ； 32;
引物 Π  Primer Π
5'-CGCGGATCCTATTTCTTTTCAATAACAACCTTTG-3', 链长： 5'-CGCGGATCCTATTTCTTTTCAATAACAACCTTTG-3 ', chain length:
34 。 34.
选取质粒（载体） ， 将经扩增步骤后得到的完整葡激酶基因 DNA 小片段引入该质粒， 实现对该质粒的重组。 在质粒重组中， 该基因 DNA片段与载体质粒， 分别以 EcoRI和 BamHI酶切； 再将酶切的上 述质粒和葡激酶基因 DNA小片段，用 T4DNA连接酶连接获得重组质 粒。 重组后的质粒可以直接转入宿主细胞， 以完成工程菌的抅建。 也 可以将该重组质粒中的葡激酶基因 DNA片段切下， 用于重组另一质 粒， 再将后重组的质粒转入宿主细胞， 抅建工程菌。 质粒和宿主的选 择范围在上文中已有表述。优选的质粒为 pUC19 , pBV220 ；优选的 宿主为大肠杆菌 DH5a 、 TGI和 TG2 。 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. In plasmid recombination, 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.
因而， 一种高表达工程菌 TG2(pHK408) , 高效表达本发明的重组 葡激酶。该菌株已保藏于中国微生物菌种保藏管理委员普通微生物中 心， 保藏号为 CGMCC No.0333 。 该工程菌株为高表达系统， 使用该 工程菌株生产本发明的重组葡激酶， 其产率为 40 %或以上。 与同类 由金葡菌染色体 DNA克隆的 Sak基因的表达水平 (10-15%)相比提高 了近 1.7倍或以上。  Therefore, 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. Compared with similar Sak gene cloned from S. aureus chromosomal DNA (10-15%), the expression level was increased by nearly 1.7 times or more.
本发明的重组葡激酶可用于治疗心肌梗塞、 血栓疾病， 如其可用 于治疗心肌梗塞、 深度静脉血栓、 肺栓塞、 急性或亚急性外周动脉血 栓和慢性动脉栓塞以及中央视网膜动脉或静脉栓塞等疾病。 因而， 根 据本发明， 一种药物组合物， 包括有效剂量的本发明重組葡激酶和药 用赋形剂。 药用赋形剂可以是常用的赋形剂， 如： 人白蛋白、 水解胶 原蛋白、 谷氨酸、 甘氨酸和无机盐等。  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.
重组葡激酶药理药效研究  Study on Pharmacological Effect of Recombinant Staphylokinase
1 、 r-Sak—般药理研究  1. r-Sak—general pharmacological research
(1) 给小鼠静注！ "-Sak 1.5x105AU/kg 、 3xl05AU/kg和 6x l05AU/kg 三种剂量， 对小鼠行为、 自主活动均无明显影响， 表明对中枢神经系 统无兴奋和抑制作用； (1) Intravenous injection to mice! "-Sak 1.5x10 5 AU / kg, 3xl0 5 AU / kg and 6x l0 5 AU / kg three doses have no significant effect on mouse behavior and autonomous activity, indicating that it has a central nervous system No excitement and inhibition effect;
(2) r-Sak静脉给药 6.9xl04AU/kg 、 13.8xl04AU/kg两种剂量， 对 麻醉小型猪血压、 心律及心电图波均无明显改变； (2) 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;
(3) 同样， 麻醉小型猪出血及凝血时间、 血小板聚集性、 KpTT (白陶土部分凝血活酶时间） 、 ΡΤ (凝血酶原时间） 及 ΤΤ (凝血酶 时间） 均无显著改变；  (3) Similarly, there was no significant change in bleeding and clotting time, platelet aggregation, KpTT (partial thromboplastin time), PT (prothrombin time), and TT (thrombin time) in anesthetized mini-pigs;
(4) 静注 r-Sak对呼吸系统无明显影响。  (4) Intravenous injection of r-Sak has no significant effect on the respiratory system.
2 、 r-Sak药效研究  2. R-Sak efficacy study
所使用的动物模型及研究用药剂量如下：  The animal models and research doses used are as follows:
小型猪： 股动脉富红细胞血栓、 冠脉血栓、 给药后血流纤溶系统 生化分析。 冠脉血栓静脉给药 4.6xl04AU/kg和 9.2xl04AU/kg 。 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.
大鼠： 颈动脉血栓，静脉给药 1.8xl04 、 6xl04 、 1.8xl05AU/kg 。 兔: 肺栓塞， 静脉给药 3xl03 、 lxlO4 、 3xl04AU/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.
人血： 陈旧性人富纤維蛋白血栓等， 用药浓度 10〜 300AU/ml 。 药效试验结果：  Human blood: old human fibrin-rich thrombus, etc., the drug concentration is 10 ~ 300AU / ml. Pharmacological test results:
重组葡激酶是一种间接作用方式的纤溶酶原激活剂， 本研究采用 在体及离体多种模型评价了重组葡激酶的溶栓活性及纤维蛋白特异 性。 实验结果显示：  Recombinant staphylokinase is an indirect mode of plasminogen activator. In this study, in vivo and in vitro models were used to evaluate the thrombolytic activity and fibrin specificity of recombinant staphylokinase. experiment result shows:
(1) 重组葡激酶静脉给药后， 对电刺激引起的小型猪冠脉血栓产 生显著的溶栓效果； 心肌缺血程度及范围明显緩解。  (1) After intravenous administration of recombinant staphylokinase, it has a significant thrombolytic effect on mini-pig coronary artery thrombosis caused by electrical stimulation; the degree and extent of myocardial ischemia is significantly relieved.
(2) 重组葡激酶静脉给药后， 富红细胞血栓栓塞的小型猪股动脉 很快恢复流量， 血管再通； 残存血栓长度及重量均明显減少。  (2) After intravenous administration of recombinant staphylokinase, the small pig femoral artery with red blood cell thromboembolism quickly restored flow and recanalization of the blood vessel; the length and weight of the remaining thrombus were significantly reduced.
(3) 重组葡激酶静脉给药后， 对电刺激大鼠颈动脉引起的血栓产 生明显的溶栓作用。  (3) After intravenous administration of recombinant staphylokinase, it has a significant thrombolytic effect on thrombus caused by electrical stimulation of carotid arteries in rats.
(4) 在兔肺栓塞模型上， 重组葡激酶静脉给药后， 产生剂量依赖 性、 渐进性血栓溶解作用。  (4) In the rabbit pulmonary embolism model, after intravenous administration of recombinant staphylokinase, a dose-dependent, progressive thrombolytic effect was produced.
(5) 重组葡激酶对陈旧性(161 人富纤維蛋白血栓产生快速、 高效 的溶栓作用。  (5) Recombinant staphylokinase produces fast and efficient thrombolytic effects on stale (161 human fibrin-rich thrombi).
(6) 重组葡激酶溶栓吋， 不伴有明显血浆纤维蛋白原等降解， 具 有较高的纤维蛋白特异性。  (6) Thrombolysis of recombinant staphylokinase, without significant degradation of plasma fibrinogen, etc., has high fibrin specificity.
(7) 重组葡激酶较尿激酶和链激酶溶栓速度快， 效力强， 纤维蛋 白特异性高。 采用 125I-标记的放射性同位素方法 (RA法）、 HPLC分离结合放射 性检測方法 (HPLC-RA法)研究 r-Sak大鼠的药代动力学。 主要结论：(7) Recombinant staphylokinase has a faster thrombolytic rate than urokinase and streptokinase, has strong efficacy and high fibrin specificity. 125 I-labeled radioisotope method (RA method), HPLC separation and radioactivity detection method (HPLC-RA method) were used to study the pharmacokinetics of r-Sak rats. main conclusion:
(1) 大鼠静注 (iv) r-Sak 3 x 104、 9 x 104、 和 3 x 105AU/kg三 个剂量进行药代动学试验， 以 RA法检测血清总放射性， 以 HPLC-RA 法检测血清原型药物浓度、浓度-时间数据经药代动力学参数计算， 证 明 r-Sak iv给药后总放射性体内消除较快， t1/2在 134min左右,而原型药 物消除很快， t1/2在 56min左右。 二种方法所得结果均证明 AUC(药时 曲线下面积)与剂量呈显著相关。 (1) 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 The 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. The results obtained by both methods proved that AUC (Area Under the Curve) was significantly correlated with dose.
(2) 大鼠 iv 9 X 104AU/kg给药后用 RA法和 HPLC-RA法測定不 同组织的药物含量， 发现在同一时间以肾药含量最高， 脑浓度最低。 (2) 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.
(3) 大鼠 iv 9 104AU/kg给药后， 用 RA法測定尿和粪放射性总 量。在 24h内 81.54%放射性从尿中回收， 2.05%从粪中回收到， 尿粪总 排泄量达给药剂量的 83.59%, 表明排泄快也完全。 (3) 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.
4 、 安全性及毒性研究  4. Safety and toxicity studies
与 r-Sak安全性及毒性有关的研究结果列于表 2如下：  The research results related to the safety and toxicity of r-Sak are listed in Table 2 as follows:
表 2 ： 本发明 r-Sak安全性及毒性试验结果  Table 2: Safety and toxicity test results of r-Sak of the present invention
Figure imgf000011_0001
Figure imgf000011_0001
实施例一  Example one
1、 金葡菌 (Staphylococcus aureus)的分离 、 鉴定和产葡激酶 (Staphylokinase, Sak)菌株筛选：  1. Isolation, identification of Staphylococcus aureus and screening of Staphylokinase (Sak) strains:
为分离金葡菌， 从医院外科、眼科和耳鼻喉等科采集了 90多个样 品， 以稀释法或划线法， 接种在蛋黄氯化钠琼脂培养基上， 36 °C培 养 24-48小时， 挑取金黄色或柠檬色、 周围培养基呈珍珠光泽白色沉 淀的菌落， 接入普通营养培养基中， 36 °C培养过夜， 革兰氏染色、 镜 检， 选择 G+菌体呈球形并堆积成葡萄状、无芽孢菌株， 在蛋黄培养基 上作进一步纯化。 纯化菌株分别接在高盐甘露醇琼脂培养基上， 36 °C培养 24-48小吋， 菌落周围出现光亮的黄色圓环。 经分离、 纯化、 鉴定， 共得到 167株金葡菌， 其形态和培养特征如下： 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:
革兰氏染色为阳性， 菌体为球形、 堆积成葡萄状、 无芽孢， 菌体 大小通常为 0.8-0.9μπι 。 如图 2 。  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 μm. See Figure 2.
培养特征：  Training characteristics:
在甘露醇高盐琼脂培养基上， 36 °C培养 24-48小吋，菌落为金黄 色， 圆形凸起， 外围为微黄色环， 如图 3 。  Incubate on mannitol high-salt agar medium for 24-48 hours at 36 ° C. The colonies are golden-colored, round and convex, and the periphery is a yellowish ring, as shown in Figure 3.
在卵黄高盐琼脂培养基上， 36 °C培养 24-48 小吋 ， 菌落为金黄 色或柠檬色， 其周围呈白色月暈状油膜， 如图 4， 表明该菌株具有产 卵磷脂酶的能力， 此即金葡菌特征之一。  Cultured on egg yolk high-salt agar medium at 36 ° C for 24-48 hours, the colonies are golden yellow or lemon-colored, and the surroundings are white moon-shaped oil film, as shown in Figure 4, which shows that the strain has the ability to produce lecithin This is one of the characteristics of S. aureus.
在血琼脂平 上划线， 36 °C培养】8-24小吋，菌落周围呈现透明 的溶血环， 即 β型溶血环， 见图 5 。  Streak on blood agar plate, culture at 36 ° C] 8-24 hours, the colonies present a transparent hemolytic ring, that is, β-type hemolytic ring, as shown in Figure 5.
为了测定是否产耐热核酸酶，将供试菌株在普通营养培养液中 37 °C培养 18-24小吋， 菌液煮沸 10分钟， 点入甲苯胺兰 DNA琼脂平 的小孔中， 37 °C保温 3-5小吋， 阳性者均产生粉红圓环， 见图 6 。 图 6中： 1， 4为生理盐水； 2, 3, 5, 6, 7, 为 SL1.063菌株发酵上清液。  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. In FIG. 6: 1, 4 are physiological saline; 2, 3, 5, 6, 7, are fermentation supernatants of SL1.063 strain.
血浆凝固酶试验：取兔血浆:生理盐水 (1 : 1)与等体积各 株普通营 养培养液混合。 对照： 为等体积的生理盐水。 37 °C保溫过夜， 阳性 对照与试验菌株血浆均凝固， 对照管血浆流动自如。  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.
本发明使用的培养基：  Medium used in the present invention:
卵黄高盐琼脂培养基  Yolk High Salt Agar Medium
蛋白胨 10g 牛心浸液 10g  Peptone 10g Beef Heart Infusion 10g
甘露醇 10g 卵黄悬液 100g  Mannitol 10g Yolk Suspension 100g
NaCl 70g 琼脂 15g  NaCl 70g Agar 15g
LiCl 5e 蒸馏水 890g 甘露醇高盐琼脂培养基  LiCl 5e distilled water 890g mannitol high salt agar medium
蛋白胨 10g  Peptone 10g
甘露醇 10g 牛肉膏 lg 10g mannitol Beef paste lg
NaCl 75g  NaCl 75g
酚 红 0.025g  Phenol red 0.025g
琼脂 15g  Agar 15g
蒸馏水 1000ml 普通营养琼脂培养基  Distilled water 1000ml common nutrient agar medium
蛋白胨 10g  Peptone 10g
牛肉膏 3g  Beef Paste 3g
NaCl 5g  NaCl 5g
琼 脂 15g  Agar 15g
蒸馏水 1000ml  Distilled water 1000ml
以上培养基调 PH7.2 , 121 °C灭菌 20分钟。 甲苯胺兰 (Toluidine blue)DNA琼脂平  The above medium was adjusted to pH 7.2 and sterilized at 121 ° C for 20 minutes. Toluidine blue DNA agarpine
鱼精 DNA 0.03g  Fish sperm DNA 0.03g
NaCl l .OOg  NaCl l .OOg
琼脂 l .OOg  Agar l.OOg
2%甲苯胺兰 0.3ml  2% toluidine blue 0.3ml
O.OlM CaCl 0.1ml  O.OlM CaCl 0.1ml
0.05M Tris-HCl(pH9.0) 100ml  0.05M Tris-HCl (pH9.0) 100ml
121 °C灭菌 15分钟。  Sterilize at 121 ° C for 15 minutes.
为了筛选产葡激酶的金葡菌， 将 167株， 取其单菌落接在 3ml LB液 体培养基试管中， 37 °C振荡培养过夜， 离心 4000rpm 5分钟， 上清液 作为粗酶液， 取 ΙΟμΙ点在人血纤維蛋白平 中， 37 °C保溫 16 小时， 观察是否有透明圆形成。经多次重复，证明金葡菌 SL1.063 菌株产 Sak 水平最高。 为了确认这是葡激酶活性还是蛋白酶活性 ， 按同样方法 制做了两个相同的人血纤维蛋白平 一个加热 85 ：、 40分钟 。 另一个按常规作为标准平 ， 见图 7a, 打孔后， 分别点 ΙΟμΙ发酵上清 液， 37 °C保溫 16小时。 结果是： 在标准平皿上， 3, 5号为 SL1.063 粗酶液； 1 , 2号为 t-PA和尿激酶； 4号为蚓激酶， 均产生透明圈。 在加 热平 上， 见图 7b所示， 除 4号蚓激酶产生透明圈外， 3, 5号 SL1.063 粗酶液与尿激酶 t-PA—样， 均无透明圈， 结果表明： SL1.063的酶是 通过激活纤溶酶原产生纤溶酶水解纤维蛋白， 经 85 °C加热 40分钟处 理， 使纤溶酶原全部失活， 因而， SL1.063 产生的酶像尿激酶 t-PA 一样在加热纤維平 上不再产生透明圈 。 In order to screen staphylokinase-producing Staphylococcus aureus, 167 strains were collected, and a single colony was placed in a 3 ml LB liquid medium test tube, cultured with shaking at 37 ° C overnight, centrifuged at 4000 rpm for 5 minutes, and the supernatant was used as a crude enzyme solution. Spot in human fibrin level, incubate at 37 ° C for 16 hours, and observe if transparent circles are formed. Repeatedly, it was confirmed that S. aureus SL1.063 strain produced the highest Sak level. In order to confirm whether this is staphylokinase activity or protease activity, two identical human fibrin levels were prepared in the same way and heated for 85 minutes and 40 minutes. The other was used as a standard flat, as shown in Figure 7a. After punching, 10 μl of fermentation supernatant was spotted and incubated at 37 ° C for 16 hours. The results were: on standard plates, No. 3 and No. 5 were crude SL1.063 enzyme solution; No. 1 and No. 2 were t-PA and urokinase; No. 4 were lumbrokinase, all of which produced clear circles. On the heating level, as shown in Figure 7b, except for No. 4 lumbrokinase producing transparent circles, No. 3 and No. 5 SL1.063 crude enzyme solution is similar to urokinase t-PA, and there is no transparent circle. The results show: SL1. 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.
为了提高金葡菌 SL1.063 菌株产酶能力， 对其发酵条件进行了初 步试验。  In order to improve the enzyme-producing ability of Staphylococcus aureus SL1.063, preliminary tests were performed on its fermentation conditions.
首先， 观察不同的有机氮源对 SL1.063 菌株产酶的影响， 有机氮 源有胰蛋白胨 (北京海淀）、 大豆胨 (上海云州）、 乳清蛋白水解物（日本) 矛口聚月、(polypeptone, 曰本)。  First, observe the effects of different organic nitrogen sources on the enzyme production of SL1.063 strain. The organic nitrogen sources are tryptone (Beijing Haidian), soybean mash (Shanghai Yunzhou), whey protein hydrolysate (Japan), Maokou Juyue, (polypeptone, Japanese version).
培养基成分  Media composition
有机氮 1 .0  Organic nitrogen 1.0
酵母粉 0.5  Yeast powder 0.5
NaCl 0.5  NaCl 0.5
调 pH至 7.2, 分装于 250ml三角瓶中， 分装量为 25ml/瓶， 121 。C , 30分钟， 灭菌。  Adjust the pH to 7.2, and aliquot it in a 250ml triangle bottle with a volume of 25ml / bottle, 121. C, 30 minutes, sterilize.
发酵条件： 37 震荡培养（180rpm) 16小吋。  Fermentation conditions: 37 shake culture (180rpm) 16 hours.
活力测定： 纤维蛋白一琼脂平 方法， 定吋离心， 取发酵上清液 ΙΟμΙ点样， 37 °C , 16小吋。 结果如下：  Viability determination: Fibrin-agar level method, fixed centrifugation, take 10 μl of fermentation supernatant, 37 ° C, 16 hours. The results are as follows:
不同氮源 豆胨 胰蛋白胨 乳清蛋白胨 101胨 聚胨 相对酶活性(％) 100 190.0 191 .0 1 10.0 200 其中以聚胨效果最好， 其次为胰胨和乳清蛋白胨， 豆胨最差。 Different nitrogen sources Douchi Trypton 胨 Whey Protein 胨 101 胨 Poly (R) Relative enzyme activity (%) 100 190.0 191.0 0 1 10.0 200 Among them, poly 胨 has the best effect, followed by Pancreas and Whey Peptone, and 胨 is the worst.
其次， 在培养基中动物血清对 SL1 .063 菌株产酶的影响， 实验结 果表明： 加入一定量的动物血清 (4%)可提高产酶能力 2倍。  Secondly, the effect of animal serum on the enzyme production of SL1.063 strain in the culture medium, the experimental results show that: adding a certain amount of animal serum (4%) can increase the enzyme production capacity by 2 times.
动物血清有： 兔、 鸡、 羊、 牛等动物血清， 其效果相当。  Animal serums include: rabbit, chicken, sheep, bovine and other animal serums, which have comparable effects.
发酵周期： 37 震荡培养 12-16小时为宜。  Fermentation cycle: 37 shake culture for 12-16 hours is appropriate.
2 、 SL1 .063 Sak基因的克隆:  2.Clone of SL1.063 Sak gene:
(1 ) 金葡菌染色体 DNA制备：  (1) Preparation of S. aureus chromosomal DNA:
将菌株单菌落接种在 25ml LB液体培养基中， 37 °C振荡培养过 夜， 离心 4000rpm 、 8 分钟收集菌体， 菌体悬浮在 2.5mlGTE 溶液 (50mM Glucose-50mM Tris-HCl- 1 OmM EDTA,PH8.0)中。加溶菌酶终浓 度为 10 mg/ml， 37 °C保溫 60分钟， 加 300μ1的 10% SDS , RNase A(10mg/ml)和 RNase TiPOOOU/ml)各 40μ1, 37 °C保温 60分钟， 加等体 积苯酚-氯仿-异戍醇 (12: 12: 1 )抽提三次，对界面上的白色膜以等体积的 TE溶液 (20mM Tris-HCl(H8.0)-lmM EDTA)反抽提两次， 水相合并后 , 加入 1 0体积 3 M NaCl和 2倍体积的 95%乙醇 ，以玻棒搅出纤维状 的 DNA , 以 70%乙醇和无水乙醇沖洗各两次 ,赶掉乙醇。 DNA溶在 适量的 TE溶液中， 浓度约 0.2 g^l 。经 SDS-PAGE 检查， 没有 RNA 存在。 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). Add lysozyme at a final concentration of 10 mg / ml, incubate at 37 ° C for 60 minutes, add 300 μ1 of 10% SDS, RNase A (10 mg / ml) and RNase TiPOOOU / ml) each at 40 μ1, incubate at 37 ° C for 60 minutes, etc. The volume of 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.
(2) 引物设计：  (2) Primer design:
参考已知的 Sak基因的结构， 设计了若干个引物， 并引入适当的 限制酶切位点。  With reference to the known Sak gene structure, several primers were designed and appropriate restriction sites were introduced.
引物 酶切位点 核苷酸序列 （5'→3') 链长  Primer Restriction site Nucleotide sequence (5 '→ 3') Chain length
τ „ „ , CGCGAATTCATGTCAAGTTCATTC  τ „„, CGCGAATTCATGTCAAGTTCATTC
CGCGGATCCTATTTCTTTTCAATA CGCGGATCCTATTTCTTTTCAATA
BamH, 34  BamH, 34
ACAACCTTTG  ACAACCTTTG
(3) 聚合酶链反应 （PCR ) 系统： (3) Polymerase chain reaction (PCR) system:
组成 反应液 (μΐ)  Composition Reaction Solution (μΐ)
1. SL1.063模版 DNA(0.2 g^l) 3.0  1. SL1.063 template DNA (0.2 g ^ l) 3.0
2. 引物 Ρ-ΝΠ (33ριηο1/μ1) 3.0  2. Primer Ρ-ΝΠ (33ριηο1 / μ1) 3.0
3. 引物 P-C (33ρπιο1/μ1) 3.0  3. Primer P-C (33ρπιο1 / μ1) 3.0
4. 10 x PCR緩沖液 5.0  4.10 x PCR buffer 5.0
5. 4 x dNTP (2mM) 4.0  5.4 x dNTP (2mM) 4.0
6. dd¾0 31.0  6. dd¾0 31.0
7. Taq DNA聚合酶 (2.5υ/μ1) 1.0  7. Taq DNA polymerase (2.5υ / μ1) 1.0
1-6混匀，变性处理 95 °C 、 7分钟 , 然后加入 Tag DNA聚合酶混 匀 在 Progene PCR循环仅中，进行扩增。  Mix 1-6, denaturing at 95 ° C for 7 minutes, then add Tag DNA polymerase and mix well. Progene PCR cycle only, and then perform amplification.
94 °C 40秒 、 94 ° C 40 seconds,
50 。C 40秒 |·Χ 30→72 °C 延伸反应 10分钟  50. C 40 sec. · × 30 → 72 ° C Extension reaction 10 min.
72 °C 40秒  72 ° C 40 seconds
(4) DNA片段的分离、 纯化、 酶切、 连接与转化：  (4) Isolation, purification, digestion, ligation and transformation of DNA fragments
①纯化： 经 1.5%Agarose电泳检查， 在 360mn波长下， 切下扩增 ① Purification: Examined by 1.5% Agarose electrophoresis, and cut off and amplified at 360mn wavelength.
DNA片段。采用 Genelute Agarose Spin Columns方法纯化 PCR扩增的DNA fragment. Genelute Agarose Spin Columns method was used to purify PCR amplified
DNA片段。 DNA溶于 20μ1 ΤΕ(ΡΗ8.0)溶液中。 ②酶切： DNA fragment. The DNA was dissolved in 20 μl TE (PQ8.0) solution. ② enzyme digestion:
§ 切反应液 (μΐ)  § Cut reaction solution (μΐ)
DNA片段 20  DNA fragment 20
10 X緩沖液  10 X buffer
EcoRI (20υ/μ1) 2  EcoRI (20υ / μ1) 2
BamHj (12υ/μ1) 3  BamHj (12υ / μ1) 3
dd¾0 20  dd¾0 20
37 °C 、 2小时。  37 ° C for 2 hours.
再以 Genelute Agarose Spin Columns方法纯化，将 DNA片段溶于 20μ1 ΤΕ中。  Then purified by Genelute Agarose Spin Columns method, the DNA fragment was dissolved in 20μ1 TE.
③连接： pUC19质粒提取参照 Sambrook等人的方法。按上述方 法， 以 EcoRI和 Bami^酶切 pUC19 质粒,经 Genelute Agarose Spin ③ Ligation: 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.
Columns方法纯化。 与经 EcoRI和 BamH,酶切的 Sak基因连接 ， 16Columns method purification. Linked to Sak gene digested with EcoRI and BamH, 16
。C保温 12小时。 . C was held for 12 hours.
④转化 ： 根据 Sambrook等人的方法， 感受态菌株为钙离子处理 的 DH5 或 JM109， 涂在平 i上培养过夜。 以 x_gal试验检测， 挑出 白色菌落， 接入 LB-AP液体培养基中， 37 °C振荡培养过夜， 提取质 粒 DNA，以 EcoRI和 BamHi酶切，经 1.5%Agarose电泳检査 ,有 431bp DNA片段区带， 证明已经得获得 pUK408重组质粒， 如图 8所示。抅 建 pUK408重组质粒的流程参见图 9a 。  ④ Transformation: According to the method of Sambrook et al., The competent strain was calcium ion-treated DH5 or JM109, which was spread on flat i and cultured overnight. Detected by x_gal test, white colonies were picked out, inserted into LB-AP liquid medium, cultured with shaking at 37 ° C overnight, plasmid DNA was extracted, digested with EcoRI and BamHi, and checked by 1.5% Agarose electrophoresis, with a 431bp DNA fragment The band proves that the pUK408 recombinant plasmid has been obtained, as shown in FIG. 8. See Figure 9a for the process of constructing pUK408 recombinant plasmid.
(5) DNA序列測定：  (5) DNA sequence determination:
重组质粒 DNA的提取，采取碱性 SDS裂解细胞 PEG NaCl方法， 经 7%Agarose凝胶电泳检查，基本上不含 RNA , 可供 DNA序列測定 用。 DNA序列测定采用的是 T7聚合酶测序系统 (Promega公司产品 结果如前所述序列。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).
Figure imgf000016_0001
Figure imgf000016_0001
按图 9b的流程抅建高效表达质粒 pHK408 。质粒 DNA的制备参 考 Sambrook等人的方法。 取 DH5a (pUK408)质粒单菌落， 接于 LB- AP液体培养基中， 37 °C振荡培养过夜。以碱性 SDS处理裂解大肠杆 细胞， 以 PEG-NaCl沉淀掉 RNA , 得到质粒 DNA 。 经 SDS-PAGE 检查， 基本上不含 RNA 。 以 EcoRI和 BamH,酶切 pUK408 DNA , 经 1.5%Agarose电泳分离 ' 切下小片段 DNA， 按 Genelute Agarose Spin Columns方法， 得到纯化 DNA 。 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.
pBV220载体质粒， 按上述方法分离纯化。 以 EcoRI和 BamHi双 酶切。 ddH20 36μ1 The pBV220 vector plasmid was isolated and purified as described above. Digest with EcoRI and BamHi. ddH 2 0 36μ1
10 x buffer 5μ1  10 x buffer 5μ1
质粒 DNA (0.5μ^μ1) 5μ1  Plasmid DNA (0.5μ ^ μ1) 5μ1
EcoRI (20υ/μ1) 2μ1  EcoRI (20υ / μ1) 2μ1
BamHj (12υ/μ1) 2μ1  BamHj (12υ / μ1) 2μ1
37 °C保温 2小时。  Incubate at 37 ° C for 2 hours.
酶切反应液走 0.7%Agarose 电泳， 在长波长紫外灯下切下 DNA 大片段。 按 Genelute Agarose Spin Columns方法纯化。 将上述基因片 段与 pBV220质粒 DNA进行连接， 16 °C反应 12小时， 转入大肠杆 菌 TG2受体细胞中。提取重组质粒 DNA，经 EcoRI和 BamHi双酶切， 走 1.2% Agarose凝胶电泳 , 确认 DNA基因片段的插入。 将含重组质 粒的 TG2 (pHK408)菌株， 接入 LB液体培养基中， 37 'C振荡培养过 夜， 离心取上清液， 点在人血纤维蛋白平皿上 37 °C保溫 16小吋， 产 生透明圈， 确认该菌株能够表达产生葡激酶。  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.
(7) 工程菌 TG2(pHK408)的高效表达：  (7) High-efficiency expression of engineering strain TG2 (pHK408):
pH 408质粒转入不同的大肠杆菌菌抹，如 JM109 、 DH5a 、 TGI 或 TG2均有较高的表达水平。  The pH 408 plasmid was transferred into different E. coli strains, such as JM109, DH5a, TGI or TG2 with higher expression levels.
工程菌 TG2(pHK408) ,保藏号为 CGMCC NO.0333，接种于 50ml PY 培养基中（1000ml 、 16g 胰蛋白胨， 5g 酵母粉， 5g NaCl ， PH7.2)30 °C振荡培养 6小时， 立即升温至 42 °C继续振荡培养 5-6小 时，离心收集菌体。菌体悬浮在 5ml TSE溶液中（20mM Tns-HCl-50mM NaCl-10iTiM EDTA pH8.0) , 用 JY92- Π型超声波细胞粉碎机进行超声 处理， 离心收集上清液， 进行 SDS-PAGE电泳分析。 电泳完毕， 将电 泳凝胶板取下， 貼在预先制成的含有人纤溶酶原的纤维平板上， 拷贝 约 10-20分钟后， 取出电泳凝胶进行考马斯亮兰染色。 纤维平板室温 放置或 37 °C保温约 20分钟， 即在葡激酶处出现透明区帝。  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. After electrophoresis, remove the electrophoresis gel plate and paste it on a pre-made fiber plate containing human plasminogen. After copying for about 10-20 minutes, remove the electrophoresis gel for Coomassie blue staining. Place the fiber plate at room temperature or incubate at 37 ° C for about 20 minutes, that is, a transparent area appears at the staphylokinase.
为了考察工程菌 TG2(pHK408)表达产物 Sak在大肠杆菌可溶蛋白 ό 比例， 超声波细胞破碎上清液， 走 SDS-PAGE电泳。考马斯亮兰染 色， 褪色至背景清晰后 , 用 Pharmacia Image Master Software and Sharp J x 330 Scanner扫描仅进行凝胶扫描， 如图 10所示。 图 10中： 1, 9 为： 细胞色素 C(W.M 13370) 2， 10为： RNase A(W.M. 15000); 3为： 未 诱导 TG2 (pHK408)全细胞裂解液； 4为： 未诱导 TG2 (pHK408)细胞超 声破碎上清液； 5为： 未诱导 TG2 (pHK408)细胞超声破碎沉淀； 6为： 诱导的 TG2 (pHK408)全细胞裂解液； 7为： 诱导 TG2 (pHK408)细胞超 声破碎上清液； 8为： 诱导 TG2 (pHK408)细胞超声破碎沉淀。 葡激酶 区带在细胞破碎上清液总蛋白各区带中占 40%以上。 对图 10进一步 的分析结果列于表 3 。 实施例二 In order to examine the proportion of soluble protein Sak expressed in engineered bacteria TG2 (pHK408) in E. coli, the supernatant was disrupted by ultrasonic wave and subjected to SDS-PAGE electrophoresis. Coomassie bright blue staining, fade to clear background, use Pharmacia Image Master Software and Sharp The J x 330 Scanner scan performs gel scanning only, as shown in Figure 10. In Figure 10: 1, 9 are: Cytochrome C (WM 13370) 2, 10 are: RNase A (WM 15000); 3 is: Uninduced TG2 (pHK408) whole cell lysate; 4 is: Uninduced TG2 (pHK408) ) Cell sonication of the supernatant; 5 is: TG2 (pHK408) cells are not induced by sonication; 6 is: TG2 (pHK408) is induced by whole cell lysate; 7 is: TG2 (pHK408) is induced by sonication 8 is: Inducing TG2 (pHK408) cells to ultrasonically disrupt the pellet. The staphylokinase bands accounted for more than 40% of the total protein bands in the cell disrupted supernatant. The results of further analysis of Figure 10 are listed in Table 3. Example two
用工程菌 TG2 pHK408)发酵制备重組葡激酶。 Recombinant staphylokinase was prepared by fermentation with engineered bacteria TG2 (pHK408).
1、 发酵  1.fermentation
将 100ml工程菌 TG2(pHK408)接入 1.5L发酵罐，用无机盐培养液 培养诱导 20小时 O.D.600值为 42时放罐， 离心收集菌体，超声破碎， 收集上清液即为粗酶液。  Put 100ml of engineering bacteria TG2 (pHK408) into a 1.5L fermentation tank, incubate with an inorganic salt culture medium for 20 hours, and release the tank when the OD600 value is 42. Centrifuge to collect the bacteria, sonicate, and collect the supernatant as the crude enzyme .
2、 纯化  2. Purification
将粗酶液经 SP-Sepharose F F 柱（φ45 x 200mm)及 Phenyl- Sepharose F F柱 (φ45 x 200i丽)及 Sephacryl S-300((j)26 x 800議）三步 纯化得到精制酶液， 经浓缩、 除菌得到重组葡激酶 970mg 。  The crude enzyme solution was purified in three steps by SP-Sepharose FF column (φ45 x 200mm) and Phenyl- Sepharose FF column (φ45 x 200i) and Sephacryl S-300 ((j) 26 x 800). Concentrated and sterilized to obtain 970mg of recombinant staphylokinase.
3、 检验 3. Inspection
将得到的重組葡激酶样品用 SDS-PAGE和 HPLC检测，纯度分别 为 96 %和 100 %， 血纤维蛋白平板測定比活力为 6.5 X 104AU/mg蛋 白（相当于 1〜 1.2 X 105HU/mg蛋白） 。质谱及 SDS-PAGE測得 MW 为 15.4kD 。 N- 末 端 氨 基 酸 測 得 结 果 与 理 论 值 （N- SSSFDKGKYKKGDDA)相同。 . 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). .
表 3: 扫描分析结果 Table 3: Scan analysis results
Lane 1 Lane 1
Band // Rf Mol. Wt. Pcok Trace Relative Contour Quantity Bond Name  Band // Rf Mol. Wt. Pcok Trace Relative Contour Quantity Bond Name
KDa OD ODxmm Percent ODxmm  KDa OD ODxmm Percent ODxmm
1-1 0.772 0.45 1.100 97.3  1-1 0.772 0.45 1.100 97.3
1-2 0.830 0.05 0.036 2.7  1-2 0.830 0.05 0.036 2.7
Lane 2  Lane 2
Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name da OD ODxmm Percent ODxmm  Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name da OD ODxmm Percent ODxmm
2-1 0.298 0.03 0.040 0.45  2-1 0.298 0.03 0.040 0.45
2-2 0.389 0.15 0.229 0.6  2-2 0.389 0.15 0.229 0.6
2-3 0.585 0.08 0.108 0.6  2-3 0.585 0.08 0.108 0.6
2-4 0.738 0.74 98.2  2-4 0.738 0.74 98.2
Band * Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name Band * Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name
Kda OD ODxmm Percent ODxmm  Kda OD ODxmm Percent ODxmm
3-1 0.036 0.22 0.147 8.8  3-1 0.036 0.22 0.147 8.8
3-2 0.073 0.06 0.044 2.8  3-2 0.073 0.06 0.044 2.8
3-3 0.135 0.11 0.130 8.4  3-3 0.135 0.11 0.130 8.4
3-4 0.204 0.24 0.192 12.6  3-4 0.204 0.24 0.192 12.6
3-5 0.265 0.10 0.118 8.1  3-5 0.265 0.10 0.118 8.1
3-6 0.327 0.11 0.076 4.8  3-6 0.327 0.11 0.076 4.8
3-7 0.375 0.14 0.218 14.2  3-7 0.375 0.14 0.218 14.2
3-8 0.571 0.06 0.037 2.2  3-8 0.571 0.06 0.037 2.2
3-9 0.647 0.04 0.028 1.7  3-9 0.647 0.04 0.028 1.7
3-10 0.727 0.35 0.519 31.3  3-10 0.727 0.35 0.519 31.3
3-11 0.844 0.02 0.047 2.8  3-11 0.844 0.02 0.047 2.8
Lane4  Lane4
Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name  Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name
Kda OD ODxmm Percent ODxmm  Kda OD ODxmm Percent ODxmm
4-1 0.044 0.25 0.133 5.6  4-1 0.044 0.25 0.133 5.6
4-2 0.080 0.12 0.129 5.4  4-2 0.080 0.12 0.129 5.4
4-3 0.138 0.14 0.172 . 8.3  4-3 0.138 0.14 0.172. 8.3
4-4 0.207 0.29 0.219 9.3  4-4 0.207 0.29 0.219 9.3
4-5 0.276 0.10 0.103 5.4  4-5 0.276 0.10 0.103 5.4
4-6 0.331 0.10 0.063 3.6  4-6 0.331 0.10 0.063 3.6
4-7 0.378 0.14 0.244 10.3  4-7 0.378 0.14 0.244 10.3
4-8 0.527 0.03 0.024 1.0  4-8 0.527 0.03 0.024 1.0
4-9 0.593 0.07 0.079 3.3  4-9 0.593 0.07 0.079 3.3
4-10 0.644 0.10 0.085 3.6  4-10 0.644 0.10 0.085 3.6
4-11 0.727 0.45 0.921 42.9  4-11 0.727 0.45 0.921 42.9
4-12 0.858 0.07 0.107 4.5 Lane5 4-12 0.858 0.07 0.107 4.5 Lane5
Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name kda OD ODxnim Percent OD xmm Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name kda OD OD x nim Percent OD xmm
5-1 0.044 0.24 0.148 9.5  5-1 0.044 0.24 0.148 9.5
5-2 0.081 0.06 0.052 3.3  5-2 0.081 0.06 0.052 3.3
5-3 0.143 0.15 0.197 12.6  5-3 0.143 0.15 0.197 12.6
5-4 0.212 0.30 0.244 15.6  5-4 0.212 0.30 0.244 15.6
5-5 0.278 0.12 0.156 9.9  5-5 0.278 0.12 0.156 9.9
5-6 0.337 0.12 0.079 5.0  5-6 0.337 0.12 0.079 5.0
5-7 0.385 0.12 0.232 14.8  5-7 0.385 0.12 0.232 14.8
5-8 0.557 0.09 0.082 6.3  5-8 0.557 0.09 0.082 6.3
5-9 0.568 0.06 0.097 7.2  5-9 0.568 0.06 0.097 7.2
5-10 0.718 0.04 0.056 3.9  5-10 0.718 0.04 0.056 3.9
5-11 0.766 0.08 0.115 7.3  5-11 0.766 0.08 0.115 7.3
5-12 0.861 0.03 0.061 3.9  5-12 0.861 0.03 0.061 3.9
Lane6  Lane6
Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name  Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name
Kda OD ODxmm Percent ODxmm  Kda OD ODxmm Percent ODxmm
6-1 0.044 0.19 0.136 3.2  6-1 0.044 0.19 0.136 3.2
6-2 0.088 0.1 1 0. Π0 2.6  6-2 0.088 0.1 1 0. Π0 2.6
6-3 0.169 0.16 0.251 5.9  6-3 0.169 0.16 0.251 5.9
6-4 0.210 0.26 0.206 4.9  6-4 0.210 0.26 0.206 4.9
6-5 0.276 0.12 0.139 4.3  6-5 0.276 0.12 0.139 4.3
6-6 0.335 0.12 0.105 3.5  6-6 0.335 0.12 0.105 3.5
6-7 0.386 0.20 0.367 8.6  6-7 0.386 0.20 0.367 8.6
6-8 0.529 0.07 0.090 2.1  6-8 0.529 0.07 0.090 2.1
6-9 0.596 0.06 0.049 1.1  6-9 0.596 0.06 0.049 1.1
6-10 0.654 0.33 0.497 Π .7  6-10 0.654 0.33 0.497 Π .7
6-11 0.754 0.85 1.976 46.5  6-11 0.754 0.85 1.976 46.5
6-12 0.853 0.09 0.214 5.0  6-12 0.853 0.09 0.214 5.0
Lane7  Lane7
Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name  Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name
Kda OD ODxmm Percent ODxmm  Kda OD ODxmm Percent ODxmm
7-1 0.044 0.15 0.078 1.2  7-1 0.044 0.15 0.078 1.2
7-2 0.088 0.10 0.098 1.6  7-2 0.088 0.10 0.098 1.6
7-3 0.173 0.19 0.183 4.5  7-3 0.173 0.19 0.183 4.5
7-4 0.210 0.29 0.235 4.7  7-4 0.210 0.29 0.235 4.7
7-5 0.279 0.14 0.123 3.2  7-5 0.279 0.14 0.123 3.2
7-6 0.335 0.13 0.116 3.7  7-6 0.335 0.13 0.116 3.7
7-7 0.386 0.21 0.310 5.7  7-7 0.386 0.21 0.310 5.7
7-8 0.533 0.06 0.066 2.1  7-8 0.533 0.06 0.066 2.1
7-9 0.654 0.42 0.726 17.9  7-9 0.654 0.42 0.726 17.9
7-10 0.768 0.91 2.582 46.9  7-10 0.768 0.91 2.582 46.9
7-11 0.849 0.06 0.113 8.3 Lane8 7-11 0.849 0.06 0.113 8.3 Lane8
Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name  Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name
KDa OD ODxmm Percent ODxmm  KDa OD ODxmm Percent ODxmm
8-1 0.048 0.26 U.156 9.4  8-1 0.048 0.26 U.156 9.4
8-2 0.096 0.14 0.150 9.0  8-2 0.096 0.14 0.150 9.0
8-3 0.151 0.15 0.159 9.6  8-3 0.151 0.15 0.159 9.6
8-4 0.217 0.31 0.280 16.8  8-4 0.217 0.31 0.280 16.8
8-5 0.290 0.13 0.142 8.5  8-5 0.290 0.13 0.142 8.5
8-6 0.342 0.12 0.097 6.8  8-6 0.342 0.12 0.097 6.8
8-7 0.393 0.16 0.225 14.5  8-7 0.393 0.16 0.225 14.5
8-8 0.460 0.06 0.073 5.4  8-8 0.460 0.06 0.073 5.4
8-9 0.562 0.06 0.094 6.6  8-9 0.562 0.06 0.094 6.6
8-10 0.614 0.07 0.063 3.8  8-10 0.614 0.07 0.063 3.8
8-1 1 0.783 0.06 0.107 6.4  8-1 1 0.783 0.06 0.107 6.4
8-12 0.871 0.04 0.047 2.8  8-12 0.871 0.04 0.047 2.8
Lane9  Lane9
Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name  Band # Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name
Kda OD ODxmm Percent ODxmm  Kda OD ODxmm Percent ODxmm
9-1 0.801 0.44 1.079 99.2  9-1 0.801 0.44 1.079 99.2
Lane 10  Lane 10
Band * Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name  Band * Rf Mol. Wt. Peak Trace Relative Contour Quantity Band Name
Kda OD ODxmm Percent ODxmm  Kda OD ODxmm Percent ODxmm
10-1 0.413 0.06 0.058 6.9  10-1 0.413 0.06 0.058 6.9
10-2 0.594 0.04 0.056 6.6  10-2 0.594 0.04 0.056 6.6
10-3 0.771 0.31 0.539 86.8 10-3 0.771 0.31 0.539 86.8
序列编目 一般信息： Sequence Cataloging General Information:
(i)申请者：  (i) Applicants:
(A)姓名： 白寒三 （青岛双龙制药有限公司）  (A) Name: Bai Hansan (Qingdao Ssangyong Pharmaceutical Co., Ltd.)
(B)街道： 青岛经济技术开发区丼岗山路 6号  (B) Street: No. 6 Yangangshan Road, Qingdao Economic and Technological Development Zone
(C)城市： 山东省青岛市  (C) City: Qingdao City, Shandong Province
(D)国家: 中国  (D) Country: China
(E)邮編： 266555  (E) Postcode: 266555
(F)电话： 0532 - 6897888  (F) Phone: 0532-6897888
(G)传真 : 0532 - 6895348  (G) Fax: 0532-6895348
(ii)发明题目 ： 重组葡激酶及其高表达工程菌株  (ii) Title of the invention: Recombinant staphylokinase and its highly expressed engineering strain
(2)编号 1序列信息 (2) No. 1 sequence information
(i)序列特征：  (i) Sequence characteristics:
(A)长度： 136个氨基酸  (A) Length: 136 amino acids
(B)类型： 氨基酸  (B) Type: Amino acid
(C)拓朴结抅： 线性  (C) Topology: Linear
(i i)分子类型： 蛋白质  (i i) molecular type: protein
(ii i)特点： (ii i) Features:
(A)名称 /关键： 氨基酸  (A) Name / Key: Amino acid
(B)位置： 第 34位为甘氨酸  Position (B): Glycine at position 34
(C)其它信息： 不同于 Sak Star (丝氨酸）  (C) Other information: different from Sak Star
(iii)特点：  (iii) Features:
(A)名称 /关键： 氨基酸  (A) Name / Key: Amino acid
(B)位置： 第 36位为甘氨酸  Position (B): Glycine at position 36
(C)其它信息： 不同于 Sak 42D和 Sak SY (二者均为精氨酸） (iii)特点：  (C) Other information: Different from Sak 42D and Sak SY (both are arginine) (iii) Features:
(A)名称 /关键： 氨基酸  (A) Name / Key: Amino acid
(B)位置： 第 43位为精氨酸 (C)其它信息：不同于 Sak Star和 Sak S-29 (二者均为組氨酸） (iii)特点： Position (B): Arginine at position 43 (C) Other information: Different from Sak Star and Sak S-29 (both are histidine) (iii) Features:
(A)名称 /关键： 氨基酸  (A) Name / Key: Amino acid
(B)位置： 第 92位为酪氨酸  Position (B): Tyrosine at position 92
(C)其它信息： 不同于 SakS-29 (苯丙氨酸）  (C) Other information: different from SakS-29 (phenylalanine)
(iii)特点：  (iii) Features:
(A)名称 /关键： 氨基酸  (A) Name / Key: Amino acid
(B)位置： 第 133位为甘氨酸  Position (B): Glycine at position 133
(C)其它信息： 不同于 SakS-29 (精氨酸）  (C) Other information: Different from SakS-29 (Arginine)
(3)编号 2序列信息 (3) No. 2 sequence information
(i)序列特征：  (i) Sequence characteristics:
(A)长度： 408个核苷酸  (A) Length: 408 nucleotides
(B)类型： 核苷酸  (B) Type: Nucleotide
(C)拓朴结抅： 线性  (C) Topology: Linear
(ii)分子类型： 脱氧核糖核酸  (ii) Molecular type: DNA
(iii)特点： (iii) Features:
(A)名称 /关键： 核苷酸  (A) Name / Key: Nucleotide
(B)位置： 第 100位、 105位和 128位为鸟苷酸， 第 399位为 胸月象嘧啶核苷酸  Position (B): Positions 100, 105, and 128 are guanylic acid, and position 399 is thoracic pyrimidine nucleotides
(C)其它信息： 不同于 SakStar , 这四个位置均为腺苷酸； (iii)特点：  (C) Other information: Unlike SakStar, all four positions are adenylate; (iii) Features:
(A)名称 /关键： 核苷酸  (A) Name / Key: Nucleotide
(B)位置： 第 105位、 106位为鸟苷酸， 第 399位为胸腺嘧 核苷酸  Position (B): Positions 105, 106 are guanylic acid, and position 399 is thymidine
(C)其它信息： Sak42D第 105 、 106和 399均为腺苷酸； (iii)特点：  (C) Other information: Sak42D 105, 106 and 399 are adenylate; (iii) Features:
(A)名称 /关键： 核苷酸  (A) Name / Key: Nucleotide
(B)位置： 第 33位为腺苷酸； 第 105位和 128位为鸟苷酸, 第 399位为胸腺嘧啶核苷酸 (C)其它信息： SakS(|)C第 33位为鸟苷酸， 第 105位、 128 位和 399位均为腺苷酸； (B) Position: Adenosine at position 33; Guanylic acid at positions 105 and 128; Thymine nucleotide at position 399 (C) Other information: SakS (|) C is guanylic acid at position 33, and adenylic acid is at positions 105, 128, and 399;
(iii)特点： (iii) Features:
(A)名称 /关键： 核苷酸  (A) Name / Key: Nucleotide
(B)位置： 第 105和 128位为鸟苷酸， 第 275位为腺苷酸， 第 398和 399位为胸腺嘧啶核苷酸  Position (B): Guanylic acid at positions 105 and 128, adenylate at position 275, and thymine nucleotides at positions 398 and 399
(C)其它信息： Sak S-29第 105 、 128 、 399位为腺苷酸， 第 275位为胸腺嘧啶核苷酸， 第 398位为鸟苷酸； (C) Other information: Sak S-29 is adenylate at positions 105, 128, and 399, thymidine at position 275, and guanylate at position 398;
(iii)特点： (iii) Features:
(A)名称 /关键： 核苷酸  (A) Name / Key: Nucleotide
(B)位置： 第 105位和 106位为鸟苷酸， 第 346和 399位为 胸腺嘧啶核苷酸  Position (B): Guanylic acid at positions 105 and 106 and thymidine at positions 346 and 399
(C)其它信息： Sak SY第 105 、 106 、 346和 399位均为腺 苷酸； (C) Other information: Sak SY positions 105, 106, 346 and 399 are adenylate;
(iii)序列说明 （ Sequence description ) (iii) Sequence description
序列号 （ Sequence II> No.) ： 1 Sequence number (Sequence II> No.): 1
Seri Ser Ser Phe Asp5 Lys Gly Lys Tyr Lys'0 Lysn Gly Asp Asp Ala15 Ser Tyr Phe Glu Pro2o Thr2, Gly Pro Tyr Leu25 Met Val Asn Val Thr30 Seri Ser Ser Phe Asp 5 Lys Gly Lys Tyr Lys' 0 Lysn Gly Asp Asp Ala 15 Ser Tyr Phe Glu Pro 2 o Thr 2 , Gly Pro Tyr Leu 25 Met Val Asn Val Thr 30
GIy3i Val Asp Glv Lys35 Glv Asn Glu Leu Leu o GIy 3 i Val Asp Glv Lys 35 Glv Asn Glu Leu Leu o
34 36  34 36
Ser" Pro Tyr VaUs Glu Phe Pro He LyssoSer "Pro Tyr VaU s Glu Phe Pro He Lysso
Figure imgf000025_0001
Figure imgf000025_0001
Pro51 Gly Thr Thr Leu55 Thr Lys Glu Lys Ile6 Pro 51 Gly Thr Thr Leu 55 Thr Lys Glu Lys Ile 6
Glu i Tyr Tyr Val Glues Trp Ala Leu Asp Ala7 Glu i Tyr Tyr Val Glues Trp Ala Leu Asp Ala 7
Thr71 Ala Tyr Lys Glu7S Phe Arg Val Val Glug0 Thr 71 Ala Tyr Lys Glu 7S Phe Arg Val Val Glu g0
Leu8i Asp Pro Ser Alass Lys He Glu Val Thr90 Leu 8 i Asp Pro Ser Alass Lys He Glu Val Thr 90
Tyr91 Tyr Asp Lys Asn95 Lys Lys Lys Glu Glu Tyr 91 Tyr Asp Lys Asn 95 Lys Lys Lys Glu Glu
92  92
Thr101 Lys Ser Phe rows He Thr Glu Lys Gl i Thr 101 Lys Ser Phe rows He Thr Glu Lys Gl i
Phem Val Val Pro Asp115 Leu Ser Glu His Phe m Val Val Pro Asp 115 Leu Ser Glu His
Lysi2i Asn Pro Gly Phei2s Asn Leu lie Thr Lys 130 Lysi2i Asn Pro Gly Phei 2 s Asn Leu lie Thr Lys 130
Valisi Val lie Glu Lysi3S Lysis Valisi Val lie Glu Lysi 3S Lysis
133 133
(iii) A序列说明 （ Sequence ： ion ) ： (iii) A sequence description (Sequence: ion):
列号 （ Sequence ID No. ) ：  Column number (Sequence ID No.):
TCA AGT TCA TTC GAC AAA GGA AAA TAT AAA  TCA AGT TCA TTC GAC AAA GGA AAA TAT AAA
1  1
AAA GGC GAT GAC GCG AGT TAT TTT GAA CCA  AAA GGC GAT GAC GCG AGT TAT TTT GAA CCA
31 33  31 33
ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT  ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT
61  61
GGA GTT GAT GGT AAG GGA AAT GAA TTG CTA  GGA GTT GAT GGT AAG GGA AAT GAA TTG CTA
91 100 105 106  91 100 105 106
TCC CCT CGT TAT GTC GAG TTT CCT ATT AAA  TCC CCT CGT TAT GTC GAG TTT CCT ATT AAA
121 128  121 128
CCT GGG ACT ACA CTT ACA AAA GAA AAA ATT  CCT GGG ACT ACA CTT ACA AAA GAA AAA ATT
151  151
GAA TAC TAT GTC GAA TGG GCA TTA GAT GCG  GAA TAC TAT GTC GAA TGG GCA TTA GAT GCG
1S1  1S1
ACA GCA ΤΛΤ ΛΛΛ GAG TTT AGA GTA GTT GAA  ACA GCA ΤΛΤ ΛΛΛ GAG TTT AGA GTA GTT GAA
211  211
GAT CCA AGC GCA AAG ATC GAA GTC ACT  GAT CCA AGC GCA AAG ATC GAA GTC ACT
TAT TAT GAT AAG AAT AAG AAA AAA GAA GAA TAT TAT GAT AAG AAT AAG AAA AAA GAA GAA
271 275  271 275
ACG AAG TCT TTC CCT ATA ACA GAA AAA GGT  ACG AAG TCT TTC CCT ATA ACA GAA AAA GGT
301  301
TTT GTT GTC CCA GAT TTA TCA GAG CAT ATT  TTT GTT GTC CCA GAT TTA TCA GAG CAT ATT
331 346  331 346
AAA AAC CCT GGA TTC AAC TTA ATT ACA AAG  AAA AAC CCT GGA TTC AAC TTA ATT ACA AAG
361  361
GTT GTT GAA AAG AAA  GTT GTT GAA AAG AAA
391 391
参考文献 、 萨姆布鲁克等编著 《分子克隆——实验指南》 第二版， 1996 。 、 Sneath, P.H.A. et al. Bergey's manual of systematic bacteriology,References, Sambrook et al., Molecular Cloning-A Guide to Experiments, Second Edition, 1996. , Sneath, P.H.A. et al. Bergey's manual of systematic bacteriology,
1986. 、 Holt, J.G. Bergey's manual of determinative bacteriology, 8th, 1986的 方法。 1986., Holt, JG Bergey's manual of determinative bacteriology, 8 th, 1986 of the method.

Claims

杈 利 要 求 书 Request for Profit
1、 一种重組葡激酶， 其氨基酸序列为序列 1 。 1. A recombinant staphylokinase whose amino acid sequence is sequence 1.
2、 杈利要求 1 的重组葡激酶， 其中编码该酶的基因具有序列 2 的核苷酸序列。  2. The recombinant staphylokinase of claim 1, wherein the gene encoding the enzyme has the nucleotide sequence of sequence 2.
3、 一种载体， 含有编码杈利要求 1重组葡激酶的基因。  3. A vector containing a gene encoding a recombinant staphylokinase.
4、 杈利要求 3的载体， 选自 ： pUC19和 pBV220 。  4. The carrier of claim 3 is selected from: pUC19 and pBV220.
5、 杈利要求 4的载体， 为 pBV220 。  5. The carrier of requirement 4 is pBV220.
6、 一种宿主， 含有能表达杈利要求 1重组葡激酶的载体。  6. A host containing a vector capable of expressing recombinant staphylokinase.
7、 杈利要求 6的宿主为大肠杆菌。 7. The requirements for the benefits of 6 are E. coli.
8、 杈利要求 7 的宿主， 其中该大肠杆菌选自 C600 、 JM83 、 JM109 、 DH5 、 TGI 、 TG2和 MC1061 。  8. The host of claim 7, wherein the E. coli is selected from the group consisting of C600, JM83, JM109, DH5, TGI, TG2 and MC1061.
9、 杈利要求 8的宿主， 选自 DH5a 、 TGI和 TG2 。  9. The host of claim 8 is selected from DH5a, TGI and TG2.
10、 一种分离和纯化的产葡激酶的金葡菌 SL1.063 , 该葡激醇具 有序列 1的氨基酸序列， 其保藏号为 CGMCC No.0353 。  10. An isolated and purified staphylokinase-producing Staphylococcus aureus SL1.063, the glucosinol has the amino acid sequence of sequence 1 and its deposit number is CGMCC No.0353.
11、 一种工程菌 pHK408(TG2) , 生产杈利要求 1 的重組葡激酶， 其保藏号为 CGMCC No.0333 。  11. An engineering bacterium pHK408 (TG2), which produces recombinant staphylokinase of claim 1, and its accession number is CGMCC No. 0333.
12、 一种抅建生产葡激酶的工程菌的方法， 包括：  12. A method for constructing engineered bacteria producing staphylokinase, including:
1)筛选产葡激酶的金葡菌，  1) Screening S. aureus producing staphylokinase,
2)以该产葡激酶的金葡菌染色体 DNA为模板进行 PCR扩增， 所用的引物为：  2) PCR is performed using the staphylokinase-producing Staphylococcus aureus chromosomal DNA as a template. The primers used are:
引物 I  Primer I
5'-CGCGAATTCATGTCAAGTTCATTCGACAAAGG-3', 链长： 32 ； 引物 Π  5'-CGCGAATTCATGTCAAGTTCATTCGACAAAGG-3 ', chain length: 32; primer Π
S'-CGCGGATCCTATTTCTTTTCMTAACMCCTTTG-S1, 链长： 34 。S'-CGCGGATCCTATTTCTTTTCMTAACMCCTTTG-S 1 , chain length: 34.
3) 将扩增步骤所得的 DNA 片段引入一质粒， 该质粒选自 ： pUC19和 pBV220 ， 3) introducing the DNA fragment obtained in the amplification step into a plasmid selected from: pUC19 and pBV220,
4) 将该重组质粒转入宿主细胞， 该宿主为大肠杆菌。  4) The recombinant plasmid is transferred into a host cell, and the host is E. coli.
13、 杈利要求 12的方法， 其中该宿主为大肠杆菌 DH5ot 、 TG1 或 TG2菌株。  13. The method of claim 12, wherein the host is an E. coli DH5ot, TG1 or TG2 strain.
14、 一种药物组合物， 含有有效量的杈利要求 1 的重組葡激酶 及药用赋形剂。 14. A pharmaceutical composition containing an effective amount of recombinant staphylokinase according to claim 1 And medicinal excipients.
杈利要求 1 的重组葡激酶用于治疗心肌梗塞和血栓疾病。 杈利要求 1的重组葡激酶用于治疗动脉血栓。 Recombinant staphylokinase requires treatment of myocardial infarction and thrombotic diseases. Recombinant staphylokinase is required for the treatment of arterial thrombosis.
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