WO2009039704A1 - A method for producing human kallikrein 1 - Google Patents

Abstract

An isolated polynucleotide encoding human kallikrein 1, a method for producing the encoded kallikrein 1, a vector comprising the polynucleotide and a host cell comprising the vector or a host cell into which the polynucleotide is integrated are disclosed. The polynucleotide can highly express in the host cell. The purity of the human kallikrein 1 after purification is at least 98%.

Description

 Method for preparing human kallikrein 1

 Technical field

 The present invention is in the field of genetic engineering, and more particularly, the present invention relates to a polynucleotide encoding human kallikrein 1 and a method for large-scale expression of recombinant human kallikrein 1. Background technique

 hKl belongs to the peptidase class S1 family of serine proteases (or kallikrein subfamily). It specifically hydrolyzes peptide bonds between Met-Lys or Arg-Ser amino acid residues in low molecular weight or high molecular weight kininogen molecules, releasing Lys-bradykinin or vasodilating hormone, respectively. (Kallidin). The kinins released by these hydrolysis will regulate the body's blood pressure, electrolyte balance, inflammation and cell proliferation. For example, Chinese Patent No. CN02116783.4 discloses that hK1 can be used for the preparation of a medicament for the treatment and prevention of cerebral infarction.

 At present, there are two main methods for preparing human kallikrein-1: One method is to directly extract hK or hKl components from human urine or animal tissues, and the preparation cost is high and the quality of raw materials cannot be guaranteed. Another method is to express human kallikrein 1 in microorganisms or cells such as E. coli or yeast using genetic recombination techniques. However, the current gene recombination studies on human kallikrein 1 are mainly based on the expression of Pro-hKl or Pr-ro-hKl or Met-hKl, and when the direct secretion of hKl protein is secreted, the signal peptide sequence is found. Can not be removed smoothly. See, for example, Angermann, A. et al., "Cloning and expression of human salivary-gland kallikrein in E. coli". Biochem J 1989, 262 (3) ) : 787-793; Jing Wang, Julie Chao et al., "Purification and characterization of recombinant tissue kallikrein from E. coli and yeast." Biochem. J 1991, 276:63-71; Lu HS, Hsu Yr et al. "Preparation and characterization of human tissue kallikrein in E. coli: release of pro-kallikrein and Met-kallikeptin with no enzymatic activity Isolation and characterization of human tissue kallikrein produced in E. coli: biochemical comparison to the enzymatically inactive prokallikrein and methionyl kallikrein) Protein Expr Purif. 1996, 8(2):215- 26; Lu HS et al Human "Expression and characterization of human tissue kallikrein and kallikrein isoforms in CHO cells" (Purification and characteriza Ttion of human tissue prokallikrein and kallikrein isoforms expressed in Chinese hamster ovary cells) . Protein Expr. Purif. 1996, 8 (2): 227-37; Hedy Chan et al. "Expression and characterization of human tissue kallikrein mutants" ( Expression and Characterization of Human Tissue Kallikrein Variants.. Protein Expr & Purif. 1998, 12: 361-370. The existing recombinant production of rhK1 has the following disadvantages: (a) low expression yield (200 μg/L~30 mg/L); (b) recombinant rhK1 is unglycosylated or heterogeneous in degree of glycosylation or signal The peptide can not be naturally excised, requires additional protease digestion, and the product is not uniform. The molecular weight distribution range is large (usually 27~40kDa); (c) Due to low product yield, heterogeneity, complicated purification process and low effective yield.

 In summary, it is extremely difficult to prepare rhKl in large quantities in terms of yield, preparation cost, and activity. Summary of the invention

 It is an object of the present invention to provide an isolated polynucleotide encoding human kallikrein 1, i.e., hK1.

Another object of the present invention is to provide a large-scale acquisition of recombinant human kallikrein by a methanol yeast expression system. -1 (Recombinant Human Kallikrein 1, rhKl) method.

 In a first aspect of the invention, there is provided an isolated polynucleotide encoding human kallikrein 1, i.e., hK1, which has the nucleotide sequence set forth in SEQ ID NO: 1.

 In a second aspect of the invention, a vector is provided, the vector comprising the polynucleotide.

 In a third aspect of the invention, a host cell comprising the vector or the polynucleotide of the host cell in which the polynucleotide is integrated is provided.

 In another preferred embodiment, the host cell is a yeast. More preferably, the host cell is methanol yeast. In another preferred embodiment, the expression level of recombinant human kallikrein l (rhKl) expressed by the host cell is 1. 3~

2. 7 g / L fermentation broth, more preferably 2. 2 ~ 3g / L fermentation broth.

 In another preferred embodiment, the specific activity of the expressed rhK1 is not less than 5. 2 AU/mg.

 In a third aspect of the invention, there is provided a method of preparing human kallikrein 1, hK1, comprising the steps of:

(a) cultivating said host cell to express rhKl;

 (b) separating or purifying rhKl from the culture.

 In another preferred embodiment, the whole salt medium is used for the fermentation in the step (a), and the pH of the induction phase is 6. 0 ± 0. 5 (preferably 60 ± 0.2), and the temperature is 25. ± 5 ° C, induction time 65 ± 15 hours (preferably, induction time is 65 ± 10 hours, more preferably, induction time is 65 ± 7 hours).

 In another preferred embodiment, purification is carried out in step (b) using a method selected from the group consisting of hydrophobic chromatography, anion exchange chromatography and gel filtration chromatography.

In another preferred embodiment, the step (b) comprises: sequentially performing hydrophobic chromatography, Ni ²⁺ and Cu ²⁺ chelate chromatography, and anion exchange chromatography to obtain purified hK1.

In another preferred embodiment, the purified hK1 has a purity greater than 95%, more preferably greater than 98%, and most preferably greater than 99%. In another preferred embodiment, the hydrophobic chromatography is performed using a Phenyl Sepharose 6 FF (HS) filler, and the Ni ²⁺ and Cu ²⁺ chelate chromatography is performed using a Chelating Sepharose FF filler, the anion exchange chromatography. Q Sepharose FF packing was used.

 In another preferred embodiment, in step (b):

(i) The treated fermentation broth was applied to a Phenyl Sepharose 6 FF chromatography cartridge under the conditions of: equilibration buffer of 20 mM Ι, Ι.ΟΜ Na ₂ SO ₄ , pH 7.0, elution of the target protein hKl Is 20mM PB, 0.23M Na ₂ SO ₄ , pH7.0);

(ii) chromatographically extracting the hK1 eluate obtained in step (i) with M ²⁺ -Chelating Sepharose FF, using 20 mM PB, pH 7.0 eluent to hKl; and/or upper Cu ²⁺ -Chelating Sepharose FF layer Analysis, elution of hKl with 20 mM PB, 10 mM imidazole, pH 7.0;

 (iii) The eluate obtained in the step (ii) was subjected to Q Sepharose FF chromatography, and eluted with 20 mM PB, 0.25 M NaCl, pH 7.0 to obtain a high-purity hK1 protein.

 In another preferred embodiment, the activity yield of hK1 after purification is higher than 22%.

In another preferred embodiment, the hK1 produced in the step (b) has the following characteristics: the molecular weight range is 28 to 32 kDa, all of the molecules are N-type sugar chain modifications of methanol yeast, and the sugars account for 8 of the entire glycoprotein. 8 ±1. 3。 The 2, the isoelectric point (pi) of the protein is 4. 2 ± 0. 3. In a fourth aspect of the invention, the use of the polynucleotide or the protein encoded thereby for the preparation of a medicament for preventing or treating cerebral infarction is provided.

 In another preferred embodiment, the medicament is further for: increasing cerebral blood flow in a mammal, reducing cerebral vascular resistance in a mammal, increasing blood flow in the neck and vertebral arteries, reducing vascular resistance, and increasing blood flow in the femoral artery.

 Other aspects of the invention will be apparent to those skilled in the art from this disclosure. DRAWINGS

 Figure 1 shows SDS-PAGE electrophoresis of reduced (R) and non-reduced (NR) induced expression in six high-resistant Zeocin clones screened on 24-well plates in vitro.

 Figure 2 shows the SDS-PAGE reduction electrophoresis pattern of rhKl produced in the 30L fermentation induction phase.

 Figure 3 shows the SDS-PAGE electrophoresis of Phenyl-S-printed harose FF chromatography (A) and Superdex 75 molecular sieve chromatography (B).

 Figure 4 3D Scheyl-Sepharose FF (HS), Chelating-Sepharose FF and Q_SepharoseFF chromatograms of reduction (R) and non-reduction (NR) SDS-PAGE of each target peak.

 Figure 5 shows the results of isoelectric point measurement of rhKl.

 Fig. 6 is a mass spectrum of different degree of glycosylation r-hKl purified by gel filtration chromatography, wherein r-hKl-A is a mass spectrum of rhKl with low molecular weight, and r-hKl-B is high molecular weight during electrophoresis. The mass spectrum of r_hKl.

 Figure 7 shows the SDS-PAGE electrophoresis (A) and mass spectrogram (B) of PNGaseF removal of N-glycosylation of two glycosylated rhKl.

 In addition, the molecular weight standard of each protein used in SDS-PAGE electrophoresis in the picture is LMW Marker Kit (Amersham Pharmacia Biotech), and the molecular weight of each band is from upper (large) to lower (small): 97. OkDa, 66. OkDa, 45. 0 kDa, 30. 0 kDa, 20. 1 kDa, 14. 4 kDa. detailed description

 After long-term research, the present inventors unexpectedly discovered a polynucleotide encoding human Kallikrein (hKl), which is a sequence of a polynucleotide encoding hK1. Differently, the expression of rhK1 in the host cell, especially methanol yeast, can obtain very high protein expression. The expression product has a good glycosylation modification mode, high specific activity, uniform product and easy purification. . The present invention also provides a method of expressing the rhK1, which is simple in process, easy to control expression conditions, and low in cost. The present invention has been completed on this basis.

 The present invention separates and obtains the polynucleotide from the human kidney cDNA library, and performs recombinant expression, directly obtaining a high expression amount of high activity protein secreted in the form of hKl, and the product is uniform, the purification process is simple, and the effective yield is high, The expression yield of rhKl can reach 2. 5mg/ml fermentation broth in 64 hours after fermentation induction; it has similar efficacy compared with the protein directly isolated from urine, but overcomes the animal tissue or human The direct extraction of hKl components in urine is costly and the quality of the raw materials cannot be guaranteed very well.

The singularity of the whole glycoprotein is 8. 8~17. 2% 5。 The isoelectric point (pi) of the protein is 4. 2 ± 0.3. As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .

 The term "about" refers to an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which depends in part on the measurement or measurement of the value, that is, on the limits of the measurement system. For example, in the present context, "about" can mean that the standard deviation of each practice is within 1 or exceeds 1. Alternatively, "about" when referring to the purity of the composition of the invention may mean the following range of specific values: preferably at ± 0.5%, more preferably at ± 0.25% and still more preferably at ± 0 . 1%.

 The term "kallikrein" refers to an alcohol-insoluble protease that hydrolyzes kininogens in the body to release kinins, which causes a decrease in blood pressure, including, but not limited to, urokininogenase, urinary kallikrein, and pancreatic Peptidase, pancreatic kallikrein, tissue kallikrein.

 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The polynucleotide of the present invention is preferably provided in an isolated form, more preferably purified to homogeneity.

 The polynucleotide of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

 Once the relevant sequences are obtained, the polynucleotide can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

 In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.

 At present, the polynucleotide sequence can be obtained completely by chemical synthesis. This sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

 A method of amplifying DNA/RNA by PCR technique (Saiki et al., Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method can be preferably used.

(RACE-cDNA end rapid amplification method), primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragment can be isolated and purified by a conventional method such as by gel electrophoresis.

 The invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered with the vectors of the invention or the polynucleotide sequences of the invention, and methods for producing rhKl by recombinant techniques.

 The polynucleotide sequence of the present invention can be used to express or produce rhK1 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally, the following steps are carried out: (1) using a polynucleotide of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide; (2) culturing in a suitable medium Host cells; (3). Isolation and purification of proteins from culture media or cells.

In the present invention, the polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to Bacterial plasmids, yeast plasmids or other vectors well known in the art. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element. As a preferred mode of the present invention, the recombinant expression vector is a vector suitable for expression in yeast or E. coli; and most preferred is a vector suitable for expression in methanol yeast.

 Vectors comprising appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells to enable expression of the protein.

 The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. In a preferred embodiment of the present invention, the host cell is selected from the group consisting of: yeast or Escherichia coli; more preferably, the host cell is methanol yeast. The polynucleotide of the present invention is introduced into a methanol yeast through a suitable vector, and the obtained recombinant methanol yeast is particularly suitable for expressing hK1, and the expression amount is high, and the rhK1 has a good glycosylation pattern and is excellent in biological activity.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl ₂ . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.

 The obtained transformants are cultured to express the polypeptide encoded by the polynucleotide of the present invention.

As a preferred mode of the present invention, the expressed protein is purified by hydrophobic chromatography, anion exchange chromatography, or gel filtration chromatography in sequence; or, hydrophobic chromatography, Ni ²⁺ and Cu ^{2+ are} sequentially used. Chelating chromatography, and anion exchange chromatography. The present inventors have found that a high purity protein can be obtained by the above purification method, and the biological activity of the protein can be well retained.

 After long-term experimental research, the present inventors obtained the gene sequence corresponding to the hKl mature protein by direct PCR amplification from the human kidney cDNA library through the synthesized primers. As a preferred embodiment of the present invention, the hK1 gene fragment obtained by PCR of the present invention is inserted into the pPICZ α Α vector to construct a pPICZ a -hK1 expression vector, which is linearized and transformed into a methanol yeast host strain X33, and finally realized. The expression of hK1 is secreted in methanol yeast. It was confirmed by measurement that the N-terminus of the recombinant product was completely identical to the naturally extracted hK1, and also the methanol yeast glycosylation modification mode. This is the recombinant human kallikrein-1 (rhKl).

 Further, the present invention provides the use of the polynucleotide of the present invention or a protein encoded thereby for the preparation of a medicament for preventing or treating cerebral infarction. The inventors found through animal experiments that rhKl can significantly reduce the area of cerebral infarction area and the weight of cerebral infarction area, which shows that it has a good therapeutic effect on cerebral infarction. Further animal studies have also found that rhKl can significantly increase cerebral blood flow in mammals and significantly reduce cerebral vascular resistance. Moreover, rhKl can significantly increase the blood flow in the neck and vertebral arteries, reduce vascular resistance, and exhibit significant vasodilatation; femoral artery flow also increases to varying degrees. The selectivity of rhKl to cerebral blood vessels is about 3 times stronger than that of peripheral blood vessels. Intravenous administration of rhKl has a tendency to lower blood pressure, especially in diastolic blood pressure, but has no significant effect on mammalian heart rate.

 The invention produces the protein drugs traditionally produced by the biological extraction method through modern bioengineering technology, which is a concentrated expression of technological progress in the field of biopharmaceuticals. The quality of the product can be fundamentally controlled to minimize contamination of the product by bacterial viruses of human and animal origin. Therefore, the advantages of the present invention for producing rhKl in methanol yeast are:

1 The production process is simple: the secretory expression of methanol yeast, using conventional hydrophobic, chelated, anion exchange chromatography media, High-purity rhKl (95%) can be purified from the fermentation supernatant on a large scale with low production costs. At present, hKl, which is routinely extracted from human urine, uses a self-crosslinking high-cost affinity chromatography medium, and its product uniformity is not high. At present, the stability study of rhKl's water-needle formulation is underway. It is preliminarily judged that the variety can be used in the form of water injection, which not only reduces the production components, but also makes the clinical use more convenient.

 2 The specific activity of the product is high: the expression of methanol yeast is secreted, rhKl has glycosylation modification, and the specific activity is high (5.2 AU/mg). The specific activity of the biological extract from urine is only 3~4AU/mg, and the difference between batches is obvious.

 3 The quality controllability of the product is good: The genetic engineering product has little dependence on the environment, has few external pollution sources, and can be well controlled, and the product quality is controllable. When extracted from the urine, the quality control of raw materials (urine) will be greatly affected by the seasons. At the same time, in practice, it is impossible to ensure that the urine of healthy people is collected, and the quality control is difficult.

The average total activity unit is calculated according to the specific activity of 5. 2 AU / mg, the specific activity unit is calculated according to the specific activity of 5. 2 AU / mg. There are 13 000 AU (2. 5 X 1, 000 X 5. 2 = 1, 3000) ₀ Refer to the similar products currently on the market, Kylikone® (Ulyclin for Injection), with a quantity of 0 per load. Calculated by 15 AU, then a pure batch of purified supernatant from a batch of 30 L fermenter can produce about 86,000 (13, 000 / 0.15 = 86, 666).

 5 The efficacy is better than or at least equivalent to extracting from the pancreas or human urine: The drug made by the pig's pancreas extract from the blood of Tianjin Pharmaceutical College and the Kaitekang of Guangzhou Tianpu Co., Ltd. Pharmacodynamic studies conducted in animals that are officially carried out in animals have shown that the efficacy is comparable when using the same dose. The acute, chronic and reproductive toxicity tests commissioned by the School of Pharmacy of the Second Military Medical University showed that rhKl is safe and non-toxic. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.

 Example 1 Construction of hKl engineering strain

 1. Acquisition and expression vector of hKl gene pPICZa-hKl construction

 Refer to the complete sequence information of hKl gene published in GeneBank, design and synthesize (Shanghai Shengong, Sangon) two primers that can amplify mature hKl gene Kal5 (5'-primer, SEQ ID NO: 3) Kal3 (3' - Primer, SEQ ID NO: 4): Kal5: 5. -cat etc gag aaa aga att gtg gga ggc tgg gag tgt gag-3.;

 Kal3: 5. -cat gcg gcc get tag gag ttc tec get atg gtg tec tc_3.;

 The hKl gene was obtained by PCR using Panomics' human kidney cDNA library (P/N: 7202, L/N: P0260602) as a template. The primer Kal5 can add the DNA restriction endonuclease Xhol site (CTCGAG) at the 5'-end of the hK1 gene, and the codon AAA AGA corresponding to the recognition sequence Lys-Arg of the Kex2 protease, which will ensure the expression of the inserted hK1 gene during secretion. The α_signal peptide can be successfully excised to obtain the hKl protein molecule with the natural Ν-terminal sequence; the primer Kal3 is added to the 3'-end stop codon TAA and the DNA restriction endonuclease Notl site (GCGGCCGC).

 PCR amplification was carried out using DNA polymerase (Pyrobest), and the system was as follows:

 Human kidney cDNA 1 μ 1

Kal5 (5, -primer) 2 μ 1 Kal3 (3' - Primer) 2 μ 1

 dNTP Mix 1 μ 1

 lO X Pyrobest Buffer 2 μ 1

 Pyrobest 0. 25 μ 1

 Dd3⁄40 U. 75 μ 1

 Total 20 μ 1

 PCR conditions: Denaturation at 94 °C for 3 minutes, followed by circulation at 94 °C, 30 seconds / 55 °C, 30 seconds / 72 °C, 1 minute. After the end of PCR, 2 μl of the amplified product was electrophoresed on 1.5% agarose, and a length of about 730 bp was obtained, indicating that the PCR was successful.

 The PCR product was purified by a column recovery kit (Sangon), and the PCR product recovered by the column was double-cleaved with restriction enzymes Xhol and Notl, and the hK1 gene was recovered by gelation. The pPICZ a A vector (Invitrogen) was double-cut with Xhol and Notl, and a large fragment of the vector was recovered as a vector for inserting the gene of interest, and the recovered hK1 gene fragment was ligated with the pPICZ a vector as follows:

 pPICZ a vector (Xhol- Notl) 2 μ 1

 hKl gene (Xhol- Notl) 4 μ 1

 10 X Ligation Buffer 1 μ 1

 T4 DNA Ligase (ligase) 0. 5 μ 1

 ddH20 2. 5 μ 1

 Total 10 μ 1

The reaction was carried out at 22 °C for about 1 hour. The above-mentioned ligation product 5 μ 1 was mixed with NovaBlue (Novagen) competent 100 μ 1 prepared by CaCl ₂ method, 4 ° C, 30 min, 42 ° C, heat. Shock for 90 seconds, put on ice for 3 to 5 minutes, then add 500 μ ΐ LB medium, incubate at 37 ° C for 200 minutes at 45 rpm, and take 100 μl of bacteria solution coated with LZ (LB+Ze _0C) In 25 μg/ml) + l. 5% agarose plates, overnight culture at 37 ° C, positive clones that were successfully transformed were grown. Six single colonies were randomly picked from 5 ml LZ medium, cultured overnight at 37 ° C, and plasmids were extracted with plasmid DNA extraction kit (BioAsia), and screened by Xhol and Notl double digestion. Cloning of the approximately 750 bp fragment was sequenced to confirm the sequence. The sequencing result is as SEQ ID NO: 1, and its corresponding amino acid sequence is SEQ ID NO: 2.

 2. Engineering strains obtained

The host cell competent cells of methanol yeast X-33 (purchased from Invitrogen) were prepared according to the method of Invitrogen Ρ·methanolica Expression Kit. The plasmid expression vector was linearized by Sacl digestion, and the protein was removed by phenol-chloroform extraction. The linearized vector after ion water dissolution was mixed with X33 competent cells, electrotransformed, and plated on a plate containing Zeocin 500 ug/ml YPD (1% yeast extract, 2% polypeptone, 2% glucose), 30 Incubate at °C for 2 to 3 days until a yeast single colony grows. According to the description in the Easyselect Pichia Expression Kit, the recombinants were first screened by increasing the resistance of the Zeocin antibiotics, and 100 recombinants were picked and seeded in a 24-well plate of 400 μl YPD (Zeocin 600 μg/ml) medium. Incubate overnight at 30 °C on a shaker, and screen out the high-resistant Zeocin wells (6 eligible clones) and select them for transfer to the tubes. Add 2 ml of YPD medium to continue the proliferation, and collect them 24 hours later. The cells were induced to express at 30 °C with methanol containing 0.5% BMMY medium, and methanol was added for 24 hours. The final concentration in the bacterial solution was 0.5%, and the induced state was maintained for 48 hours. The supernatant of the bacterial liquid was collected by centrifugation, and the comparison was made before and after the induction by SDS-PAGE electrophoresis, and it was found that there was a 30 kDa protein strip in the fermentation supernatant after the induction. Bring it out. Since it is difficult to accurately judge the expression yield by electrophoresis observation of the protein band type change, the pre-induction supernatant was used as a blank control, and the chromogenic substrate S-2266 was used to accurately determine the rhKl activity unit yield in the induced fermentation supernatant. Finally, one of the highly expressed hKl engineering strains was determined for fermentation and a seed bank was established. Figure 1 is a SDS-PAGE electrophoresis map of reduced (R) and non-reduced (NR) induced expression of six high anti-Zeocin clones screened in 24-well plates in a test tube; Lane 0 is the fermentation before induction of expression. Supernatants; Lanes 1 to 6 were 48-hour results for cloning induction of high anti-Zeocin resistance (500 μg/ml). Example 2 Fermentation of rhKl engineering bacteria

 Seed preparation

Take the working seed glycerol cryotube, melt and take 1ml to 500ml YPD medium (1% yeast powder, 2% peptone, 2% glucose), and incubate for 30 hours to 0D600 at 30 °C, 300rpm shaker. At 6. 0 ± 1. 0, the microscopic examination was performed to obtain the seed solution for inoculation. Prepare the basic salt medium BSM1 for fermentation (K ₂ S0 ₄ 60. 7 g, MgS0 ₄ 24. 2 g, CaS0 ₄ · 23⁄40 3. 9 g, H ₃ P0 ₄ 89 ml, K0H 13. 8 g, PTM1 14 ml 400 g of glycerin, 2 ml of foaming enemy, taking 10 L as an example, taking 1 liter of trace element medium PTM1 as an example, wherein the content of each component is: CuS0 ₄ ·5Η ₂ 0 6 · 0 g, Nal 0. 008 g, MnS0 ₄ 3 · 0 g, NaMo0 ₄ 0 · 2 g, H ₃ B0 ₃ 0 · 02 g, ZnS0 ₄ 20. 0 g, CoCl ₂ 0. 5 g, FeS0 ₄ · 7H ₂ 0 65. 0 g 2, 2 g, H ₂ S0 ₄ 5 ml, with water to a volume of 1 liter), and then sterilized in a can. Sterilization conditions were 121 ° C for 20 minutes and then cooled to 30 ° C later. Inoculate at a ratio of 1:15 (V/V, seed solution: base tank-based medium BSM1).

 2. Fermentation process

 The fermentation temperature of the initial cell proliferation stage is 30. 0 ± 0. 5 ° C, pH 5. 00 ± 0. 3, the initial rotation speed is 300 rpm, the ventilation is 0. 5wm, the dissolved oxygen (DO) value is 100%, and the PTM1 is added. . At this stage, about 20 hours, the dissolved oxygen value is decreasing, but the DO value should be maintained at no less than 20%. When the carbon source is consumed, the dissolved oxygen value rises rapidly, and the wet weight of the bacteria reaches about 100g/L. Enter the rate-limiting growth phase. During the first 2 hours of this phase, add 50% glycerol solution at a rate of 240 ml/hour and maintain a D0 value of not less than 20%. After 2 hours of feeding, the feed rate was increased to 360 ml/hour. The D0 value is maintained at a level of not less than 20% by adjusting the stirring speed, air flow rate, and tank pressure (<0.8 bar). After adding about 4 hours, when the wet weight of the cells is about 180 to 220 g/L, the feeding is stopped and the value of D0 rises. The increase in the value of D0 indicates that the carbon source in the medium is depleted, and the carbon source and the inducer methanol may be fed, which enters the critical stage of the fermentation to induce the expression phase: the pH is controlled to 6. 0 ± 0. 2 with ammonia water. , start adding methanol. The initial addition amount of methanol was controlled at 30 ml/hr, and the amount of methanol added was slowly increased, and the feed rate was set to 60 ml/hr after about 2 hours. After 4 hours of induction, the methanol feed rate was set to 120 ml/hr. At this time, methanol induction was successful, and the cells could use methanol as a carbon source at a normal high speed. Maintain D0 value not less than 20%, temperature 30 ° C, pH value of 6.0 ± 0.2, perform fermentation expression, take samples every 8 hours during the whole fermentation period, determine activity (Table 1) and SDS- PAGE electrophoresis, as shown in Figure 2. Figure 2 shows the SDS-PAGE reduction electrophoresis of rhK1 produced during the 30 L fermentation induction phase: samples were taken every 8 hours at the induction period, and the numbers below each lane indicate the number of hours induced by methanol. After the end of the fermentation process, the supernatant was collected by centrifugation, and the expression yield was measured by SDS-PAGE electrophoresis and S-2266 chromogenic substrate method.

In the whole fermentation induction stage, 1 ml of fermentation broth was used as the measurement standard, and the fermentation was measured at the induction stage of methanol. The wet weight of the bacteria in the liquid increased, the fermentation supernatant decreased, and the expression product rhKl protein increased significantly. It can be seen that the longer the induction time, the better, because the sampling will reduce the total volume of the fermentation broth during the fermentation process. The large increase of the bacteria will greatly reduce the supernatant and the long-term induction time will greatly increase the heteroprotein, and the rhKl degradation will increase. These factors then set the induction time of the fermentation to 64 to 72 hours.

 Table 1. Comparison of bacterial wet weight, supernatant volume and expression yield during fermentation induction

Example 3 Purification of rhKl protein

 Method I: Hydrophobic, anion exchange and gel filtration chromatography

 The purification process consists of three steps: hydrophobic, anion exchange and gel filtration chromatography. Hydrophobic chromatography selects Phenyl Sepharose FF, anion exchange medium is Q_S printed harose FF, and gel filtration chromatography uses Superdex75. The specific process is as follows:

 1. Pretreatment of fermentation broth

To the fermentation supernatant, 0. 2M PB, pH 6. 0 and 3. 0M (NH ₄ ) ₂ S0 ₄ solution to a final concentration in the fermentation broth of 20 mM and 1.0 M, respectively, adjusted pH to 6. 0, after standing for 1 hour, the 0. 45 micron filter is filtered to form a treated fermentation broth (sample solution) containing 20 mM PB, 1. 0M (NH ₄ ) ₂ S0 ₄ , pH 6.0. process.

 2. Phenyl-Sepharose FF hydrophobic chromatography

The above treated fermentation broth was applied to a Phenyl-S-printed harose FF column with a column type of Index 70/500, a bed volume of 500 ml, an equilibration buffer of 20 mM PB, 1. 0 M (NH ₄ ) ₂ S0 ₄ , pH 6 0。 The elution buffer is 20 mM PB, 0. 7M (NH ₄ ) ₂ S0 ₄ , pH 6. 0, 20 mM PB, 0. 4M (NH ₄ ) ₂ S0 ₄ , pH 6. 0 and 20 mM PB, pH 6. 0 , collect each elution peak. The target protein rhKl was mainly in the elution peak of 20 mM PB, pH 6.0 (Fig. 3A). Figure 3A is an SDS-PAGE electrophoresis of Phenyl_S-printed harose FF chromatography. The Phenyl-S-printed Harose FF chromatography medium was equilibrated with 20 mM PB, 1.0 M (NH ₄ ) ₂ S0 ₄ , pH 6.0 buffer; wherein: Lane 1 was 200 mM PB, pH 6.0 and 3M (NH ₄ ) ₂ S0 _{4 The} mother liquor is added to the fermentation broth, and the final concentration in the fermentation broth is 20 mM PB, 1.0 M (NH ₄ ) ₂ S0 ₄ , pH 6.0, and then filtered using a membrane of 0. 45 μm. Lane 2 is the effluent; Lane 3 is 20 mM PB, 0.7 M (NH ₄ ) ₂ S0 ₄ , elution peak at pH 6.0; Lane 4 is 20 mM PB, 0. 35M (NH ₄ ) ₂ S0 ₄ , pH 6 0 0 eluted peaks, wherein the molecular weight of the larger and slightly lower hKl each accounted for about 50%; Lane 5 was 20 mM PB, pH 6.0 elution peak. 3. Q-Sepharose FF anion exchange chromatography

 The hydrophobic chromatography elution main peak collection solution was adjusted to pH 7.5 with 1.0 M NaOH, diluted conductance 8. OmS/cm, upper QS printed harose FF anion exchange chromatography column, column size 5 X 10 cm, column bed The respective elution peaks were collected in a volume of 200 ml, an equilibration buffer of 20 mM PB, a pH of 7.5, an elution buffer of 20 mM PB, 0.2 M NaCl, pH 7.5 and 20 mM PB, 0.5 M NaCl, pH 7.5. The target protein rhKl is mainly in the elution peak of 20 mM PB, 0.5 M NaCl, pH 7.5.

 4. Superdex 75 gel filtration chromatography

The equilibrium and elution were carried out using 20 mM PB, 0.15 M NaCl, pH 7.5 buffer, and the anion exchange chromatography eluate was batched onto Superdex 75. The column size was 6. OX 60 cm prepacked column, and the bed volume was 1700ml, each time the sample loading is not more than 5% of the bed volume (about 85ml), the main peak is collected in stages, and the purity is determined by SDS-PAGE electrophoresis, and the parts that meet the requirements are combined. The filter is sterilized, which is the rhKl stock solution. Figure 3B is an SDS-PAGE electrophoresis of Superd _ex 75 molecular sieve chromatography purification process.

The balance and elution buffer of the Superdex 75 molecular sieve chromatography are 20 mM PB, 0.15 M NaCl, pH 7.5, and the elution peaks appearing at a volume of 0.5 V column are collected in stages, and are divided into 1, 2, 3, Sections 4, 5 and 6 have the largest molecular weight and a small amount. The actual molecular weight measured by mass spectrometry r-hKl-B is 32871. 16Da, the fourth segment is the main peak of hKl, the largest amount, and the mass spectrometry r-hKl-A determines its The actual molecular weight is 28975. 79 Dalton. Method II: Hydrophobic chromatography, Ni ²⁺ and Cu ²⁺ chelate chromatography, and anion exchange chromatography

 Purification according to Method I, the molecular sieve step is limited for large-scale production, the same result can be obtained according to the method, which is particularly advantageous for scale-up production of the process scale, wherein the second and third steps are adjusted and completely stabilized in the fermentation conditions. After the step can be omitted, if the two steps are carried out in sequence, the product purity is higher, and the fermentation broth can be used for different conditions, even the crude extract of biological tissues, and the hKl or hKl analog in the urine of human or animal. You can purify the extraction by following the steps below.

 First step, hydrophobic chromatography

Phenyl Sepharose 6FF (HS) filler was used. The equilibration buffer was 20 mM PB, 1.0 M Na ₂ SO ₄ , pH 7.0, and the elution buffer was 20 mM PB, 0.58 M Na ₂ S0 ₄ , pH 7.0, 20 mM PB, 0.23 M Na ₂ SO ₄ , pH 7. 0, water was injected into the column, and 20 mM PB, 0.23 M Na ₂ SO ₄ , pH 7.0 eluate was collected.

Second step, Ni ²⁺ metal chelate chromatography

The Chelating Sepharose FF filler was used. Hang M ²⁺ , equilibration buffer is 20 mM PB, 0.5 M NaCl, pH 7.0, elution buffer is 20 mM PB, pH 7.0, 50 mM EDTANa 3⁄4lM NaCl, pH 8.0, collect 20 mM PB, pH 7.0 eluate .

The third step, Cu ²⁺ metal chelate chromatography

The Chelating Sepharose FF filler was used. Cu ^{2+ was} suspended, the equilibration buffer was 20 mM PB, 0.15 M NaCl, pH 7.0, and the elution buffer was 20 mM PB, pH 7.0 and 20 mM PB, 10 mM imidazole, pH 7.0, 50 mM hydrazine, ΙΜ NaCl, pH 8 .0, 20 mM PB, 10 mM imidazole, pH 7.0 eluate was collected.

 The fourth step, anion chromatography

 Use Q Sepharose FF packing. The equilibration buffer was 20 mM PB, pH 7.0, and the elution buffer was 20 mM PB, 0.15 M NaCl, pH 7.0 20 mM PB, 0.25 M NaCl, pH 7.0, and collected 20 mM PB, 0.25 M NaCl, pH 7.0. Deliquoring.

Purification (R) and non-reduction (NR) SDS-PAGE electrophoresis of each target peak in the whole process of purification according to the above process The results are shown in Figure 4, in which lane 1 is the pretreated fermentation supernatant, lane 2 is the target eluate for Phenyl Sepharose FF (HS) chromatography, and lane 2 is the destination eluate for Ni ²⁺ _Chelating Sepharose FF chromatography. Lane 4 is the target eluate for Cu ²⁺ -Chelating Sepharose FF chromatography, and lane 5 is the desired eluate (stock solution) for QS-printed harose FF chromatography.

 The purification process was verified three times in a batch of fermentation supernatant, and the results are shown in Table 2.

 Table 2

 * The average activity of the average activity was 22.3%, and the average specific activity of the target protein rhKl was 5.2 AU/mg. Example 4 confirming the nature of rhKl

 1. rhKl specific activity assay

 1. 1 Determination of the concentration of rhKl

 The purified rhKl was subjected to protein purity (SDS-PAGE electrophoresis, RP-HPLC), and the concentration of rhKl having a purity of not less than 98% was accurately determined by the Lorry method.

 1. 2 Determination of the number of active units (AU) using S-2266 (Chromogenix) chromogenic substrate

The sample to be tested is diluted with a 20 mM Tris-Cl, pH 8.0 buffer to a series of multiples, such as 100, 200, 300, etc., based on the estimated concentration of the sample to be tested. Add 400 μ ΐ 0. 2Μ ρΗ8. 0 Tris-Cl buffer to a clean 2ml vial, add 20 μl of the diluted sample solution, and add 20 μl of the dilution buffer to the control. The temperature of the solution in each measuring tube was kept constant at 37 °C for 5 minutes in a water bath, and then 40 μl of the chromogenic substrate S-2266 was added, mixed, accurately timed, and reacted in a water bath at 37 ° C for 15 minutes. Then, the reaction sample was added with 40 μl of 50% acetic acid solution to terminate the reaction, and the spectrophotometer was adjusted to zero by the control, and the absorption value Α ⁴ ° ⁵ was measured at a wavelength of 405 nm, and the value of Α ⁴ ° ⁵ should be 0.1. Between ~0. 2, when not in this range, increase or decrease the dilution factor and repeat. The free ρΝΑ Α ⁴ ° ⁵ molar extinction coefficient is 9,600 L mol - ¹ cm - ¹ , according to the meaning of hKl activity unit: 1AU means at 37 ° C, Tris-Cl, pH 8.0 buffer system, 1 The amount of enzyme that can hydrolyze the substrate S-2266 to release Ι μ πιοΐ free ρ 分钟 in a minute is an hKl activity unit (AU). According to Beer-Lambert's law, the unit of hKl activity per ml of sample to be tested can be calculated as follows: AU/ml = 0. 1736 XA ⁴⁰⁵ X dilution factor.

The determination of hKl activity by S-2266 chromogenic substrate is the basis of the whole research work. This method can be used to accurately and accurately quantify hKl activity in the sample (fermentation solution, stock solution, purified intermediate sample, etc.). 5 μg/ml (equivalent to 0. 017~) 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 In the range of 0. 034PNAU/ml), this will reduce the dilution of the sample. The amount of work is easy to get results quickly.

 Using the activity unit (AU) / protein concentration (mg), the specific activity of rhKl can be obtained, and the actual result is about 5. 2 AU/mg.

2. Mass spectrometry and N-terminal and C-terminal sequencing of rhKl protein

 The rhKl protein was collected by gel filtration chromatography and the molecular weight was low and the molecular weight was high. The two groups were entrusted to the Proteomics Research Center of the Institute of Biochemistry and Cell Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. N-terminal sequence analysis showed that although there were differences in molecular weight between the two, there was no significant difference in the in vitro activity of the S-2266 chromogenic substrate method, and the N-terminal sequences of their polypeptide chains were completely identical. The first 15 amino acid residues at the N-terminus are: I le-Val-Gly-Gly-Trp-Glu-Cys-Glu-Gln-His-Ser-Gln-Pro-Trp-Gln. The mass spectrum of each peptide was determined by trypsin digestion of rhKl. The 12-amino acid residue at the carboxyl end was determined by mass spectrometry to be -Val-Lys-Trp-I le-Glu-Asp-Thr-I le-Ala-Glu. -Asn-Ser. This indicates that we use the methanol yeast system to secrete rhK1, but there is no N-terminal heterogeneity described by previous researchers. Electrophoresis shows that the molecular heterogeneity is caused by different degrees of glycosylation, but the sugar can be separated by purification. RhKl protein molecules with different degrees of basement.

 3. Determination of isoelectric point of rhKl protein

The isoelectric point of rhKl protein was determined by isoelectric focusing method. The pi standard protein used was GE Pharma Life Science's Broad pi Kit pH 3~10. The pi of high glycosylation and low glycosylation modified rhK1 protein was not significantly different. In the intermediate region of the pi standard protein 3. 9~4. 5 position, as shown in Fig. 5, Fig. 5 shows the isoelectric point measurement result of rhKl, and the bands in the lane M (Br ₀ ad pi Kit) are from the top. The next step is Amylglucosidase (3. 50), Trypsin inhibitor (4. 55), β-lactoglobulin a (β-Lactoglobul in a) (5. 20), cattle. Bovine carbonic anhydrase b (5. 85), Human carbonic anhydrase b (6.55), Acidoglobin (acid band) (6.85), Alkali Myoglobin (basic band) (7. 35), Lenti l lectin, acidic (8. 15), Lenti l lectin (middle) (8. 45) , Lenti lectin, basic (8. 65), Trypsinogen (9. 30). Lane r-hKl-A is a protein molecule with a low degree of glycosylation; lane r-hKl-B is a protein molecule with a high degree of glycosylation. 2。。 The pi is about 4. 2 ± 0.3.

 4. Deglycosylation test of rhKl protein

 Theoretically, there are three possible N-type glycosylation sites in the amino acid sequence of hKl protein. The molecular weight calculated from the amino acid sequence should be 26405. 74Dalton, while the actually purified rhKl has a high molecular weight and a slightly lower molecular weight. The two kinds can be seen by SDS-PAGE electrophoresis (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 7A). More precisely, the molecular weight of the purified rhKl protein is determined by mass spectrometry, according to Fig. 6 (for coagulation). The mass spectrum of rhKl with different glycosylation degree was purified by gel filtration chromatography. r-hKl-A is the low molecular weight of r-hKl, which is the highest in the whole secreted protein. r-hKl-B is SDS-PAGE. The molecular weight of the higher part of the electrophoresis is lower. The mass spectral molecular weight of rhKl is 28975. 79Dalton and 31871. 16Dalton, which is a net increase of 2570. 05~5465. 42Dalton, which is based on the theoretical calculation of the amino acid sequence theory. Yeast secreted expression results in post-translational modification of the protein.

RhKl was treated with PNGase F protease (New England BioLabs) according to the standard procedure in the manual. SDS-PAGE electrophoresis showed that the rhK1 molecule became smaller after removal of the N-glycan (Fig. 7A: where r_hKl_A and r_hKl_B were not subjected to desugating At the time of treatment, De-rhKl is treated with PNGaseF, and rhKl with high glycosylation and low glycosylation modification can be seen. The molecular weights are both small and uniform in size, and there are a small amount of rhKl that has not been cleaved. The N-type sugar chain-removed rhKl (De-rhKl) was subjected to mass spectrometry (Fig. 7B: The molecular weight of De-rhKl was determined by mass spectrometry, and the result was 26404. 35 Dalton, which is very close to the theoretically calculated 26405.74Dalton, which </ br></br></br></br></br></br></br></br></br> %. Example 5 Effect of rhKl on focal cerebral ischemia in rats

 Experimental material

 1. 1 Test drug: The r_hKl stock solution of the present invention with the batch number of 20060218 has a purity of 98%, a protein concentration of 4. 8 mg/ml, and an active concentration of 25 PNAU/ml. Dilute to the desired concentration with physiological saline before use. Positive control drug: Nimodipine injection (Shandong Xinhua Pharmaceutical Co., Ltd., product batch number 0411022), containing 10 mg of nimodipine per 50 ml injection. Solvent Control: 0. 9% sodium chloride injection (Shanghai Baite Medical Products Co., Ltd., product batch number A6B11307).

 1. 2 Reagents: Red tetrazolium (TTC) (Shanghai Shenggong Bioengineering Co., Ltd., batch number 2803B19), prepared in a concentration of 2% with PBS buffer before use.

 1. 3 experimental animal male SD rats, weighing 280-350 grams, were purchased from Shanghai Slack Laboratory Animals Co., Ltd. 1. 4 Instrument Image Analysis System (Germany, Leica Microsystems, Type DM LB2).

 2. Experimental methods

 2. 1 Local cerebral ischemia model preparation

 Male rats were anesthetized by intraperitoneal injection of 15% chloral hydrate (300 mg/kg). The supine position is fixed, the midline of the neck is incision, the muscles and fascia are separated along the inner edge of the sternocleidomastoid muscle, and the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) and occipital artery are carefully separated. , hang the line at the telecentric end of the CCA and near the heart and ECA. The ICA was temporarily clamped with a micro-arterial clamp, and then CCA and ECA were ligated proximally. Cut a small opening 4 mm from the CCA bifurcation and insert the sacral line into the ICA. Then gently tie the thread with a thin wire around the telecentric end of the CCA. Use the ophthalmology to gently push the sacral line and calculate the distance from the vascular bifurcation. When the insertion depth is 20 mm, tightly fasten the thin line of the CCA telecentric end and suture the incision layer by layer.

 2. 2 jugular vein cannulation

 The external jugular vein was separated and threaded two times for use. The distal end of the heart is ligated, and the proximal end of the artery is clamped. Cut the mouth obliquely and insert a 45-degree angle into the venous tube filled with liquid or saline. The proximal end of the catheter is ligated to remove the arterial clip. A slight re-examination was successful, and was administered at 0.1 ml/100 g body weight 30 min after the cerebral infarction model was caused. Another thread is placed under the catheter, the end of the catheter is ligated, the catheter is pulled out, and the layers are sutured layer by layer. Return to the cage. The room temperature is strictly controlled at 24~25 °C. After 24 h, the head was decapitated and the area of the infarct area and the weight of the infarct area were measured.

 2. 3 experimental group and dose

 The experiment was divided into three groups (model group, positive control group, test group), and about 15 male SD rats in each group. The model group was modeled and given an equal volume of physiological saline 0.1 ml/lOOg. The positive drug control group was given nimodipine injection 0.5 mg / kg. The test group was given r-hKl 30 X 10 PNAU/kg, 0.1 ml/100 g.

 2. 4 observation indicators

At 24 h after surgery, the animal's death was observed and recorded. Then, the animal was decapitated and the brain was removed. The completely excised brain was frozen at -20 °C. As shown in Tables 3 and 4, the mortality of rats in the nimodipine group and the r-hK1 group was significantly lower than that in the model group within 24 hours, suggesting that r-hKl and nimodipine can reduce the death of acute cerebral infarction animals.

 Table 3 Death counts of animals in each group within 24 hours Table 4 Percentage of cerebral infarction in the nimodipine group

 Table 5 Percentage of cerebral infarction in model group Table 6 Percentage of cerebral infarction in r-hKl group

 Rat number method (%)

 1 40,5 41,7

 $ , 4? 2-3.0

 16

 6.6-0 5,11

 ,s 1S,5

 3,40

 32 24,7

 3S 14. ί ~.1:3

 4Z S.70 !&.§

 46 5. iO 5, "ι5

 As shown in Tables 5 and 6, the positive control drug nimodipine significantly reduced the area of cerebral infarction and the weight of the cerebral infarction area (Ρ < 0.05) compared with the model group. Compared with the model group, the test drug r-hKl significantly reduced the area of cerebral infarction and the weight of cerebral infarction (P<0.01). The experimental results show that: similar to nimodipine, r-hKl has a good therapeutic effect on acute ischemic cerebral infarction. Example 6 Comparison of the effects of hKl and rhKl extracted from human urine on cerebral blood flow in Beagle dogs

 Test drug and test animal

 1. 1 test drug

 hKl (batch number: 20060218) and Ureklin for injection (trade name Kailikang ®, purified from human urine, referred to as uK, batch number 31051201)

 1. 2 animals

 Beagle Dog, provided by Weiguang Experimental Animal Center, Fuyang City, Anhui Province, License No. SCXK (皖) 06-001. Feeding Observed for more than two weeks.

 2. Experimental method

Adult healthy Beagle dog, weighing 10. 4 ± 0. 9 (9~12) kg, both male and female, pre-test animals fasted for 12 h, from Anesthetized by drinking water, 3% sodium pentobarbital (30 mg/kg) on the day of the experiment. The animal is placed on the operating table in the supine position, the skin is cut in the side of the neck, and the right conical artery is separated by about 3 cm. The appropriate electromagnetic flowmeter probe (1.5 to 2.0 mm) is attached to the MFV-3200 electromagnetic flowmeter. Record the cone arterial blood flow (VBF); separate the right common carotid artery, and separate the internal carotid artery and the external carotid artery branch, thread and ligation under the external carotid artery, and fit the electromagnetic flowmeter on the common carotid artery. Probe (3.0~4.0 mm), the internal carotid artery blood flow (ICBF) was measured by MFV-3200 electromagnetic blood flow meter; the left femoral artery was separated, and the appropriate electromagnetic flowmeter probe (2.0~2.5 mm) was attached to the MFV. On the -3200 electromagnetic flowmeter, record femoral artery blood flow (FBF); separate the right femoral artery, insert a plastic tube filled with 0.5% heparin, connect to the TP-400T pressure transducer, and measure the contraction through the AP-641G blood pressure amplifier. Pressure (SAP), diastolic blood pressure (DAP), and mean arterial pressure (MAP); needle insertion into the needle electrode, recording of the II lead electrocardiogram (ECGII) through the AB-601G ECG amplifier, used to measure heart rate (HR). The right femoral vein was isolated and cannulated for administration and rehydration. The above various analog signals are input to the MP-100 data acquisition system via the body-6300 multi-channel physiological recorder, and continuous real-time A/D conversion, data acquisition (sampling frequency 300 Hz), and digital signals are stored in the computer. Data analysis was performed using Acqknowledge v.3.5.7 software.

After the operation is completed and the indicators are stable, the measured values of each parameter are recorded as pre-dose values. Then, the infusion pump was used for constant-vein intravenous administration, and the volume was 30 mL/mouse, and the speed was 1 mL/min. Were divided into 5 groups, each group of six dogs, saline control group, 3 groups given the test drug rhKl, doses of ^{1.25X10- 3, 2.5X10- 3, 5.0 X} 10- 3 AU / kg, positive drug Kallikrein in administration group (uK), a dose of ^{2.5X10- 3 AU / kg, 5,} 10, 20, 30, 40, 50, 60, 90, 120 minutes These various parameter values measured after administration, and in accordance with Standard company calculations.

 3. Experimental results

 3.1 Effects on cerebral blood flow

Intravenous administration of rhKl significantly increased cerebral blood flow, and the mean flow during the doses of 1.25 X 10- ³ , 2.5X 10- ³ , 5X 10 - ³ AU/kg increased by 4.7%, 13.4%, 20.9%, respectively, compared with before administration. ; can significantly reduce cerebral vascular resistance, decreased by 5.1%, 12.2%, 19.2%. Equal doses of rhKl and uK increased cerebral blood flow (13.4% vs 9.9%) and decreased cerebrovascular resistance (12. 2% vs 11.8%).

 3.2 Selectivity of cerebral vessels and peripheral blood vessels

Intravenous rhKl can significantly increase the internal carotid and vertebral artery blood flow, reduced vascular resistance, showed significant ^{vasodilatation, 1.25X10- 3, 2.5X10- 3, 5} X 10- 3 AU / kg administered during carotid average The flow rate increased by 5.1%, 13.6%, and 21.6%, respectively. The vertebral artery flow increased by 2.6%, 12.8%, and 18.1%. The femoral artery flow also increased to different degrees, increasing by 3.5%, 4.7%, and 6.3%, respectively. . The selectivity to cerebral blood vessels is about 3 times stronger than that of peripheral blood vessels.

 3.3 Effects on system blood pressure and heart rate

Intravenous rhKl tends to lower blood pressure, diastolic blood pressure decreased more obvious especially after 5X10- ³ AU / kg administered diastolic blood pressure decreased significantly, the maximum decrease of about 6 mmHg, soon after drug discontinuation. Intravenous administration of rhKl had no significant effect on heart rate in anesthetized dogs. The intensity of action of rhKl is comparable to that of uK. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

 An isolated polynucleotide encoding human kallikrein 1, i.e., hK1, characterized in that said polynucleotide has the nucleotide sequence shown in SEQ ID NO: 1.

A vector, characterized in that the vector contains the polynucleotide of claim 1.

A host cell comprising the vector of claim 2 or the polynucleotide of the host cell in which the polynucleotide of claim 1 is integrated.

4. The host cell of claim 3, wherein the host cell is a yeast.

5. A method for preparing human kallikrein 1, hKl, comprising the steps of:

 (a) cultivating the host cell of claim 3 to express rhKl;

 (b) separating or purifying rhKl from the culture.

The method of claim 5, wherein, in step (a), the whole salt medium is used for the fermentation, and the pH of the induction phase is 6. 0 ± 0. 5, the temperature is 25 ± 5 °C, induction time 65 ± 15 hours.

7. The method according to claim 5, wherein in the step (b), the purification is carried out by a method selected from the group consisting of hydrophobic chromatography, anion exchange chromatography and gel filtration chromatography.

8. The method according to claim 5, wherein in the step (b), the method comprises: sequentially performing hydrophobic chromatography, Ni ²⁺ and Cu ²⁺ chelate chromatography, and anion exchange chromatography to obtain purification. hKl.

9. The method according to claim 5, wherein the hK1 obtained in the step (b) has the following characteristics: a molecular weight range of 28 to 32 kDa, all of which are N-type sugar chain modifications of methanol yeast, and 5。 The sugar is the total glycoprotein of 8. 8~1. 2%, the isoelectric point of the protein is 4. 2 ± 0.3.

The use of the polynucleotide of claim 1, or a protein encoded thereby, for the preparation of a medicament for preventing or treating cerebral infarction.