CN1712418A - Production of low-molecular heparin - Google Patents

Abstract

The invention discloses a method for preparing low molecular weight heparin. The method for preparing low-molecular-weight heparin of the present invention uses heparin as a substrate, and uses maltose-binding protein-heparanase I fusion protein to degrade heparin to obtain low-molecular-weight heparin; the maltose-binding protein-heparanase I fusion protein is A protein having the amino acid residue sequence of SEQ ID No. 1 in the sequence listing. The method of the invention adopts maltose-binding protein-heparanase I fusion protein with high enzyme activity to prepare heparin, and obtains low-molecular-weight heparin oligosaccharides with ideal average molecular weight and narrow molecular weight distribution by controlling the degradation time. The invention provides a new way for the industrialized production of low molecular weight heparin by enzymatic method.

Description

A method for preparing low molecular weight heparin

technical field

The invention relates to a method for preparing low-molecular-weight heparin, in particular to a method for preparing low-molecular-weight heparin by using heparanase I fusion protein MBP-HepA.

Background technique

Heparin is a mucopolysaccharide formed by hexuronic acid (L-iduronic acid, D-glucuronic acid) and D-glucosamine sulfate with alternating 1→4 glycosidic bonds, and has a linear chain of six- or eight-saccharide repeating units Like structure, its molecular weight is between 3000-37000, and the average molecular weight is 15000. Since heparin was first discovered in 1916, its application in medicine as an anticoagulant and antithrombotic agent has attracted more and more attention. In addition, heparin also has various biological functions such as anti-inflammation, anti-allergy, anti-virus, anti-cancer, and blood lipid regulation. However, because heparin has anticoagulant activity, a large amount of heparin will cause side effects such as bleeding and induction of thrombocytopenia, which greatly limits the clinical application of heparin.

Low molecular weight heparins (low molecular weight heparin oligosaccharides) (LMWHs for short) (Robert J.Linhardt, PH.D.AndNur Sibel Guany, M.S, Seminar in Thrombosis and Hemostasis, 1999, 25(3): 5-16) are isolated Some low molecular weight components obtained from unfractionated heparin, or small molecular fragments produced after heparin cleavage, are about 1/3 of the length of unfractionated heparin. The molecular weight of LMWHs is between 3000-8000Da, and the average molecular weight is about 5000Da. Compared with unfractionated heparin, it was found through in vivo and in vitro experiments that at the same dose, the anticoagulant effect of LMWHs was less than that of heparin, but its antithrombotic effect in vivo and in vitro was significantly stronger than that of heparin. In addition, LMWHs also has some other advantages, such as small molecular weight, high bioavailability, and long plasma half-life; it does not bind to heparin-binding protein, so there is a more stable dose-effect relationship, and the administration is based on body weight, dose control, and no experiments are required Laboratory monitoring; less combined with platelets, less likely to cause thrombocytopenia. Therefore, LMWHs can not only effectively prevent thrombosis, but also reduce bleeding and other adverse reactions. It is a safe and effective antithrombotic drug and can be used as a substitute for heparin. Heparin oligosaccharides with different degrees of polymerization (dp) can interact with different protein factors, thus presenting different biological effects. The molecular weight distribution range is narrow, and the relatively uniform LMWHs have better drug efficacy. In terms of quality control of LMWHs, each production unit has specified its average molecular weight and molecular weight distribution range. Therefore, it is of great significance to produce LMWHs with desired average molecular weight and heparin oligosaccharides with narrow molecular weight distribution range.

At present, the preparation methods of LMWHs (Zhang Wanzhong, Wang Yunshan, Ma Runyu, Su Zhiguo, National Journal of Biochemical Medicine, 2001, 22(1): 48-51) mainly include chemical cleavage and enzymatic degradation. Chemical degradation method is commonly used in industry, mainly including nitrous acid degradation method, β-elimination degradation method, hydrogen peroxide degradation method, periodic acid, hypochlorous acid, sulfuric acid-chlorosulfonic acid and γ-irradiation method, etc. However, the chemical cleavage of heparin reacts violently, so that some functional groups in the heparin molecule are more or less destroyed during the reaction process, and thus some biologically active functions are more or less destroyed in different degrees. The enzymatic degradation method has become a research hotspot for many glycobiology researchers because of its mild reaction conditions, high yield and non-toxicity to the environment. U.S. Patent (Nielsen, US 5106734, 1992) utilizes the absorbance value at 232nm to control product quality, and can prepare low-molecular-weight heparin products with ideal average molecular weight. Yu Guangli et al. (Yu Guangli, Wang Qun, Guan Huashi, Xu Jiamin, Robert J.Linhardt, Qingdao Ocean University Journal, 2002, 32(2): 231-235) used heparinase to control bovine lung heparin The pure oligosaccharides with a degree of polymerization of 2-20 were obtained. Gao Ningguo et al. (Gao Ningguo, Cheng Xiulan, Yang Jing, Zhang Shuzheng, Acta Microbiology, 1999, 39 (1): 64-67) have screened out a kind of sphingosine hepatic bacteria that can produce heparanase, and utilize the produced A series of heparin oligosaccharides with anti-smooth muscle hyperplasia activity and low anticoagulant activity were obtained by enzymatically degrading heparin. However, the heparanase used in these methods needs to go through multi-step purification steps, and the yield is low, causing the cost of the enzyme to be very expensive (the price of the commercial heparinase produced by Heparin Flavia bacterium is 40 U.S. dollars/U), This limits the development of enzymatic preparation of low molecular weight heparin. The use of recombinant strains to produce heparanase I is a very promising approach, but usually recombinant heparanase I is very easy to form inclusion bodies and requires a complex refolding process to form active proteins.

Contents of the invention

The purpose of the present invention is to provide a method for preparing low molecular weight heparin.

The method for preparing low-molecular-weight heparin provided by the present invention uses heparin as a substrate, and uses maltose-binding protein-heparanase I fusion protein (MBP-hepA) to degrade heparin to obtain low-molecular-weight heparin; the maltose-binding protein-hepA Primease I fusion protein (MBP-hepA) is the protein with the amino acid residue sequence of SEQ ID N: 1 in the sequence listing.

Sequence 1 in the sequence listing consists of 756 amino acid residues.

The maltose-binding protein-heparanase I fusion protein (MBP-hepA) can be prepared according to the following method: pMal-hepA is transformed into Escherichia coli TB1 to obtain recombinant Escherichia coli TB1 (pMal-hepA) containing pMal-hepA, and the recombinant Escherichia coli TB1 (pMal-hepA), induced expression, obtains maltose binding protein-heparanase I fusion protein (MBP-hepA); The pMal-hepA will have the DNA sequence of SEQ ID No.: 2 in the sequence table The recombinant vector obtained by inserting the coding gene of maltose binding protein-heparanase I fusion protein (MBP-hepA) between the recognition sites of BamHI and PstI of pMal-p2x or pMal-c2x vector.

The DNA sequence in Sequence 2 consists of 2271 deoxynucleotides, and the coding sequence of the gene is from the 1st to the 2271st deoxynucleotides at the 5' end.

The heparin can be obtained from commercial sources, and can also be synthesized according to existing methods.

The initial concentration of said heparin is 1-100 g/l, preferably 25 g/l.

The solvent used to dissolve the heparin may be 0.1M NH ₄ COOCH ₃ buffer containing 3.5 mM Ca(CH ₃ COO) ₂ and 0.05% NaN ₃ at a pH of 6.5-8.0.

The dosage of the maltose binding protein-heparanase I fusion protein is 1.875-187.5 IU/g substrate, preferably 7.5 IU/g substrate.

In the method, the reaction temperature may be 10-45°C, preferably 15-20°C.

In the method, the reaction time is 6-12 hours.

The reaction time is preferably 9-12 hours; especially preferably 9 hours.

In the method, the low-molecular-weight heparin can be purified according to the following method: after the reaction is terminated, the mixture is subjected to ultrafiltration with a molecular weight cut-off of 10,000 Da, and the obtained filtrate is subjected to gel filtration chromatography using a TSK-GEL G2000SW column, and the collection retention time is 18 -20 minutes of elution peak, low molecular weight heparin is obtained; the buffer used in the gel filtration chromatography is NH ₄ COOCH ₃ containing 0.05% NaN ₃ with a mass percentage content of 0.1M at a pH of 7.0 Buffer, the flow rate of the buffer is 0.5ml/min.

The method of the present invention adopts maltose-binding protein-heparanase I fusion protein with relatively high enzyme activity to prepare heparin, and since maltose-binding protein has the ability of affinity adsorption with maltose, the recombinant expression of MBP-HepA is beneficial to the separation of heparinase Purification, MBP-HepA with a purity of about 95% can be obtained in one step of purification, which can greatly reduce the cost of separation and purification of the enzyme; using the affinity adsorption capacity of maltose-binding protein (MBP), the orientation of heparanase I can be easily realized Immobilization makes it possible to use the enzyme repeatedly, thereby improving the efficiency of the enzyme reaction and reducing the cost of using the enzyme; thereby reducing the production cost of low molecular weight heparin. The invention obtains low-molecular-weight heparin (average molecular weight 5000-6000) with narrow molecular weight distribution range by controlling the reaction time of enzymatic hydrolysis. Since the cost of production, separation, purification and use of MBP-HepA can be greatly reduced, the method of using the fusion protein to produce low molecular weight heparin with ideal average molecular weight and narrow molecular weight distribution range has great industrial application value.

Description of drawings

Figure 1 is a schematic diagram of the construction process of the expression vector pMal-hepA

Fig. 2 is the electrophoresis pattern of heparanase I gene obtained by PCR amplification from Flavobacterium heparinus

Figure 3 is a standard curve diagram of molecular weight and retention time in gel chromatography

Figure 4 is a gel chromatogram of heparin oligosaccharides obtained by degrading heparin with MBP-HepA for 6 hours

Figure 5 is the gel chromatogram of heparin oligosaccharides obtained by degrading heparin with MBP-HepA for 9 hours

Figure 6 is the gel chromatogram of heparin oligosaccharides obtained by degrading heparin with MBP-HepA for 0.5 hours

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified. The percentages in the following examples are all mass percentages unless otherwise specified.

Embodiment 1, the acquisition of maltose binding protein-heparanase I fusion protein (MBP-HepA)

1. Construction of the expression vector pMal-hepA containing the gene encoding heparanase I The construction process of the expression vector pMal-hepA is as shown in Figure 1, and the specific process is as follows: Genomic DNA from Flavobacterium heparinum (purchased from IAM) Amplify the heparanase I gene, the upstream and downstream primers used are 5'GCCT GGATCC CAGCAAAAAAAATCCGGTAAC 3' (the underlined base is the enzyme recognition site of BamHI), 5'GCTT CTGCAG TCTGGCAGTTTCGCTGTAC 3' (the underlined base The base is the PstI enzyme recognition site), and BamHI and PstI enzyme recognition sites were introduced respectively, and the 50 μL amplification reaction system was: 50ng template DNA, 100pmol of each primer, 1× amplification buffer (Beijing Tianwei Biotechnology Co., Ltd.) , 200 μmol/L of each dNTP, 1 unit of high-retention Pfu enzyme; the amplification program is: denaturation at 95 degrees Celsius for 5 minutes, primer annealing at 50-60 degrees Celsius for 45 seconds, primer extension at 72 degrees Celsius for 90 seconds, and after 30 cycles, extension at 72 degrees Celsius for 5 seconds minutes to end the reaction. The PCR result is shown in FIG. 2 , indicating that a 1.1 kb heparanase I gene fragment was amplified. In Figure 2, 1-6 are the amplification results of the primer annealing temperature at 50, 51, 53, 55, 58 or 59°C respectively, 7 is the molecular weight marker 15kb, and the arrow points to the 1.1kb target fragment.

The pMal-p2x or pMal-c2x vector (purchased from NEB Company)) and the PCR product were double-digested with BamHI and PstI respectively, ligated with T4 DNA ligase, and transformed into JM109, using 5'GCCTGGATCCCAGCAAAAAAAATCCGGTAAC3' and 5'GCTTCTGCAGTCTGGCAGTTTCGCTGTAC 3' as primers , Transformants were screened by colony PCR, and the plasmids in the transformants that could obtain 1.1kb PCR products were extracted and verified by double digestion with BamHI and PstI. The plasmid of 1.1 kb fragment obtained by double digestion with BamHI and PstI was sequenced, and the plasmid containing the heparanase I fusion protein encoding gene MBP-HepA having the nucleotide sequence of sequence 2 in the sequence listing was named pMal-hepA.

pMal-hepA was transformed into Escherichia coli TB1 to obtain two strains containing correctly connected recombinant plasmid vectors, one of which was named recombinant Escherichia coli TB1 (pMal-hepA).

2. Expression of heparanase I fusion protein MBP-HepA

Recombinant Escherichia coli TB1 (pMal-hepA) was cultured in LB medium (containing 100 μg/ml Amp) at 37°C for 3 hours, added 1 mM IPTG, and induced at 15°C and 200 rpm for 21 hours, and the obtained culture solution was grown at 4°C, Centrifuge at 10000rpm for 10min, collect the cells, wash the cells twice with Tris buffer solution containing 0.017M (0.017mol/L) pH 7.0 and 0.2M NaCl, add 1ml of Tris buffer solution for every 2ml cells obtained by centrifugation The bacteria were suspended in the same buffer, and ultrasonically disrupted in an ice bath (output power 300W, 99 times of ultrasonication for 3 seconds and intermittent 3 seconds). The cell disruption solution was centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant was taken to obtain a cell-free crude enzyme solution. The enzyme activity of the crude enzyme solution was measured, and the detection of the enzyme activity was detected by the light absorption method at 232nm. The enzyme activity of 1IU was defined as the amount of protein required to degrade 1mg of the substrate heparin per hour at 30°C. The results showed that the specific activity was 10.69IU/mg culture solution.

3. The one-step purification of heparanase I fusion protein MBP-HepA: with pH 7.0, concentration is 0.017M Tris buffer solution containing 0.2MNaCl to balance the amylose (amylose) affinity separation column of 2ml, and the supernatant is washed with 0.5ml Pass through the affinity column at a speed of 1/min, then elute with 10 mM Tris buffer containing 10 mM maltose, collect the part with enzyme activity, and obtain 2 ml of enzyme solution.

4. Measurement of the activity of the heparinase I fusion protein MBP-HepA: the detection of the enzyme activity adopts the 232nm light absorption method, and the enzyme activity of 1IU is defined as the amount of protein required to degrade 1mg of the substrate heparin per hour at 30°C. Take 0.5ml of the heparin substrate solution, add the enzyme solution purified in step 3, supplement the other volume with Tris buffer, the final volume of the reaction solution is 1.5ml, measure the absorbance change ΔA ₂₃₂ at 232nm per unit time. Extinction coefficient ε=3800M ^-1 . The measurement results showed that the specific activity of the purified maltose binding protein-heparanase I fusion protein was 15.57IU/mg culture solution. After one-step purification in step 3, MBP-HepA with a purity of 95% was obtained.

Embodiment 2, the preparation of low molecular weight heparin oligosaccharide

1. Preparation of standard curve

The assay method of low molecular weight heparin oligosaccharide average molecular weight is as follows: with dextran standard substance (sigma), by gel filtration chromatography (TSK-GEL G2000SW, TOSOH company, Japan), obtain the standard curve of molecular weight and retention time, as Fig. 3, (standard product is dextran standard product, molecular weight is respectively 1000Da, 5000Da, 12000Da, 25000Da). Wherein, the buffer used in the gel filtration chromatography is NH ₄ COOCH ₃ buffer containing 0.05% NaN ₃ by mass percentage, pH 7.0 and concentration 0.1M; the flow rate of the buffer is 0.5ml/ min.

The formula for calculating the average molecular weight is

$\mathrm{<span\; class="notranslate">\; Mr.</span>}<span\; class="notranslate">\; =</span>\frac{\underset{\mathrm{<span\; class="notranslate">\; i</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; m</span>}{\mathrm{<span\; class="notranslate">\; w</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; )</span>}{\underset{\mathrm{<span\; class="notranslate">\; i</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>$

Wherein, Mr is the average molecular weight, Mwi is the molecular weight of each peak corresponding substance, and Ai is the area of each peak.

2. Production of low molecular weight heparin

Commercial heparin (Beijing Dingguo, potency 150U/mg) was used as the raw material, and the heparin solution used was to add commercial heparin to 3.5mM Ca(CH ₃ COO) ₂ , 0.05% NaN ₃ , and the concentration of pH 7.0 was 0.1 In the NH ₄ COOCH ₃ buffer of M, the concentration of heparin was 25 g/l. Take 495 μl of heparin solution, add 5 μl of one-step purified MBP-HepA enzyme solution (18.6IU/ml), and react the reaction solution on a shaker at 15°C for 6 hours and 9 hours respectively. At this time, the absorbance values A ₂₃₂ are 0.638 and 0.908 respectively , Stop the enzyme reaction in a metal bath at 100°C for 3-5min. During the reaction process, 15 μl of the reaction solution was taken every 0.5 h, and 1485 μl of 30 mM HCl was added to terminate the reaction, and A ₂₃₂ was measured to monitor the reaction process. After the reaction solution was filtered through an ultrafiltration membrane with a molecular weight cut-off of 10,000 to remove macromolecular heparin and enzyme protein, its molecular weight distribution was analyzed by gel filtration chromatography (TSK-GEL G2000SW, TOSOH, Japan). The buffer used in the gel filtration chromatography is 0.1M NH ₄ COOCH ₃ buffer containing 0.05% NaN ₃ by mass, pH 7.0, and the flow rate of the buffer is 0.5 ml/min. A ₂₃₂ was measured with an ultraviolet detector, and the chromatograms of the 6h reaction solution and the 9h reaction solution were respectively obtained. Three main peaks appeared in the chromatogram (Figure 4) of the heparin-oligosaccharide mixture obtained after the 6h reaction. According to the molecular weight and retention time The standard curve of each peak was obtained to obtain the molecular weight corresponding to each peak (as in Table 1). It can be seen from Table 1 that in the mixture, there are mainly two peaks with a molecular weight less than 8000, accounting for 18.13% and 31.15% of the peak area, and the sum of the two accounts for half of the total peak area. According to the formula (1), the average molecular weight of the heparin oligosaccharides obtained after 6 hours of reaction is 6176.7.

The main parameters corresponding to each peak in the heparin oligosaccharide chromatogram in Figure 4 after 6 hours of reaction in Table 1 retention time (min) Molecular weight (Da) Peak area% peak 1 17.85 9609.1 18.13 peak 2 18.45 7470.1 31.15 peak 3 19.42 4022.6 47.60 The average molecular weight is 6176.7

Two main peaks appeared in the chromatogram ( FIG. 5 ) of the heparin-oligosaccharide mixture obtained after reacting for 9 hours, and the molecular weights corresponding to each peak are shown in Table 2. It can be seen from Table 2 that the molecular weights of these two peaks are both less than 8000, being 7202.7 and 3933.5 respectively, and the peak areas occupied are 37.51% and 61.11% respectively. According to the formula (1), the average molecular weight of the heparin oligosaccharide obtained after reacting for 9 hours is about 5176.8.

Table 2 Parameters corresponding to each peak in Figure 5 heparin oligosaccharide chromatogram after reaction for 9h Residence time (min) Molecular weight (D) Peak area% peak 1 18.53 7202.7 37.51 peak 2 19.44 3933.5 61.11 The average molecular weight is 5176.8

And for the chromatogram (Fig. 6) of the heparin oligosaccharide mixture that reacts 0.5h to obtain, four main peaks have occurred, and peak time is all earlier than Fig. 4, 5, shows that degradation reaction is not sufficient, and some molecular weight Heparin oligosaccharides were not degraded to lower molecular weight heparin oligosaccharides. Therefore, controlling the enzyme reaction time is one of the main factors to obtain the heparin-oligosaccharide mixture with the ideal average molecular weight.

This example shows that MBP-HepA can degrade heparin as effectively as commercial heparinase, and low molecular weight heparin oligosaccharides with ideal average molecular weight can be obtained by controlling the enzyme degradation reaction time.

Sequence Listing

<160>2

<210>1

<211>756

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>1

Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys

1 5 10 15

Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr

20 25 30

Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe

35 40 45

Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala

50 55 60

His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile

65 70 75 80

Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp

85 90 95

Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu

100 105 110

Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys

115 120 125

Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly

130 135 140

Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro

145 150 155 160

Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys

165 170 175

Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly

180 185 190

Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp

195 200 205

Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala

210 215 220

Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys

225 230 235 240

Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser

245 250 255

Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro

260 265 270

Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp

275 280 285

Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala

290 295 300

Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala

305 310 315 320

Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln

325 330 335

Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala

340 345 350

Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn

355 360 365

Ser Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile

370 375 380

Glu Gly Arg Ile Ser Glu Phe Gly Ser Gln Gln Lys Lys Ser Gly Asn

385 390 395 400

lle Pro Tyr Arg Val Asn Val Gln Ala Asp Ser Ala Lys Gln Lys Ala

405 410 415

Ile Ile Asp Asn Lys Trp Val Ala Val Gly Ile Asn Lys Pro Tyr Ala

420 425 430

Leu Gln Tyr Asp Asp Lys Leu Arg Phe Asn Gly Lys Pro Ser Tyr Arg

435 440 445

Phe Glu Leu Lys Ala Glu Asp Asn Ser Leu Glu Gly Tyr Ala Ala Gly

450 455 460

Glu Thr Lys Gly Arg Thr Glu Leu Ser Tyr Ser Tyr Ala Thr Thr Asn

465 470 475 480

Asp Phe Lys Lys Phe Pro Pro Ser Val Tyr Gln Asn Ala Gln Lys Leu

485 490 495

Lys Thr Val Tyr His Tyr Gly Lys Gly Ile Cys Glu Gln Gly Ser Ser

500 505 510

Arg Ser Tyr Thr Phe Ser Val Tyr Ile Pro Ser Ser Phe Pro Asp Asn

515 520 525

Ala Thr Thr Ile Phe Ala Gln Trp His Gly Ala Pro Ser Arg Thr Leu

530 535 540

Val Ala Thr Pro Glu Gly Glu Ile Lys Thr Leu Ser Ile Glu Glu Phe

545 550 555 560

Leu Ala Leu Tyr Asp Arg Met Ile Phe Lys Lys Asn Ile Ala His Asp

565 570 575

Lys Val Glu Lys Lys Asp Lys Asp Gly Lys Ile Thr Tyr Val Ala Gly

580 585 590

Lys Pro Asn Gly Trp Lys Val Glu Gln Gly Gly Tyr Pro Thr Leu Ala

595 600 605

Phe Gly Phe Ser Lys Gly Tyr Phe Tyr Ile Lys Ala Asn Ser Asp Arg

610 615 620

Gln Trp Leu Thr Asp Lys Ala Asp Arg Asn Asn Ala Asn Pro Glu Asn

625 630 635 640

Ser Glu Val Met Lys Pro Tyr Ser Ser Glu Tyr Lys Thr Ser Thr Ile

645 650 655

Ala Tyr Lys Met Pro Phe Ala Gln Phe Pro Lys Asp Cys Trp Ile Thr

660 665 670

Phe Asp Val Ala Ile Asp Trp Thr Lys Tyr Gly Lys Glu Ala Asn Thr

675 680 685

Ile Leu Lys Pro Gly Lys Leu Asp Val Met Met Thr Tyr Thr Lys Asn

690 695 700

Lys Lys Pro Gln Lys Ala His Ile Val Asn Gln Gln Glu Ile Leu Ile

705 710 715 720

Gly Arg Asn Asp Asp Asp Gly Tyr Tyr Phe Lys Phe Gly Ile Tyr Arg

725 730 735

Val Gly Asn Ser Thr Val Pro Val Thr Tyr Asn Leu Ser Gly Tyr Ser

740 745 750

Glu Thr Ala Arg

755

<210>2

<211>2271

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>2

atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60

ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120

ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180

atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240

accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300

aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360

gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420

aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480

ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540

gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600

aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660

ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720

gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780

ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840

ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900

ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960

actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020

tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080

gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140

aacctcggga tcgagggaag gatttcagaa ttcggatccc agcaaaaaaa atccggtaac 1200

atcccttacc gggtaaatgt gcaggccgac agtgctaagc agaaggcgat tattgacaac 1260

aaatgggtgg cagtaggcat caataaacct tatgcattac aatatgacga taaactgcgc 1320

tttaatggaa aaccatccta tcgctttgag cttaaagccg aagacaattc gcttgaaggt 1380

tatgctgcag gagaaacaaa gggccgtaca gaattgtcgt acagctatgc aaccaccaat 1440

gattttaaga aatttccccc aagcgtatac caaaatgcgc aaaagctaaa aaccgtttat 1500

cattacggca aagggatttg tgaacagggg agctcccgca gctatacctt ttcagtgtac 1560

atacccctcct ccttccccga caatgcgact actatttttg cccaatggca tggtgcaccc 1620

agcagaacgc ttgtagctac accagaggga gaaattaaaa cactgagcat agaagagttt 1680

ttggccttat acgaccgcat gatcttcaaa aaaaatatcg cccatgataa agttgaaaaa 1740

aaagataagg acggaaaaat tacttatgta gccggaaagc caaatggctg gaaggtagaa 1800

caaggtggtt atcccacgct ggcctttggt ttttctaaag ggtattttta catcaaggca 1860

aactccgacc ggcagtggct taccgacaaa gccgaccgta acaatgccaa tcccgagaat 1920

agtgaagtaa tgaagcccta ttcctcggaa tacaaaactt caaccattgc ctataaaatg 1980

ccctttgccc agttccctaa agattgctgg attacktttg atgtcgccat agactggacg 2040

aaatatggaa aagaggccaa tacaattttg aaacccggta agctggatgt gatgatgact 2100

tataccaaga ataagaaacc acaaaaagcg catatcgtaa accagcagga aatcctgatc 2160

ggacgtaacg atgacgatgg ctattacttc aaatttggaa ttacagggt cggtaacagc 2220

acggtcccgg ttacttataa cctgagcggg tacagcgaaa ctgccagatg a 2271

Claims (9)

1. A method for preparing low-molecular-weight heparin, which uses heparin as a substrate and degrades heparin with maltose-binding protein-heparanase I fusion protein to obtain low-molecular-weight heparin; the maltose-binding protein-heparanase I fusion protein, It is a protein having the amino acid residue sequence of SEQ ID No. 1 in the sequence listing. 2. The method according to claim 1, characterized in that: the maltose binding protein-heparanase I fusion protein is prepared according to the following method: transform the plasmid pMal-hepA into Escherichia coli TB1 to obtain recombinant large intestine containing pMal-hepA bacillus TB1(pMal-hepA), culture recombinant Escherichia coli TB1(pMal-hepA), induce expression, and obtain maltose binding protein-heparanase I fusion protein; The pMal-hepA is to insert the gene encoding the maltose binding protein-heparanase I fusion protein having the DNA sequence of SEQ ID №2 in the sequence listing into the BamHI and PstI recognition sites of the pMal-p2x or pMal-c2x vector obtained recombinant vectors. 3. The method according to claim 1 or 2, characterized in that the initial concentration of said heparin is 1-100 g/l; preferably 25 g/l. 4. The method according to claim 3, characterized in that: the solvent for dissolving heparin is NH 3.5mM Ca(CH ₃ COO) ₂ and 0.05% NaN ₃ with a pH of 6.5-8.0 and a concentration of 0.1M ₄ COOCH ₃ buffer. 5. The method according to claim 1 or 2, characterized in that: the amount of the maltose binding protein-heparanase I fusion protein is 1.875-187.5 IU/g substrate; preferably 7.5 IU/g substrate. 6. The method according to claim 1 or 2, characterized in that in the method, the reaction temperature is 10-45°C, preferably 15-20°C. 7. The method according to claim 1 or 2, characterized in that: in the method, the reaction time is 6-12 hours. 8. The method according to claim 7, characterized in that: the reaction time is 9-12 hours; preferably 9 hours. 9. The method according to claim 1 or 2, characterized in that in the method, the low molecular weight heparin is purified according to the following method: after the reaction is terminated, the mixture is subjected to ultrafiltration with a molecular weight cut-off of 10000 Da, and the obtained filtrate is purified by TSK - GEL G2000SW column for gel filtration chromatography, collecting the elution peak with a retention time of 18-20 minutes to obtain low molecular weight heparin; the buffer used in the gel filtration chromatography is 0.05% by mass NaN ₃ , pH 7.0, concentration 0.1 M NH ₄ COOCH ₃ buffer solution, the flow rate of the buffer solution is 0.5 ml/min.

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