CN1712418A - Production of low-molecular heparin - Google Patents

Abstract

Production of low-molecular weight heparin is carried out by using heparin as substrate , degrading heparin from malt sugar combined protein-heparinase I fusion protein, which is amino acid residual sequence protein with SEQ ID No:1, and obtaining low-molecular weight heparin. It achieves controllable degradation time, ideal average molecular weight and higher enzyme activity.

Description

A kind of method for preparing low molecular weight heparin

Technical field

The present invention relates to a kind of method for preparing low molecular weight heparin, particularly a kind of method of utilizing Heparinase I fusion rotein MBP-HepA to prepare low molecular weight heparin.

Background technology

Heparin is the mucopolysaccharide that is alternately formed with 1 → 4 glycosidic link by hexuronic acid (L-iduronic acid, D-glucuronic acid) and D-Glucosamine Sulphate, linear chain-like structure with six sugar or eight sugared repeating units, its molecular weight is between 3000-37000, and molecular-weight average is 15000.Since 1916 found heparin first, its application as anti-freezing reagent and anti-bolt reagent aspect medical more and more was subjected to people's attention.In addition, heparin also have anti-inflammatory, antianaphylaxis, antiviral, anticancer, transfer various biological function such as blood fat.But, because heparin has anticoagulating active, thus use heparin to cause bleeding in a large number and side effect such as induced platelet minimizing, thus limited heparin application clinically greatly.

Low molecular weight heparin (low-molecular-weight heparin oligose) (being called for short LMWHs) (Robert J.Linhardt, PH.D.AndNur Sibel Guany, M.S, Seminar in Thrombosis and Hemostasis, 1999,25 (3): 5-16) be some lower-molecular-weight components that when separating unfractionated heparin, obtain, or the small molecule segment that produces after the heparin cracking, length is about 1/3 of unfractionated heparin.The LMWHs molecular weight is between 3000-8000Da, and molecular-weight average is about 5000Da.Compare with unfractionated heparin, find by the inside and outside experiment of body, under Isodose, the anticoagulation of LMWHs is less than heparin, but its body is interior and external anti thrombotic action obviously is better than heparin.In addition, LMWHs also has some other advantages, and is little as molecular weight, the bioavailability height, and plasma half-life is long; Do not combine with heparin-binding protein, therefore more stable dose-effect relationship is arranged, by the body weight administration, control dosage does not need the chamber of experimentizing monitoring; Lessly combine, be difficult for causing thrombopenia with thrombocyte.So LMWHs can effectively prevent thrombosis, can reduce the hemorrhage untoward reaction of Denging again, be a kind of antithrombotic reagent safely and effectively, can be used as the surrogate of heparin.The Heparin Oligosaccharides energy and the different protein factor effects of different polymerization degree (dp), thus present different biological actions.Range of molecular weight distributions is narrow, and relatively the drug effect of the LMWHs of homogeneous is better.LMWHs is being carried out aspect the quality control, each production unit has all been stipulated its molecular-weight average and range of molecular weight distributions.So the LMWHs and the narrow Heparin Oligosaccharides of range of molecular weight distributions of producing required molecular-weight average have great importance.

At present, the preparation method of LMWHs (Zhang Wanzhong, Wang Yunshan, Ma Runyu, Su Zhiguo, state's biochemical drug magazine, 2001,22 (1): 48-51) mainly contain chemical cracking method and enzyme liberating method.Chemical degradation method is the method for industrial normal employing, mainly contains nitrous acid edman degradation Edman, β-cancellation edman degradation Edman, hydrogen peroxide degradation method, Periodic acid, hypochlorous acid, sulfuric acid-chlorsulfonic acid and gamma-irradiation method etc.But chemical cracking heparin reaction is violent, makes that some functional group in the heparin molecule is destroyed more or less in reaction process, thereby some bioactive functions is in various degree by more or less destruction.And the enzyme liberating method reaches the environment nontoxicity owing to reaction conditions gentleness, productive rate height, becomes many glycobiology research workers' research focus.United States Patent (USP) (Nielsen, US 5106734,1992) utilizes the absorbance control quality product at 232nm place, can prepare the low molecule heparin product with ideal average molecular weight.(Yu Guangli, Wang Qun, Guan Huashi, Xu Jiamin, Robert J.Linhardt, Qingdao Marine University's journal, 2002,32 (2): 231-235) utilize heparinase that ox lung heparin is controlled enzymolysis, obtained the pure product of oligosaccharides of the polymerization degree 2～20 such as Yu Guangli.(high Ningguo such as high Ningguo, Cheng Xiulan, Yang Jing, Zhang Shuzheng, the microorganism journal, 1999,39 (1): 64-67) filtered out a kind of sheath amine alcohol liver bacterium that can produce heparinase, and utilize the enzyme liberating heparin produced, obtained a series ofly having anti-proliferation of smooth muscle activity and the anti-freezing very low Heparin Oligosaccharides of living.But used heparinase need be through the purification step of multistep in these methods, and yield is lower, cause the cost of enzyme very expensive (price of the commodity heparinase that the yellow liver bacterium of heparin produces be 40 dollars/U), limited the development that enzyme process prepares Low molecular heparin.Utilizing recombinant bacterial strain to produce Heparinase I is an extremely promising approach, but the as easy as rolling off a log formation inclusion body of the Heparinase I of generally recombinating needs complicated renaturation process could form activated protein.

Summary of the invention

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

The method for preparing low molecular weight heparin provided by the present invention is to be substrate with the heparin, with maltose binding protein-Heparinase I fusion rotein (MBP-hepA) degraded heparin, obtains low molecular weight heparin; Described maltose binding protein-Heparinase I fusion rotein (MBP-hepA) is the protein with the amino acid residue sequence of the SEQ ID N:1 in the sequence table.

Sequence 1 in the sequence table is made up of 756 amino-acid residues.

Described maltose binding protein-Heparinase I fusion rotein (MBP-hepA) can prepare in accordance with the following methods: with pMal-hepA transformed into escherichia coli TB1, obtain containing the recombination bacillus coli TB1 (pMal-hepA) of pMal-hepA, cultivate recombination bacillus coli TB1 (pMal-hepA), abduction delivering obtains maltose binding protein-Heparinase I fusion rotein (MBP-hepA); Described pMal-hepA will have SEQ ID № in the sequence table: the recombinant vectors that obtains between the BamHI of the described maltose binding protein of 2 dna sequence dna-Heparinase I fusion rotein (MBP-hepA) encoding gene insertion pMal-p2x or pMal-c2x carrier and PstI recognition site.

Dna sequence dna in the sequence 2 is made up of 2271 deoxynucleotides, and the encoding sequence of this gene is from the 1st to the 2271st deoxynucleotide of 5 ' end.

Described heparin can obtain from commercial channels, also can be synthetic according to existing method.

The starting point concentration of described heparin is 1-100g/l, is preferably 25g/l.

The solvent that is used to dissolve described heparin can be and contains 3.5mM Ca (CH ₃COO) ₂And 0.05%NaN ₃PH be the NH of 0.1M for 6.5-8.0 concentration ₄COOCH ₃Damping fluid.

The consumption of described maltose binding protein-Heparinase I fusion rotein is the 1.875-187.5IU/g substrate, is preferably the 7.5IU/g substrate.

In the described method, temperature of reaction can be 10-45 ℃, is preferably 15-20 ℃.

In the described method, the reaction times is 6-12 hour.

Reaction times is preferably 9-12 hour; Especially be preferably 9 hours.

In the described method, purifying low molecular weight heparin in accordance with the following methods: after the termination reaction mixture carried out the ultrafiltration that molecular weight cut-off is 10000Da, utilize TSK-GEL G2000SW post to carry out gel permeation chromatography the filtrate that obtains, the collection retention time is 18-20 minute a elution peak, obtains low molecular weight heparin; Used damping fluid is that to contain the quality percentage composition be 0.05% NaN in the described gel permeation chromatography ₃, pH is that 7.0 concentration are the NH of 0.1M ₄COOCH ₃Damping fluid, the flow velocity of described damping fluid are 0.5ml/min.

Method employing of the present invention has high enzyme maltose binding protein-Heparinase I fusion rotein alive and prepares heparin, because maltose binding protein has the ability with the affine absorption of maltose, therefore recombinant expressed MBP-HepA helps the separation and purification of heparinase, it is about 95% MBP-HepA that single step purification just can obtain purity, thereby can reduce the separation and purification cost of enzyme greatly; Utilize the affine adsorptive power of maltose binding protein (MBP), can be easy to realize the directed immobilization of Heparinase I, make the use repeatedly of enzyme become possibility, thereby improve enzyme reaction efficient, reduce the use cost of enzyme; Thereby reduce the production cost of low molecular weight heparin.The present invention has obtained the narrow low molecular weight heparin (molecular-weight average is at 5000-6000) of range of molecular weight distributions by the control enzyme digestion reaction time.Because production, separation and purification and the use cost of MBP-HepA can significantly reduce, the method for therefore utilizing this fusion rotein production to have the narrow low molecular weight heparin of ideal average molecular weight and range of molecular weight distributions has huge industrial application value.

Description of drawings

Fig. 1 is the building process synoptic diagram of expression vector pMal-hepA

Fig. 2 is the Heparinase I gene electrophoretogram that pcr amplification obtains from the heparin Flavobacterium

Fig. 3 is molecular weight and the canonical plotting of the residence time in the gel chromatography

Fig. 4 is the gel chromatography figure of 6 hours gained Heparin Oligosaccharides of MBP-HepA degraded heparin

Fig. 5 is the gel chromatography figure of 9 hours gained Heparin Oligosaccharides of MBP-HepA degraded heparin

Fig. 6 is the gel chromatography figure of 0.5 hour gained Heparin Oligosaccharides of MBP-HepA degraded heparin

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

The acquisition of embodiment 1, maltose binding protein-Heparinase I fusion rotein (MBP-HepA)

1, contain the Heparinase I encoding gene expression vector pMal-hepA construction of expression vector pMal-hepA building process as shown in Figure 1, detailed process is as follows: amplification Heparinase I gene from the genome DNA of heparin Flavobacterium Favabacterium heparinum (buying from IAM), used upstream and downstream primer is respectively 5 ' GCCT GGATCCCAGCAAAAAAAATCCGGTAAC 3 ' (base of band underscore is the enzyme recognition site of BamHI), 5 ' GCTT CTGCAGTCTGGCAGTTTCGCTGTAC 3 ' (base of band underscore is the PstI enzyme recognition site), introduce BamHI and PstI enzyme recognition site respectively, 50 μ L amplification reaction systems are: the 50ng template DNA, every kind of primer of 100pmol, 1 * amplification buffer (sky, Beijing is a Bioisystech Co., Ltd), every kind of dNTP of 200 μ mol/L, the high Pfu enzyme of protecting of 1 unit; Amplification program is: 95 degrees centigrade of sex change 5 minutes, and 50-60 degree centigrade of primer annealing 45 seconds, 72 degrees centigrade of primer extensions 90 seconds, after 30 circulations, 72 degrees centigrade are extended and finished reaction in 5 minutes.This PCR result shows that amplification obtains the Heparinase I gene fragment of 1.1kb as shown in Figure 2.Among Fig. 2, it is 50,51,53,55,58 or 59 ℃ of amplifications that 1-6 is respectively the primer annealing temperature, and 7 is molecular weight marker 15kb, and arrow indication place is a 1.1kb target segment.

With pMal-p2x or pMal-c2x carrier (available from NEB company)) and the PCR product use BamHI and PstI double digestion respectively, connect with the T4DNA ligase enzyme, transform JM109, with 5 ' GCCTGGATCCCAGCAAAAAAAATCCGGTAAC3 ' and 5 ' GCTTCTGCAGTCTGGCAGTTTCGCTGTAC 3 ' is primer, by bacterium colony PCR screening transformant, extraction can obtain plasmid in the transformant of 1.1kb PCR product by BamHI and the checking of PstI double digestion.To obtain the segmental plasmid of 1.1kb by BamHI and PstI double digestion and check order, will contain the plasmid called after pMal-hepA of the Heparinase I fusion rotein encoding gene MBP-HepA of nucleotide sequence with sequence 2 in the sequence table.

With pMal-hepA transformed into escherichia coli TB1, obtain the recombinant plasmid vector bacterial strain that two strains contain correct connection, wherein a strain called after recombination bacillus coli TB1 (pMal-hepA).

2, the expression of Heparinase I fusion rotein MBP-HepA

Recombination bacillus coli TB1 (pMal-hepA) was cultivated 3 hours for 37 ℃ at LB substratum (containing 100 μ g/ml Amp), add 1mM IPTG, in 15 ℃, 200rpm carried out inducing culture 21 hours, the nutrient solution that obtains is at 4 ℃, the centrifugal 10min of 10000rpm, collect thalline, with pH is that 7.0 concentration are the Tris damping fluid washing thalline 2 times that 0.017M (0.017mol/L) contains 0.2M NaCl, the ratio that adds the 1mlTris damping fluid in the centrifugal thalline that obtains of every 2ml bacterium liquid, thalline is suspended in the identical damping fluid ultrasonication in ice bath (output rating is 300W, each ultrasonic 3 seconds and intermittently 3 seconds processing 99 times).Cytoclasis liquid is at 4 ℃, and the centrifugal 30min of 13000rpm gets supernatant liquor and gets the acellular enzyme liquid of slightly carrying.Measure the enzyme activity of slightly carrying enzyme liquid, the optical absorption method of 232nm is adopted in the detection that enzyme is lived, and the enzyme of 1IU is lived and is defined as 30 ℃ of needed protein contents of 1mg substrate heparin of per hour degrading.The result shows that than vigor be the 10.69IU/mg nutrient solution.

3, the single step purification of Heparinase I fusion rotein MBP-HepA: with pH is that 7.0 concentration are the affine separator column of amylose starch (amylose) that 0.017M contains the Tris damping fluid balance 2ml of 0.2MNaCl, supernatant liquor passes through affinity column with the speed of 0.5ml/min, use the Tris buffer solution elution of the 10mM that contains 10mM maltose again, collection has the enzymic activity part, gets 2ml enzyme liquid.

4, the measurement of Heparinase I fusion rotein MBP-HepA vigor: the optical absorption method of 232nm is adopted in the detection that enzyme is lived, and the enzyme of 1IU is lived and is defined as 30 ℃ of needed protein contents of 1mg substrate heparin of per hour degrading.Taking heparin substrate solution 0.5ml, the enzyme liquid of purifying in the adding step 3, other volume replenishes with the Tris damping fluid, and final reaction solution volume is 1.5ml, surveys the absorbancy changes delta A of inherent 232nm of unit time ₂₃₂Extinction coefficient epsilon=3800M ^-1Measurement result shows that maltose binding protein-Heparinase I fusion rotein that purifying obtains is the 15.57IU/mg nutrient solution than vigor.Obtaining purity through the single step purification of step 3 is 95% MBP-HepA.

The preparation of embodiment 2, low-molecular-weight heparin oligose

1, the making of typical curve

The measuring method of low-molecular-weight heparin oligose molecular-weight average is as follows: with dextran standard (sigma), by gel permeation chromatography (TSK-GEL G2000SW, TOSOH company, Japan), obtain the typical curve of the molecular weight and the residence time, as Fig. 3, (standard substance are dextran standard, and molecular weight is respectively 1000Da, 5000Da, 12000Da, 25000Da).Wherein, used damping fluid is that to contain the quality percentage composition be 0.05% NaN in this gel permeation chromatography ₃, pH is that 7.0 concentration are the NH of 0.1M ₄COOCH ₃Damping fluid; The flow velocity of this damping fluid is 0.5ml/min.

The calculation formula of molecular-weight average is

$\mathrm{Mr}=\frac{\underset{i}{\mathrm{\Σ}}\left(M{w}_i{A}_i\right)}{\underset{i}{\mathrm{\Σ}}{A}_i}---\left(1\right)$

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

2, produce low molecular weight heparin

With commodity heparin (Beijing ancient cooking vessel state, 150U/mg tires) is raw material, and used heparin solution contains 3.5mM Ca (CH for the commodity heparin is joined ₃COO) ₂, 0.05%NaN ₃, the concentration of pH7.0 is the NH of 0.1M ₄COOCH ₃In the damping fluid, the concentration that makes heparin is 25g/l.Get 495 μ l heparin solutions, add the MBP-HepA enzyme liquid (18.6IU/ml) of 5 μ l single step purifications, reaction solution after reacting 6 hours and 9 hours respectively on 15 ℃ of shaking tables, this moment absorbance A ₂₃₂Be respectively 0.638 and 0.908,3-5min stops enzyme reaction in 100 ℃ metal bath.Every 0.5h, get 15 μ l reaction solutions in the reaction process, add the HCl termination reaction of 1485 μ l 30mM, measure A ₂₃₂With monitoring reaction course.Reaction solution is after 10000 ultra-filtration membrane filters macromole heparin and zymoprotein, to utilize gel permeation chromatography (TSK-GEL G2000SW, TOSOH company, Japan) to analyze its molecular weight distribution through molecular weight cut-off.Used damping fluid is that to contain the quality percentage composition be 0.05% NaN in this gel permeation chromatography ₃, pH is that 7.0 concentration are the NH of 0.1M ₄COOCH ₃Damping fluid, the flow velocity of this damping fluid are 0.5ml/min.Measure A with UV-detector ₂₃₂, having obtained the color atlas of 6h reaction solution and 9h reaction solution respectively, three main peaks have appearred in the color atlas (Fig. 4) of the Heparin Oligosaccharides mixture that reaction 6h obtains, according to the typical curve of the molecular weight and the residence time, draw each peak corresponding molecular weight (as table 1).As can be seen from Table 1, in mixture, molecular weight mainly contains two less than 8000 peak, shared peak area per-cent 18.13% and 31.15%, and both summations account for half of total peak area.According to formula (1), the molecular-weight average that draws the Heparin Oligosaccharides that obtains of reaction 6h is 6176.7.

The significant parameter of each peak correspondence in the Heparin Oligosaccharides color atlas of Fig. 4 behind the table 1 reaction 6h

	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
Molecular-weight average is 6176.7

Two main peaks occurred in the color atlas (Fig. 5) of the Heparin Oligosaccharides mixture that reaction 9h obtains, each peak corresponding molecular weight is as shown in table 2.As can be seen from Table 2, the molecular weight at these two peaks is respectively 7202.7,3933.5 all less than 8000, and shared peak area is respectively 37.51% and 61.11%.According to formula (1), the molecular-weight average that draws the Heparin Oligosaccharides that obtains of reaction 9h is about 5176.8.

Each peak corresponding parameters in Fig. 5 Heparin Oligosaccharides color atlas behind the table 2 reaction 9h

	The residence time (min)	Molecular weight (D)	Peak area %
				Peak 1	??18.53	??7202.7	??37.51
Peak 2	??19.44	??3933.5	??61.11
				Molecular-weight average is 5176.8

And for the color atlas (Fig. 6) of the Heparin Oligosaccharides mixture that obtains of reaction 0.5h, four main peaks have appearred, and appearance time all wants Zao than Fig. 4,5, shows that DeR is insufficient, and the Heparin Oligosaccharides that number molecular weight is bigger is not degraded into the lower Heparin Oligosaccharides of molecular weight.So control is to obtain one of principal element of the Heparin Oligosaccharides mixture of ideal average molecular weight the enzyme reaction time.

The same effective degraded heparin with the commodity heparinase of present embodiment explanation MBP-HepA energy by the control enzyme liberating reaction times, can access the low-molecular-weight heparin oligose with ideal average molecular weight.

Sequence table

<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?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

ataccctcct?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?attacttttg?atgtcgccat?agactggacg???2040

aaatatggaa?aagaggccaa?tacaattttg?aaacccggta?agctggatgt?gatgatgact???2100

tataccaaga?ataagaaacc?acaaaaagcg?catatcgtaa?accagcagga?aatcctgatc???2160

ggacgtaacg?atgacgatgg?ctattacttc?aaatttggaa?tttacagggt?cggtaacagc???2220

acggtcccgg?ttacttataa?cctgagcggg?tacagcgaaa?ctgccagatg?a????????????2271

Claims (9)

1, a kind of method for preparing low molecular weight heparin is to be substrate with the heparin, with maltose binding protein-Heparinase I fusion rotein degraded heparin, obtains low molecular weight heparin; Described maltose binding protein-Heparinase I fusion rotein is the SEQ ID № that has in the sequence table: the protein of 1 amino acid residue sequence.

2, method according to claim 1, it is characterized in that: described maltose binding protein-Heparinase I fusion rotein prepares in accordance with the following methods: with plasmid pMal-hepA transformed into escherichia coli TB1, obtain containing the recombination bacillus coli TB1 (pMal-hepA) of pMal-hepA, cultivate recombination bacillus coli TB1 (pMal-hepA), abduction delivering obtains maltose binding protein-Heparinase I fusion rotein;

Described pMal-hepA will have SEQ ID № in the sequence table: the recombinant vectors that obtains between the BamHI of the described maltose binding protein of 2 dna sequence dna-Heparinase I fusion rotein encoding gene insertion pMal-p2x or pMal-c2x carrier and PstI recognition site.

3, method according to claim 1 and 2 is characterized in that: the starting point concentration of described heparin is 1-100g/l; Be preferably 25g/l.

4, method according to claim 3 is characterized in that: describedly be used to dissolve the solvent of heparin for containing 3.5mMCa (CH ₃COO) ₂With 0.05% NaN ₃PH be the NH of 0.1M for 6.5-8.0 concentration ₄COOCH ₃Damping fluid.

5, method according to claim 1 and 2 is characterized in that: the consumption of described maltose binding protein-Heparinase I fusion rotein is the 1.875-187.5IU/g substrate; Be preferably the 7.5IU/g substrate.

6, method according to claim 1 and 2 is characterized in that: in the described method, temperature of reaction is 10-45 ℃, is preferably 15-20 ℃.

7, method according to claim 1 and 2 is characterized in that: in the described method, the reaction times is 6-12 hour.

8, method according to claim 7 is characterized in that: the described reaction times is 9-12 hour; Be preferably 9 hours.

9, method according to claim 1 and 2, it is characterized in that: in the described method, purifying low molecular weight heparin in accordance with the following methods: after the termination reaction mixture carried out the ultrafiltration that molecular weight cut-off is 10000Da, utilize TSK-GEL G2000SW post to carry out gel permeation chromatography the filtrate that obtains, the collection retention time is 18-20 minute a elution peak, obtains low molecular weight heparin; Used damping fluid is that to contain the quality percentage composition be 0.05% NaN in the described gel permeation chromatography ₃, pH is that 7.0 concentration are the NH of 0.1M ₄COOCH ₃Damping fluid, the flow velocity of described damping fluid are 0.5ml/min.

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