CN1312183C - Heparinase I fusion protein and genes encoding same and expression method thereof - Google Patents

Abstract

The invention discloses a heparanase I fusion protein, its coding gene and its expression method. The amino acid sequence of the heparanase I fusion protein provided by the present invention is shown in SEQ ID No.:2. The present invention realizes for the first time that more than 90% of heparanase I exists in the form of active and correctly folded soluble protein in Escherichia coli through the fusion protein; the enzyme activity can reach 270U/L fermentation broth (108UL ^-1 OD ₆₀₀ ^-1 ), The protein amount can reach 150mg/L, and the one-step purification of the fusion protein can be realized by affinity separation. The present invention can be widely used in the production of heparanase I.

Description

A kind of heparanase I fusion protein and its encoding gene and expression method

technical field

The invention relates to a heparanase I fusion protein, its encoding gene and its expression method in the fields of genetic engineering and fermentation engineering.

Background technique

Heparinase (heparinase) is a class of polysaccharide lyase that acts on heparin, found in many kinds of microorganisms, including Corynebacterium sp. .39:64-67), Sphingobacterium sp. (Gao Ningguo et al., Production of Sphingobacterium heparanase, Acta Microbiology 2003 Vol.43:813-816), Bacillus subtilis (Wang Zhongyan etc. Screening of heparanase-producing bacteria and its crude enzyme properties, Sichuan University Journal (Natural Science Edition) 2002 Vol.39: 777-779), Bacillus circulans (Yasutaka Tahara et al., Purification and characterization of heparinasethat degrades both heparin and heparin sulfate from Bacillus circulansBioSci.Biotechnol.Biochem.2002 Vol.66：1181-1184)、解肝素拟杆菌Prevotellaheparinolytica(Kazuyuki Sugahara et al.，Characterization of heparinasefrom an oral bacterium Prevotella heparinolytica J.Biochem .1998Vol.123:283-288), Bacteroides stercoris HJ-15 (Dong Hyuu Kim et al., Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15 J.Biochem.2000 Vol.128:323-328) and Flavabacterium heparinum (Sasiekharan, R. 1991 Ph.D. Thesis, Havard University). But heparanase from Flavobacterium heparinus is the only commercial source. There are three main types of heparanases from Flavobacterium heparinus, named as heparanase I (EC 4.2.2.7), II (NO EC code) and III (EC 4.2.2.8) (RobertJ.Linhardt et al., Purification and Characterization of heparin lyases from Flavobacterium heparinum JBC 1992 Vol.267: 24347-24355).

The research of heparinase has very important significance (Sasiekharan, R.1991 Ph.D.Thesis, HavardUniversity; Sasiekharan, R.et al., A comparative analysis of the primary sequences and characteristics of heparinase I, II, and III from Flavobacterium heparinum Biochemical and Biophysical Research Communication 1996Vol.229: 770-777): Heparanase is a kind of polysaccharide lyase, which is used to study the interaction between heparanase and its substrate polysaccharide heparin, which helps to clarify the function of polysaccharide lyase Mechanism of action; heparanase can be used to analyze the structure and biological functions of complex mucopolysaccharides such as heparin; heparanase can be used to analyze the coagulation and anticoagulation mechanism in the human body; heparanase can be used to prepare low molecular The anticoagulant drug low molecular weight heparin; heparinase can be used to remove heparinization of clinical blood to prevent bleeding after surgery; heparinase is used for the treatment of blood products before PCR reaction.

Heparanase I is usually purified from the fermentation broth of Flavobacterium heparinus, usually requires multi-step chromatographic purification, and the yield is relatively low. The growth rate of Flavobacterium heparin is slow, and the production stability of heparanase is poor. Moreover, the production of heparanase requires expensive heparin induction, which increases the cost of the enzyme. Therefore, currently, the cost of producing heparanase by using Flavobacterium is extremely high, which limits the application and development of heparanase. Ram Sasiekharan (1993) (Sasiekharan, R. et al., Cloning and expression of heparinase I gene from Flavobacterium heparinum Proc. Natl. Acad. Sci. USA 1993 Vol.90: 3660-3664) firstly through amino acid sequencing, design primers to amplify The gene sequence of heparanase I was obtained and recombined to realize the expression of heparanase in Escherichia coli (Table 1); but the expression product could not be correctly folded to form an active protein. Ram Sasiekharan (1996) (Sasiekharan, R.et al., Expression in Escherichia coli, Purification and characterization of heparinase I from Flavobacterium heparinum Biochem.J.1996 Vol.315: 589-597) improved protein expression (14.4 mg/L fermentation broth) (Table 1), but the recombinant heparanase I still forms inclusion bodies, which needs renaturation to form active protein. Oded Shoseyov (1999) (Etai Shpigel, et al., Immobilization of recombinant heparinase I fused to cellulose-binding domain Biotechnology and Bioengineering 1999 Vol.65: 17-23) utilizes cellulose-binding protein (CBD) to fuse with heparinase, using fusion The protein realized fusion expression of CBD-hep and hep-CBD, separated by affinity through cellulose column, and increased the expression level of heparanase I (150mg/L fermentation broth) (Table 1); but despite low IPTG ( 0.5mM), the fusion protein still forms inclusion bodies and requires renaturation to form active recombinant heparanase I.

Table 1. Research status of recombinant expression of heparanase I

Recombinant expression research Expression amount (mg/L fermentation broth)   host, carrier Is it soluble Whether to secrete Is it a fusion protein Ram Sasiekharan(1993)Ram Sasiekharan(1996) 0.114.4 E.coli BL21(DE3)(pET3a-hepA)E.coli BL21(DE3)(pET3a, pET12a, pET15b, pET28a-hepA) A small amount of soluble no no no no Oded Shoseyov(1999) 150 E.coli BL21(DE3)(pET3d CBD-hepA, pET3d hepA-CBD) no no CBD fusion protein

The natural maltose-binding protein MBP from Escherichia coli can specifically adsorb maltose and participate in the transport and utilization of maltose by Escherichia coli. MBP can not only combine with amylose to achieve affinity separation, but also combine with potato starch to achieve affinity separation (Lian Dejun et al., an improved fusion protein affinity chromatography purification method, Progress in Biochemistry and Biophysics 1998 Vol. 25:283-284; Usha Srinivasan et al., Aconvenient method for affinity purification of maltose binding proteinfusions Journal of Biotechnology 1998 Vol.62:163-167), which can greatly reduce the cost of separation and purification of enzymes.

Invention content

The purpose of the present invention is to provide a heparanase I fusion protein and its coding gene.

The heparanase I fusion protein provided by the present invention, named MBP-HepA, is a protein having the amino acid residue sequence of SEQ ID No.: 2 in the sequence table, or the amino acid residue sequence of SEQ ID No.: 2 is passed through a A protein derived from SEQ ID №: 2 having the same activity as the amino acid residue sequence of SEQ ID №: 2 by substitution, deletion or addition of several amino acid residues.

SEQ ID No. 2 in the sequence listing consists of 756 amino acid residues.

The gene encoding heparanase I fusion protein, named MBP-HepA, has one of the following nucleotide sequences:

1) The DNA sequence of SEQ ID №: 1 in the sequence listing;

2) A polynucleotide encoding the protein sequence of SEQ ID №: 2 in the sequence listing;

3) A DNA sequence that has more than 95% homology with the DNA sequence defined by SEQ ID №: 1 in the sequence listing and encodes the same functional protein.

The DNA sequence in Sequence 1 consists of 2271 bases, and the open reading frame of the gene is from base 1 to base 2271 at the 5' end.

Expression vectors, cell lines and engineering bacteria containing the genes of the present invention, such as pMal-hepA and E. coli TB1 (pMAL-hepA) containing pMal-hepA, all belong to the protection scope of the present invention.

The pair of primers for amplifying any fragment of the gene encoding the fusion protein is also within the protection scope of the present invention.

The second object of the present invention is to provide a method for expressing heparanase I fusion protein.

The method for expressing the heparanase I fusion protein provided by the present invention is to introduce the recombinant expression vector containing the gene encoding the heparanase I fusion protein into an expression host bacterium to express the heparanase I fusion protein.

Wherein, the recombinant expression vector of the gene encoding the heparanase I fusion protein is pMal-hepA.

pMal-hepA is the heparanase I fusion protein coding gene obtained by inserting the full sequence of the heparanase I coding gene into pMal p2x and pMalc2x (physical map shown in Figure 7, purchased from NEB company) expression vector through BamHI and PstI Recombinant expression vector. Wherein, the full sequence of the heparanase I coding gene is obtained by using the genomic DNA of Flavobacterium heparinus as a template, using 5'GCCTGGATCCCAGCAAAAAAAATCCGGTAAC3' and 5'GCTTCTGCAGTCTGGCAGTTTCGCTGTAC3' as primers, and using a conventional PCR method.

The host bacteria can be Escherichia coli, and the Escherichia coli can be the following strains: B121, JM109, DH5α, TB1 and the like.

The induction expression condition of heparanase I fusion protein can be 0.01-1mM IPTG 10-42 degrees Celsius induction for 16 hours; the yeast extract concentration in the medium used can be 1g/L-20g/L, and can also Add heat shock substances such as 1%-5% ethanol and/or 0.05-2mg/L kanamycin and/or 0.01-2.0mg/L chloramphenicol. Wherein, the expression condition is preferably that engineering bacteria are cultivated at 37 degrees Celsius for 3 hours, and then induced by adding 0.3mM IPTG at 15 degrees Celsius for 16 hours: the medium used is preferably NaCl 10g/L, yeast extract is 7.5g/L, peptone 10g/L L, 1% (mass percentage) ethanol, 0.6mg/L chloramphenicol.

The invention constructs a fusion expression vector that prevents inclusion body formation, and realizes one-step affinity separation through amylose resin. For the first time, more than 90% of heparanase I in E. coli exists in the form of active, correctly folded soluble protein through fusion protein; IPTG, the induction temperature is 15 degrees Celsius, the concentration of yeast powder in the medium is 0.75%, and the concentration of ethanol is 1%. The activity of heparanase produced can reach 270U/L (108 UL ^-1 OD600 ^-1 ) fermentation broth, and the expression level can reach 150mg/L L fermentation broth, and one-step purification of the fusion protein was achieved by affinity separation. The invention improves the yield of the fusion protein and the heparinase activity by changing the concentration of IPTG, the time of adding IPTG, the induction temperature, the composition of the culture medium and the like. The present invention will play an important role in the production of heparanase I.

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 the electrophoretic pattern of transformant colony PCR and double enzyme digestion verification

Figure 4a is the protein electrophoresis pattern of E.coli TB1(pMAL-hepA) at different induction times at 37°C

Figure 4b is the protein electrophoresis pattern of E.coli TB1(pMAL-hepA) at different induction times at 21°C

Figure 5 is the SDS-PAGE electrophoresis pattern of the fusion protein MBP-HEPA through amylose resin affinity separation

Figure 6 is the physical map of pMal-hepA

Figure 7 is the physical map of pMal p2x, pMal c2x

Detailed ways

Embodiment 1, expression of heparanase I fusion protein MBP-HepA

1. Construction of expression vector pMal-hepA

The construction process of the expression vector pMal-hepA is shown in Figure 1. The specific process is as follows: the heparanase I gene was amplified from the genomic DNA of Flavobacterium heparinus, and the upstream and downstream primers used were 5'GCCT GGATCC CAGCAAAAAAAATCCGGTAAC3' (with The underlined base is the restriction site of BamHI), 5'GCTT CTGCAG TCTGGCAGTTTCGCTGTAC3' (the underlined base is the restriction site of PstI), respectively introduce the restriction site of BamHI and PstI, the amplification reaction system is: 50ng of template DNA, 100pmol of each primer, 1×amplification buffer, 200μmol/L of each dNTP, 1 unit of high-retention Pfu enzyme; the amplification program is: denaturation at 95°C for 5 minutes, primer annealing at 50-60°C for 45 seconds, 72 The primers were extended for 90 seconds at 100°C, and after 30 cycles, the reaction was terminated by extending for 5 minutes at 72°C. 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 and pMal-c2x vectors and PCR products were digested with BamHI and PstI respectively, ligated with T4 DNA ligase, transformed into JM109, and 5'GCCTGGATCCCAGCAAAAAAAATCCGGTAAC3' and 5'GCTTCTGCAGTCTGGCAGTTTCGCTGTAC3' were used as primers to screen transformants by colony PCR. The re-produced plasmid was verified by double digestion with BamHI and PstI. The verification results are shown in Figure 3. The colony PCR results show that the target gene fragment can be effectively amplified at about 55°C. Escherichia coli TB1 was transformed to obtain two strains containing correctly connected recombinant plasmid vectors, one of which was named recombinant Escherichia coli TB1 (pMal-hepA). In Figure 3, 1 and 8 are the 2k and λ/HindIII markers respectively; 5-7 are the transformants verified by PCR, and 2-4 are the electropherograms after double enzyme digestion of 5-7, and the arrow points to the heparanase I gene strip Bands; 6 and 7 are correctly linked 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, and then induced by adding 1 mM IPT6 at 37°C and 21°C, respectively. Take the bacterial solution at regular intervals for whole-cell SDS-PAGE electrophoresis. After induction, take 40ml of the bacterial solution and centrifuge at 10000rpm for 10min, wash twice with 20mM Tris-HCl, resuspend in 4ml of 20mM Tris. 300W, 99 times of sonication for 3 seconds and 3 seconds interval each time), and 50 μl of the supernatant was taken for SDS-PAGE analysis. The results are shown in Figure 4a and Figure 4b, the effect of different induction temperatures on protein activity is more obvious. Induced at 37°C, the target protein existed in the form of inactive inclusion bodies, while at 21°C, the amount of soluble protein increased significantly. In Figure 4a, 1, 9 are protein markers, 2-4 are whole cell electrophoresis images at induction time 0, 6, and 16 hours, 5, 6 are 3, 4 cells are broken and precipitated, 7, 8 are 3, 4 above The supernatant, the point indicated by the arrow is the fusion protein MBP-hepA (94KDa). In Figure 4b, 1-6 are the whole cell electrophoresis graphs at induction time t=0, 2, 3, 4, 9, 21, 7 is the molecular weight maerker; 8, 9 are the precipitates after the cells of 5 and 6 are broken, 10, 11 are the supernatants of 8 and 9, and the arrow points to the fusion protein MBP-hepA (94KDa).

Recombinant Escherichia coli TB1 (pMal-hepA) was cultured in LB medium (containing 100 μg/ml Amp) at 37°C for 3 hours, and then induced by adding 1 mM IPTG at 37°C, 21°C and 15°C for 16 hours. The cells were centrifuged, washed twice with Tris buffer pH 7.0, and then resuspended in 4ml Tris buffer for cell disruption. After centrifuging the crushed liquid, measure the heparinase activity in the supernatant. The analysis of enzyme activity adopts Azure A (Azure A) method, and 1 U of enzyme activity defines the amount of protein required to degrade 1 mg of substrate heparin per hour at 30°C. The results showed that the heparinase activities obtained at different induction temperatures were: 37°C, 14U/L fermentation broth (2.9UL ^-1 OD ₆₀₀ ^-1 ); 21°C, 89U/L fermentation broth (18.5UL ^-1 OD ₆₀₀ ^-1 ), 15°C, 270U/L fermentation broth (108UL ^-1 OD ₆₀₀ ^-1 ).

Example 2. Purification of heparanase I fusion protein MBP-HepA by amylose column

The fusion partner (fusion partner) maltose binding protein MBP utilized in the present invention can achieve one-step separation with amylose affinity adsorption. The specific steps of affinity separation are as follows: 100 ml of bacterium expressed with 0.3 mM IPTG for 16 hours was centrifuged at 10,000 rpm for 5 min; at the same time, a bacterium with no induced expression was set as a control. Then proceed with the following two options:

Option 1: Wash twice with Column buffer (20mM TrisHCl, 200mM NaCl, pH7.4), resuspend in 5ml column buffer, and perform ultrasonication (output power is 300W, each time ultrasonic 3 seconds and intermittent 3 seconds) processed 99 times).

Option 2: Osmotic pressure shock. Resuspend the bacteria in 100ml osmotic shock buffer I (20-40% sucrose, 30mM TrisHCl, 1mM EDTA) for 15min, and stir. Centrifuge at 10,000rpm for 10min, resuspend in an equal volume of 0.5mM magnesium sulfate, place on ice for 10-15min, and centrifuge at 10,000rpm for 10min.

After centrifugation, the supernatant was passed through a 2ml pre-equilibrated amylose affinity separation column at 0.5ml/min, eluted and collected by 10mM 0.5ml/min maltose.

Take 50 μl in each step for SDS-PAGE, after the target protein is adsorbed by amylose resin, the target protein can be eluted with 10 mM maltose in 1 column volume. The results are shown in Figure 5, indicating that the target protein can account for more than 95% after one-step purification with amylose resin. In Figure 5, 1 is 0.3mM IPTG induced E.coli TB1 (pMAL-hepA) whole cell electrophoresis, 2 is uninduced E.coli TB1 (pMAL-hepA) whole cell electrophoresis, 3 is molecular weight marker, 4 is available Soluble target protein MBP-HEPA, 5 is insoluble target protein, 6-8 are 1, 2, 3 tubes (1.5ml per tube) collected by amylose resin adsorption and eluted with maltose, the arrow points to the target protein .

sequence listing

<160>2

<210>1

<211>2271

<212>DNA

<213> Artificial sequence

<400>1

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

<210>2

<211>756

<212>PRT

<213> Artificial sequence

<400>2

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

Ile Pro Tyr Arg Val.Asn Val Gln Ala Asp Ser Ala Lys Gln Lys Ala

405 4l0 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

Claims (12)

1. A heparanase I fusion protein, the amino acid sequence of which is shown in SEQ ID №: 2. 2. The gene encoding the fusion protein of heparanase I is one of the following nucleotide sequences: 1) The DNA sequence of SEQ ID №: 1 in the sequence listing; 2) A polynucleotide encoding the protein sequence of SEQ ID №: 2 in the sequence listing. 3. The gene according to claim 2, characterized in that: the gene encoding the heparanase I fusion protein is SEQ ID №: 1 in the sequence listing. 4. An expression vector containing the gene of claim 2 or 3. 5. The expression vector according to claim 4, characterized in that: the expression vector is pMAL-hepA. 6. A transgenic cell line containing the gene of claim 2 or 3. 7. The engineering bacterium containing the gene of claim 2 or 3. 8. The engineering bacterium according to claim 7, characterized in that: the engineering bacterium is Escherichia coli containing the gene encoding the heparanase I fusion protein. 9. A method for expressing a heparanase I fusion protein, comprising introducing a recombinant expression vector containing a gene encoding a heparanase I fusion protein into an expression host bacterium, and expressing the heparanase I fusion protein; the heparanase I The gene encoding the fusion protein is one of the following nucleotide sequences: 1) The DNA sequence of SEQ ID №: 1 in the sequence listing; 2) A polynucleotide encoding the protein sequence of SEQ ID №: 2 in the sequence listing. 10. The method according to claim 9, characterized in that: the recombinant expression vector of the gene encoding the heparanase I fusion protein is pMAL-hepA; and the host bacteria is Escherichia coli. 11. The method according to claim 9 or 10, characterized in that: the induction expression condition of the heparanase I fusion protein is 0.01-1mM IPTG, 10-42 degrees Celsius induction for 16 hours; The concentration of yeast extract is 1g/L～20g/L; 1%-5% ethanol and/or 0.05-2mg/L kanamycin and/or 0.01-2.0mg/L chloramphenicol are also added to the medium . 12. The method according to claim 11, characterized in that: the expression condition is that the engineered bacteria are cultivated at 37 degrees Celsius for 3 hours, and then induced by adding 0.3mM IPTG at 15 degrees Celsius for 16 hours; the culture medium used contains NaCl 10g/ L, yeast extract 7.5g/L, peptone 10g/L, 1% ethanol, 0.6mg/L chloramphenicol.

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