CN105753945B - A kind of link peptide and its application - Google Patents

Abstract

The invention discloses a kind of link peptide and its applications.Link peptide of the invention is mainly used in preparation MBP fusion polysaceharide lyase, and in particular to MBP fusion heparinase I, II, III, MBP fusion chondrosulphatase B, AC.In MBP amalgamation and expression system, MBP hydrotropy is improved by the link peptide application, helps folding, increase the activity and thermal stability of target protein, the final fermentation enzyme activity obtained in Escherichia coli tentatively reaches the horizontal and MBP fusion heparinase I with outstanding thermal stability of industrial application, II, III, MBP fusion chondrosulphatase B, AC.

Description

A kind of link peptide and its application

Technical field

The invention belongs to genetic engineerings and field of fermentation engineering, and in particular to a kind of link peptide and its application.

Background technique

Heparinase (heparinase) is that one kind may act on heparin (heparin) or Heparan sulfate (heparin Sulfate) the polysaceharide lyase of the glycosidic bond of molecule.The heparinase type being currently known has I, II, III 3 kind, is from gram It is found in negative bar bacterium Flavobacterium heparinum.Three kinds of zymolyte specificity and the mode of action are different, and Heparinase I substrate is Heparin, the substrate of Heparinase I I are heparin and Heparan sulfate, and the substrate of Heparinase I II is Heparan sulfate.Heparinase There is important application value in scientific research and application field.

Chondrosulphatase (chondroitinase or chondroitin sulfaeyase, abbreviation ChSase) is a kind of The glycosaminoglycans such as chondroitin sulfate, chondroitin, hyaluronic acid can be degraded to unsaturated disaccharides.ChSase includes chondroitin sulfate Enzyme B, chondrosulphatase AC.In recent years, ChSase as it is a kind of study protein in body glycan structure and function tool with And the attention of people is increasingly subject to as a kind of novel medicinal enzyme.

In view of the significant application value of heparinase, chondrosulphatase, researcher is very early to it in microflora In recombinant expression studied, but poor effect, main technical bottleneck are to be difficult to realize heparinase, chondrosulphatase Soluble-expression, while its catalytic activity and thermal stability are relatively low, require that there are also very big distances apart from industrial application.

The soluble-expression effect of heparinase, chondrosulphatase can be improved in fusion protein strategy.In structure biology, pass through Construction of fusion protein increases the expression of soluble protein and facilitates purifying (the Hongyuan M. A self-cleavable of albumen sortase fusion for one-step purification of free recombinant proteins. Protein Expression and Purification ,2004,37(1):253-263；With Sun-ok K, Young I L. High-level expression and simple purification of recombinant human Insulin-like growth factor I. Journal of Biotechnology, 1996,48:97-105.) As a kind of effective method.Link peptide is much larger than enzyme sheet as the key element in fusion protein strategy, changeability Body, and significant impact (Cahalon L, Lider O, Schor H, et.al. Heparin can be generated to fusion enzyme performance disaccharides inhibit tumor necrosis factor-α production by macrophages and arrest immue inflammation in rodents. Int. Immunol.,1997,9:1517-1522；And Kakkar A. Low- and ultra-low-molecular-weight heparins. Best Practice & Research Clinical Haematology, 2004,17 (1): 77-87.).Link peptide with appropriate length and hard and soft degree can be two A enzyme (structural domain) is properly separated from, so as to avoid different structure and interfering with each other in folding, catalytic process.Although even Connect peptide to have a major impact the function of fusion protein, but regrettably, so far in succession peptide in heparinase, chondroitin sulfate The research of the polysaceharide lyases such as enzyme is less, and existing relevant enzyme requires also biggish distance apart from industrial application.

Summary of the invention

The present invention provides a kind of link peptide, and sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.

The link peptide is preparing MBP(maltose-binding protein) fusion polysaceharide lyase in application.

Link peptide prepare MBP fusion polysaceharide lyase in process it is as follows:

(1) permutation and combination based on rigid flexible amino acid sequence units designs link peptide L1, L2, nucleotide sequence point It Wei not SEQ ID NO.1, SEQ ID NO.2；

(2) by MBP sequence and polysaccharide in sequence SEQ ID NO.1 or SEQ ID NO.2 insertion MBP fusion polysaceharide lyase Between lyase gene sequence, obtains the MBP containing the connection peptide sequence and merge polysaceharide lyase expression vector, then carry out MBP merges the expression of polysaceharide lyase, obtains the MBP fusion polysaceharide lyase containing the link peptide.

The polysaceharide lyase gene is derived from Flavobacterium heparinum.

Polysaceharide lyase in the polysaceharide lyase gene is heparinase or chondroitin sulfate in Flavobacterium heparinum Enzyme, wherein heparinase includes heparinase I, heparinaseⅡ, heparinase III, and chondrosulphatase includes chondrosulphatase B, sulfuric acid Chondroitinase AC.

A kind of MBP fusion heparinase containing link peptide SEQ ID NO.1, including MBP fusion heparinase I, MBP merge liver Plain enzyme II, MBP fusion heparinase III, nucleotide sequence are respectively SEQ ID NO.3, SEQ ID NO.4, SEQ in sequence table ID NO.5。

A kind of MBP fusion chondrosulphatase containing link peptide SEQ ID NO.1, including MBP merge chondroitin sulfate Enzyme B, MBP merge chondrosulphatase AC, and nucleotide sequence is respectively SEQ ID NO.6 in sequence table, SEQ ID NO.7.

A kind of MBP fusion heparinase containing link peptide SEQ ID NO.2, including MBP fusion heparinase I, MBP merge liver Plain enzyme II, MBP fusion heparinase III, nucleotide sequence are respectively SEQ ID NO.8, SEQ ID NO.9, SEQ in sequence table ID NO.10。

A kind of MBP fusion chondrosulphatase containing link peptide SEQ ID NO.2, including MBP merge chondroitin sulfate Enzyme B, MBP merge chondrosulphatase AC, and nucleotide sequence is respectively SEQ ID NO.11, SEQ ID in sequence table NO.12。

The present invention designs a kind of link peptide, the molecular modification for recombinant protein.Polysaccharide in Flavobacterium heparinum is split Enzyme is solved, using the method for MBP amalgamation and expression, the catalytic activity for improving MBP fusion enzyme is redesigned and replaced to the link peptide And thermal stability.In MBP amalgamation and expression system, MBP hydrotropy is improved by the redesign and replacement of the link peptide, helps folding Folded, increase target protein activity and thermal stability.

The present invention is redesigned and is replaced to the MBP link peptide for merging polysaceharide lyase by the means of genetic engineering It changes, the final fermentation enzyme activity obtained in Escherichia coli tentatively reaches industrial application level and the MBP with outstanding thermal stability Fusion heparinase I, II, III, chondrosulphatase B, AC.

Detailed description of the invention

Fig. 1 MBP fusion heparinase I link peptide replacement front and back expression effect characterization,

Wherein T:Total Protein indicates that total protein sample, S:Soluble Protein indicate soluble protein, I:Insoluble Protein indicates insoluble albumen.

II link peptide of Fig. 2 MBP fusion heparinase replacement front and back expression effect characterization,

Wherein T:Total Protein indicates that total protein sample, S:Soluble Protein indicate soluble protein, I:Insoluble Protein indicates insoluble albumen.

III link peptide of Fig. 3 MBP fusion heparinase replacement front and back expression effect characterization,

Wherein T:Total Protein indicates that total protein sample, S:Soluble Protein indicate soluble protein, I:Insoluble Protein indicates insoluble albumen.

Fig. 4 MBP merges chondrosulphatase B link peptide replacement front and back expression effect characterization,

Wherein T:Total Protein indicates that total protein sample, S:Soluble Protein indicate soluble protein, I:Insoluble Protein, indicates insoluble albumen, and P is the purifying protein through MBPTrap HP mono- after purification.

Fig. 5 MBP merges chondrosulphatase AC link peptide replacement front and back expression effect characterization,

Wherein T:Total Protein indicates that total protein sample, S:Soluble Protein indicate soluble protein, I:Insoluble Protein, indicates insoluble albumen, and P is the purifying protein through MBPTrap HP mono- after purification.

Fig. 6 MBP fusion heparinase I link peptide replacement front and back thermal stability compares.

II link peptide of Fig. 7 MBP fusion heparinase replacement front and back thermal stability compares.

Fig. 8 MBP fusion heparinase III link peptide replacement front and back thermal stability compares.

Fig. 9 MBP fusion chondrosulphatase B link peptide replacement front and back thermal stability compares.

Figure 10 MBP fusion chondrosulphatase AC link peptide replacement front and back thermal stability compares.

The degradation of substrates mechanism parsing of Figure 11 MBP fusion heparinase I link peptide replacement front and back: product aggregate analysis,

Wherein, 11A Composition distribution；11B UV detector.

The degradation of substrates mechanism parsing of Figure 12 MBP fusion heparinase I link peptide replacement front and back: disaccharide component analysis,

Wherein, 12A:MBP-HepA；12B:MBP-R-HepA；12C:MBP-F-HepA.

The degradation of substrates mechanism parsing of II link peptide of Figure 13 MBP fusion heparinase replacement front and back: product aggregate analysis,

Wherein, 13A Composition distribution；13B UV detector.

The degradation of substrates mechanism parsing of II link peptide of Figure 14 MBP fusion heparinase replacement front and back: disaccharide component analysis,

Wherein, 14A:MBP-HepA；14B:MBP-R-HepA；14C:MBP-F-HepA.

The degradation of substrates mechanism parsing of III link peptide of Figure 15 MBP fusion heparinase replacement front and back: product aggregate analysis,

Wherein, 15A Composition distribution；15B UV detector.

The degradation of substrates mechanism parsing of II link peptide of Figure 16 MBP fusion heparinase replacement front and back: disaccharide component analysis,

Wherein, 16A:MBP-HepA；16B:MBP-R-HepA；16C:MBP-F-HepA.

Specific embodiment

The present invention is illustrated combined with specific embodiments below.

Design, the MBP of link peptide containing target fusion polysaceharide lyase vector construction and the expression of 1 link peptide of embodiment

1, the design of link peptide

(1) origin of link peptide:

Due to the length and property (hard and soft degree) property that fusion protein can be significantly affected of link peptide, this is mainly due to The space length and positional relationship in fusion protein between two elements are changed, mutual active force and space bit are caused Resistance is different, and then different degrees of influence is caused to the structure of albumen, and the biological function for finally influencing two elements is especially Biological function as main catalyzing enzyme.Influence of the length for the property of fusion protein is the most significant, however also because This reason, the variation for being difficult prediction length influence fusion protein in what degree, so applying upper desired reason for us Property design fusion protein it is very unfavorable.If on the basis of appropriate length, by the hard and soft degree of link peptide come to fusion protein Space length between two elements and positional relationship carry out " fine tuning ", then may be implemented to fusion protein physicochemical property by Step section, we, can be according to the property of different link peptides design institute on the basis of fully understanding to target protein in this way The fusion protein needed, to obtain the fusion protein of different physical and chemical attributes.

Based on above-mentioned general idea, we just include 5 amino acid the flexible peptide unit F(in nature) and just Property peptide unit R(include 5 amino acid) as we design primary element, choose FFFFF and RRRRR be study link peptide Length unit, on the basis of 5 link peptide element lengths, the hard and soft unit of opposite is replaced to be connected with heterozygosis, from And a series of heterozygosis connection peptide library that hard and soft degree have gradual change law is obtained, melted by replacing these link peptides to different In hop protein, we very light can must obtain required fusion protein.

(2) design software: " design " of link peptide is used in fact or based on rigid, flexible unit permutation and combination DiscoverStudio software carries out molecular simulation to the link peptide conformation of different permutation and combination, passes through subsequent experimental result It is compared, corrects with the analog result, obtain the link peptide for really being able to be adjusted on space length and positional relationship Candidate library and link peptide basis for selecting.It is final to obtain link peptide L1, L2, nucleotide sequence be respectively SEQ ID NO.1 or Shown in SEQ ID NO.2.

2, the MBP of link peptide containing target merges polysaceharide lyase vector construction

The MBP of link peptide containing target merges polysaceharide lyase vector construction step: expressing heparinase I fusion protein carry respectively Body pMal-hepA(patent No. CN1699424A), the heparinaseⅡ fusion protein expression vector pMal-hepB(patent No. CN101942024A), heparinase III fusion protein expression vector pMal-hepC(patent No. CN101942025A), chondroitin sulfate Plain enzyme B fusion protein expression vector pMAL-ChSase B(patent No. CN103103173A), chondrosulphatase AC fusion protein Expression vector pMAL-ChSase AC(patent No. CN103103174A) respectively with " NdeI+HindIII ", " NdeI+ PstI ", " NdeI+PstI ", " NdeI+HindIII ", " NdeI+EcoRI " carry out digestion, then respectively and with identical Link peptide L1, L2 of the first end restriction enzyme site, is attached with T4 ligase, and the fusion polysaccharide for obtaining the link peptide containing target is split Solve zymophore.

Expression, purifying and the enzyme activity analysis of 2 target protein of embodiment

The fusion polysaceharide lyase carrier expression of target link peptide will be contained: the fusion polysaccharide cracking of link peptide containing target will be built Zymophore plasmid converts Escherichia coli respectively, and conversion has the bacterial strain of recombinant plasmid in the LB training containing 100ug/mL ampicillin It supports after shaking overnight incubation (37 DEG C, 180rpm, 14-16h) in base, is inoculated into LB/M9YE fermentation training according to the ratio of 1:100 It supports in base, 37 DEG C, 180rpm shakes to logarithmic growth phase, goes to 200rpm, 15 DEG C of low temperature induction culture 20- after IPTG is added 22h。

Protein extraction and purification process: will be delayed after the Escherichia coli fermentation bacterium solution collection bacterium of 100ml mesh with 20ml protein extraction Fliud flushing is resuspended, ultrasonication thallus, and 4 DEG C of breakdown products, 10000rpm is centrifuged 30 minutes, collects supernatant, filters through 0.22 μm of film, Slowly (1mL/min) passes through MBPTrap the HP(1 ml, 5ml of GE Healthcare) affinity column, it is in charge of collection eluent, examines Survey each tubulin concentration, SDS-PAGE electrophoresis detection purification effect.Protein purification is enterprising in AKTA plus protein purification system Row, it is whole that the polysaccharide cracking MBP fusion protein purified is operated in 4 DEG C of low temperature chromatography cabinets.

Enzyme activity detection uses the optical absorption method of 232nm, the definition of the enzyme activity of 1IU be 30 DEG C of 1um generated per minute not The reaction effect of saturated bond.The GOLDS54 ultraviolet-uisible spectrophotometer of concrete operations Prism Optical Technology Co is surveyed Determine absorbance versus time curve.Scanning wavelength is set to 232 nm, and the time is 3 min.Take reaction buffer (20 mM Tris, 200 mM NaCl, after completely dissolution with the salt acid for adjusting pH value of 1 M to 7.4,4 DEG C of preservations) and a certain amount of enzyme solutions The totally 1000 specific ratios of μ L(need to be according to the big minor adjustment of enzyme solution vigor), 500 μ L substrate solutions (17 mM Tris, 44 mM NaCl, 3.5 mM CaCl2,25g/L substrates, with the salt acid for adjusting pH value of 1 M to 7.0,4 DEG C of preservations after being sufficiently stirred.) its In, I, II, III substrate of MBP fusion heparinase: 25 g/L heparin sodiums --- the enzyme activity for MBP fusion heparinase I or II is surveyed It is fixed, 25 g/L Heparan sulfate sodium --- the enzyme activity determinations for Heparinase I II；Chondrosulphatase B, AC substrate: 25 g/ L chondroitin sulfate.Then spectrophotometer is immediately placed in after quartz colorimetric utensil, mixing to start to scan, survey the unit time in The absorbance change △ A232 of 232nm.Extinction coefficient epsilon=3800 M-1 cm-1.Enzyme activity is calculated according to Beer law.

1, MBP fusion heparinase I

Heparinase I fusion protein (patent No. CN1699424A) is named as MBP-HepA as control, replacement link peptide 1 MBP fusion heparinase I is named as MBP-R-HepA；The MBP fusion heparinase I of replacement link peptide 2 is named as MBP-F-HepA.

MBP fusion heparinase I after purification, carries out SDS-PAGE electrophoresis, the result is shown in Figure 1.

It is analyzed using 5.0 software of Bandscan, the trend of replacement front and back target protein soluble-expression has indifference, target The specific gravity that albumen accounts for total protein increased, and specific gravity is increased to 61% from 48% or so.

It is that analysis measurement is carried out with supernatant soluble protein S component when analyzing enzyme activity.

The shake flask fermentation total enzyme activity measurement of 1 MBP fusion heparinase I link peptide of table replacement front and back

After link peptide is replaced, fermentation total enzyme activity of the MBP fusion heparinase I in shaking flask reaches 41230.18 IU/L, is It is currently known peak.

(2) MBP fusion heparinase II

HeparinaseⅡ fusion protein (patent No. CN101942024A) is named as MBP-HepB as control；Replacement connection The MBP fusion heparinase II of peptide 1 is named as MBP-R-HepB；The MBP fusion heparinase I of replacement link peptide 2 is named as MBP-F- HepB。

MBP fusion heparinase II after purification, carries out SDS-PAGE electrophoresis, as a result sees Fig. 2.

After link peptide replacement, the specific gravity that MBP-R-HepB target protein accounts for total protein increases by 1 times, MBP-F-HepB target egg The white specific gravity no significant difference for accounting for total protein.

The shake flask fermentation total enzyme activity measurement of 2 MBP fusion heparinase of table, II link peptide replacement front and back

After link peptide is replaced, fermentation total enzyme activity of the MBP fusion heparinase II in shaking flask reaches 3674.82 IU/L, is It is currently known peak.

(3) MBP fusion heparinase III

Heparinase III fusion protein (patent No. CN101942025A) is named as MBP-HepC as control；Replacement connection The MBP fusion heparinase II of peptide 1 is named as MBP-R-HepC；The MBP fusion heparinase I of replacement link peptide 2 is named as MBP-F- HepC。

MBP fusion heparinase III after purification, carries out SDS-PAGE electrophoresis, as a result sees Fig. 3.

After link peptide replacement, the specific gravity that MBP-R-HepC target protein accounts for total protein increases by 1.5 times, MBP-F-HepB target The specific gravity that albumen accounts for total protein increases by 1 times.

The shake flask fermentation total enzyme activity measurement of 3 heparinase III link peptide of table replacement front and back

After link peptide is replaced, fermentation total enzyme activity of the MBP fusion heparinase III in shaking flask reaches 12934.34 IU/L, It is to be currently known peak.

(4) MBP merges chondrosulphatase B

MBP merges chondrosulphatase B after purification, carries out SDS-PAGE electrophoresis, and MBP merges chondrosulphatase B connection Peptide replacement front and back expression effect is shown in Fig. 4.

After link peptide replacement, the specific gravity that MBP-R-HepB target protein accounts for total protein increases by 1 times, MBP-F-HepB target egg The white specific gravity no significant difference for accounting for total protein.

4 MBP of table merges the shake flask fermentation total enzyme activity measurement of chondrosulphatase B link peptide replacement front and back

After link peptide is replaced, MBP merges fermentation total enzyme activity of the chondrosulphatase B in shaking flask and reaches 24206.2IU/ L is to be currently known peak.

(5) MBP merges chondrosulphatase AC

MBP merges chondrosulphatase AC after purification, carries out SDS-PAGE electrophoresis, and MBP merges chondrosulphatase B connection Peptide replacement front and back expression effect is shown in Fig. 5.

After link peptide replacement, the specific gravity that MBP-R-HepB target protein accounts for total protein increases by 2.5 times, MBP-F-HepB target The specific gravity that albumen accounts for total protein increases by 1.5 times.

5 MBP of table merges the shake flask fermentation total enzyme activity measurement of chondrosulphatase AC link peptide replacement front and back

After link peptide is replaced, MBP merges fermentation total enzyme activity of the chondrosulphatase AC enzyme in shaking flask and reaches 22162IU/L is to be currently known peak.

4 link peptide of embodiment replacement front and back MBP merges polysaceharide lyase thermal stability analysis

Enzyme activity of the enzymatic activity as time point for 0 moment will be measured immediately after protein purification, by pure enzyme as detection temperature Middle water-bath, choosing different time points is sampled survey enzyme activity, is compared with 0 moment enzyme activity, when calculating the moment relative to 0 Carve the percentage of enzyme activity.Persistently the operation to enzyme activity is down to lower unknown, is thus obtained a series of warm bath time points and is remained These points are attached up to the inactivation curve of the enzyme at such a temperature by the corresponding relationship point of remaining enzyme activity, also can be to this The stability of enzyme at such a temperature is evaluated.

Enzyme heat stability is analyzed after the shake flask fermentation of 1.MBP fusion heparinase I link peptide replacement front and back

After link peptide is replaced, enzyme activity half-life period of the MBP fusion heparinase I at 30 DEG C is increased to the left side 35min from 10min As a result Fig. 6 is seen on the right side.

2, enzyme heat stability is analyzed after the shake flask fermentation of II link peptide of MBP fusion heparinase replacement front and back

After link peptide is replaced, enzyme activity half-life period of the MBP fusion heparinase II at 30 DEG C is increased to the left side 250h from 80h As a result Fig. 7 is seen on the right side.

3, enzyme heat stability is analyzed after the shake flask fermentation of III link peptide of MBP fusion heparinase replacement front and back

After link peptide is replaced, enzyme activity half-life period of the MBP fusion heparinase III at 30 DEG C from 60h be increased to 250h with On, as a result see Fig. 8.

4, enzyme heat stability is analyzed after MBP merges chondrosulphatase B link peptide replacement front and back shake flask fermentation

After link peptide is replaced, MBP fusion chondrosulphatase B thermal stability measuring the results are shown in Table 6；MBP fusion Enzyme activity half-life period of the chondrosulphatase B at 30 DEG C from the left and right 500h or more is increased to for 24 hours, as a result sees Fig. 9.

6 MBP of table merges chondrosulphatase B stability contrast table

5, enzyme heat stability is analyzed after MBP merges chondrosulphatase AC link peptide replacement front and back shake flask fermentation

After link peptide is replaced, MBP fusion chondrosulphatase AC thermal stability measuring the results are shown in Table 7；MBP fusion Enzyme activity half-life period of the chondrosulphatase AC under 30 degree is increased to 100h or so, the result is shown in Figure 10 from 8.3h.

7 MBP of table merges chondrosulphatase AC stability contrast table

Influence of 5 link peptide of the embodiment replacement front and back MBP fusion polysaceharide lyase to degradation of substrates mechanism

Choosing MBP fusion polysaceharide lyase is research object, passes through Bacillus coli expression and protein extraction, purifying Operating procedure obtains the sterling of the enzyme.Corresponding substrate is chosen, pure enzyme is added in substrate, carries out bottom under suitable conditions Object degradation reaction, using the absorbance value under 232nm as Testing index, as reaction end when no longer being changed using the absorption value.It will be anti- Product is answered to pass through the impurity such as the albumen a series of purification steps removals the inside such as be centrifuged, take supernatant, filtering, by the oligosaccharides group of collection Divide the analysis for carrying out HPLC, obtains the map of the enzyme catabolite, different control samples is chosen according to different purposes, such as Compare link peptide replacement front and back catabolite difference just replaced using link peptide before fusion enzyme catabolite as compare. Specific relatively parameter includes appearance time and peak area, and only the two numerical value are almost the same, can judge two fusion enzymes It is whether identical to the degradation mechanism of substrate.The detection type of HPLC mainly includes the spectrum analysis and drop of catabolite principal component The spectrum analysis of disaccharide component in product is solved, Detection wavelength used in the two detections is different, therefore in order to rigorous Property, need the two wavelength to be all measured.

1, MBP fusion heparinase I link peptide replacement front and back is to degradation of substrates mechanism impact analysis

Component after thoroughly degrading to the MBP fusion heparinase I of link peptide replacement front and back to heparin substrate is analyzed, and is tied Fruit such as Figure 11.Figure 12 is shown in the analysis of catabolite disaccharide component.

2, II link peptide of MBP fusion heparinase replacement front and back is to degradation of substrates mechanism impact analysis

Component after thoroughly degrading to the MBP fusion heparinase II of link peptide replacement front and back to heparin substrate is analyzed, As a result such as Figure 13.Figure 14 is shown in the analysis of catabolite disaccharide component.

3, III link peptide of MBP fusion heparinase replacement front and back is to degradation of substrates mechanism impact analysis

Component after thoroughly degrading to the MBP fusion heparinase II of link peptide replacement front and back to heparin substrate is analyzed, As a result such as Figure 15.Figure 16 is shown in the analysis of catabolite disaccharide component.

From the foregoing, it will be observed that by HPLC means in overall degradation product and product disaccharide component analysis shows, MBP melts Close that heparinase I, MBP fusion heparinase II, the replacement of link peptide does not influence heparinase to the drop of substrate in MBP fusion heparinase III Solution mechanism.

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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 tgaggctgcc gcaaaggaag cggcagcgaa agaggcggcc 1140

gcaaaagagg cagcggcgaa agaagctgcg gccaagcaaa cgaaagcgga cgtcgtctgg 1200

aaagatgtcg acggcgtgag catgcctatt ccgccgaaaa cgcacccgcg tttgtatctg 1260

cgtgaacagc aagtgccgga cctgaagaat cgcatgaacg atccgaagct gaaaaaggtt 1320

tgggccgaca tgatcaaaat gcaggaagat tggaaaccgg cggacattcc ggaagttaag 1380

gattttcgct tttacttcaa tcagaaaggt ctgacggtcc gtgttgagtt gatggcactg 1440

aattatctga tgaccaagga cccgaaagtg ggtcgtgagg cgattacgag catcatcgat 1500

accctggaaa ccgcgacctt taagccagcg ggtgatatct ctcgtggcat tggtctgttt 1560

atggttacgg gcgcaatcgt ttacgactgg tgctacgacc agctgaagcc ggaagagaaa 1620

acccgtttcg tgaaagcttt tgtccgcctg gcgaagatgt tggagtgcgg ttacccaccg 1680

gttaaggata agtcgatcgt cggtcacgcg tccgagtgga tgatcatgcg tgatttgctg 1740

agcgtgggta ttgcaattta cgatgagttc ccggagatgt acaatctggc agcgggtcgc 1800

ttcttcaaag aacacctggt tgcccgtaat tggttttatc cgtcccataa ctaccaccag 1860

ggtatgagct acctgaatgt ccgttttacg aacgatttgt tcgcgctgtg gattctggac 1920

cgtatgggtg cgggtaatgt gtttaacccg ggtcaacagt tcattttgta tgacgcgatt 1980

tacaagcgtc gcccggatgg ccagattctg gcgggtggtg acgtggacta tagccgcaag 2040

aaaccgaagt actataccat gccggccttg ctggcgggta gctactataa agacgaatac 2100

ctgaactacg agtttctgaa agacccgaat gtggaaccgc attgtaagct gttcgagttc 2160

ctgtggcgcg acacccaact gggctcccgc aaacctgacg atctgccact gtcccgttat 2220

tctggcagcc cgtttggctg gatgatcgcg cgtactggct ggggtccgga atcggtgatc 2280

gcagaaatga aagtcaacga atacagcttc ctgaatcacc agcatcaaga cgctggcgct 2340

ttccagatct actacaaggg tccgctggcc attgacgccg gcagctatac gggtagcagc 2400

ggtggttata actccccgca taacaaaaac tttttcaagc gtaccattgc gcacaatagc 2460

ctgctgattt acgaccctaa agaaaccttt agcagcagcg gctacggtgg tagcgaccac 2520

acggattttg ccgcgaacga cggtggccaa cgcctgccgg gtaagggctg gattgcccct 2580

cgtgatctga aagaaatgct ggccggtgac tttcgtacgg gcaagatcct ggcgcagggc 2640

tttggtccgg ataaccaaac cccggactat acttatctga aaggcgatat taccgcagca 2700

tatagcgcaa aagtgaaaga ggtcaaacgt agctttctgt tcctgaacct gaaagacgct 2760

aaggttccgg cagcgatgat cgttttcgat aaggtggtgg catctaatcc ggacttcaag 2820

aagttctggc tgttgcatag catcgagcaa ccggagatca aaggcaacca gattactatc 2880

aaacgtacca aaaacggtga cagcggtatg ctggttaaca ccgctctgct gccagacgct 2940

gccaatagca acatcacctc tattggtggt aagggcaaag atttctgggt gtttggcacc 3000

aattacacga atgacccgaa accgggcact gatgaggcgc tggagcgtgg cgagtggcgc 3060

gtggaaatca ccccgaagaa ggcagcggca gaggactact atttgaacgt aatccaaatt 3120

gctgataata cgcagcagaa actgcacgag gtgaagcgca ttgatggtga taaagttgtc 3180

ggtgttcaac tggcggaccg catcgtcacc ttcagcaaga ccagcgaaac ggttgatcgt 3240

ccgtttggtt ttagcgtggt tggtaaaggc accttcaagt tcgtcatgac cgatctgctg 3300

ccgggcacgt ggcaagttct gaaggatggt aagatcctgt acccggcact gagcgcgaag 3360

ggcgacgatg gtccgttgta cttcgagggt accgaaggca cctatcgttt cctgcgctaa 3420

<210> 5

<211> 3084

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 5

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 tgaggctgcc gcaaaggaag cggcagcgaa agaggcggcc 1140

gcaaaagagg cagcggcgaa agaagctgcg gccaagcaat ccagcagcat tactcgtaaa 1200

gacttcgacc acatcaacct ggaatatagc ggcttggaga aagtgaacaa ggcggtggcg 1260

gctggcaatt atgacgatgc cgcgaaagca ttgctggcgt actaccgcga gaaaagcaaa 1320

gcccgcgagc ctgactttag caatgctgag aagccggcag atattcgtca gccgattgat 1380

aaagtaaccc gcgaaatggc cgataaagcc ctggtccacc agttccagcc gcataaaggt 1440

tatggttatt tcgattatgg caaggatatc aactggcaga tgtggccggt caaggataac 1500

gaggttcgct ggcaactgca ccgtgtgaaa tggtggcagg caatggccct ggtctaccat 1560

gcgacgggtg acgagaaata cgctcgtgag tgggtttacc agtacagcga ctgggcgcgt 1620

aagaatccgc tgggtctgag ccaagacaat gacaagtttg tgtggcgtcc tctggaagtg 1680

agcgaccgtg tccaatctct gccgccgacg ttctccctgt tcgtcaactc cccggcattc 1740

acgccagcat tcctgatgga attcctgaac tcttaccacc agcaagcaga ctacctgagc 1800

acccattatg cggaacaagg taaccaccgt ttgttcgagg cgcaacgtaa tctgtttgcg 1860

ggcgtgagct tcccggagtt taaagacagc ccgcgttggc gtcaaaccgg catcagcgtg 1920

ctgaacaccg aaatcaagaa acaggtttat gcggatggta tgcaattcga actgagcccg 1980

atctaccatg ttgccgcgat cgacattttc ctgaaggcgt atggcagcgc caaacgtgtt 2040

aacctggaaa aagaatttcc gcagagctac gttcaaacgg ttgagaatat gatcatggct 2100

ctgatctcta ttagcctgcc ggactataac accccgatgt ttggtgactc gtggattacg 2160

gacaaaaact ttcgcatggc gcagtttgca agctgggcac gcgtctttcc ggcaaaccaa 2220

gcgatcaagt acttcgcgac cgatggtaag caaggtaaag caccgaactt cctgagcaag 2280

gcgctgagca atgcgggctt ctacaccttt cgtagcggct gggataagaa cgccactgtc 2340

atggtcctga aagcgagccc accgggtgaa ttccacgcgc agccggacaa cggtacgttc 2400

gagttgttta tcaaaggtcg caatttcact ccagatgcgg gtgtttttgt gtacagcggc 2460

gacgaggcca tcatgaagct gcgtaactgg tatcgccaga cgcgtattca ctcgaccctg 2520

accctggata atcagaatat ggtgattacc aaggcacgcc agaataagtg ggaaacgggt 2580

aacaacctgg acgtcctgac ctatacgaat ccgagctacc cgaatttgga tcaccagcgt 2640

tccgttctgt tcatcaataa gaaatacttc ttggtcattg accgtgcaat cggtgaggcg 2700

accggtaacc tgggtgttca ttggcagctg aaggaagata gcaatccggt ttttgataaa 2760

accaaaaacc gtgtttacac cacctaccgt gacggcaata acctgatgat tcagagcttg 2820

aatgctgatc gcaccagcct gaacgaggaa gagggcaaag tgtcttatgt atacaataag 2880

gagctgaagc gtccggcgtt cgtttttgag aagccaaaga agaatgcagg tacccaaaac 2940

tttgtgagca ttgtgtaccc gtatgatggt caaaaagctc cggagatttc catccgcgaa 3000

aacaaaggta acgattttga gaaaggcaag ctgaatttga ccctgacgat taatggcaag 3060

caacagttgg tcctggtgcc gtaa 3084

<210> 6

<211> 2622

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 6

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 tgaggctgcc gcaaaggaag cggcagcgaa agaggcggcc 1140

gcaaaagagg cagcggcgaa agaagctgcg gccaagcagg ttgttgcttc aaatgaaact 1200

ttataccagg ttgtaaagga ggtaaaaccc ggtggtctgg tacagattgc cgatgggact 1260

tataaagatg ttcagctgat tgtcagcaat tcaggaaaat ctggtttgcc catcactatt 1320

aaagccctga acccgggtaa ggtttttttt accggagatg ctaaagtaga gctgaggggc 1380

gagcacctga tactggaagg catctggttt aaagacggga acagagctat tcaggcatgg 1440

aaatcacatg gacccggatt ggtggctata tatggtagct ataaccgcat taccgcatgt 1500

gtatttgatt gttttgatga agccaattct gcttacatta ctacttcgct taccgaagac 1560

ggaaaggtac ctcaacattg ccgcatagac cattgcagtt ttaccgataa gatcactttt 1620

gaccaggtaa ttaacctgaa caatacagcc agagctatta aagacggttc ggtgggagga 1680

ccggggatgt accatcgtgt tgatcactgt tttttttcca atccgcaaaa accgggtaat 1740

gccggagggg gaatcaggat tggctattac cgtaatgata taggccgttg tctggtagac 1800

tctaacctgt ttatgcgtca ggattcggaa gcagagatca tcaccagcaa atcgcaggaa 1860

aatgtttatt atggtaatac ttacctgaat tgccagggca ccatgaactt tcgtcacggt 1920

gatcatcagg tggccattaa caatttttat ataggcaatg accagcgatt tggatacggg 1980

ggaatgtttg tttggggaag caggcatgtc atagcctgta attattttga gctgtccgaa 2040

accataaagt cgagggggaa cgccgcattg tatttaaacc ccggtgctat ggcttcggag 2100

catgctcttg ctttcgatat gttgatagcc aacaacgctt tcatcaatgt aaatgggtat 2160

gccatccatt ttaatccatt ggatgagcgc agaaaagaat attgtgcagc caataggctt 2220

aagttcgaaa ccccgcacca gctaatgtta aaaggcaatc ttttctttaa ggataaacct 2280

tatgtttacc cattttttaa agatgattat tttatagcag ggaaaaatag ctggactggt 2340

aatgtagcct taggtgtgga aaagggaatc cctgttaaca tttcggccaa taggtctgcc 2400

tataagccgg taaaaattaa agatatccag cccatagaag gaatcgctct tgatctcaat 2460

gcgctgatca gcaaaggcat tacaggaaag ccccttagct gggatgaagt aaggccctac 2520

tggttaaaag aaatgcccgg gacgtatgct ttaacggcca ggctttctgc agatagggct 2580

gcaaagttta aagccgtaat taaaagaaat aaagagcact ga 2622

<210> 7

<211> 3216

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 7

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 tgaggctgcc gcaaaggaag cggcagcgaa agaggcggcc 1140

gcaaaagagg cagcggcgaa agaagctgcg gccaagcagc agaccggtac tgcagaactg 1200

attatgaagc gggtgatgct ggaccttaaa aagcctttgc gcaatatgga taaggtggcg 1260

gaaaaggacc tgaatacgct gcagcctgac ggtagctgga aggatgtgcc ttataaagat 1320

gatgccatga ccaattggtt gccaaacaac cacctgctac aattggaaac tattatacag 1380

gcttatattg aaaaagatag tcactattat ggcgacgata aagtgtttga ccagatttcc 1440

aaagctttta agtattggta tgacagcgac ccgaaaagcc gcaactggtg gcacaatgaa 1500

attgccactc cgcaggccct tggtgaaatg ctgatcctga tgcgttacgg taaaaagccg 1560

cttgatgaag cattggtgca taaattgacc gaaagaatga agcggggcga accggagaag 1620

aaaacggggg ccaacaaaac agatatcgcc ctgcattact tttatcgtgc tttgttaacg 1680

tctgatgagg ctttgctttc cttcgccgta aaagaattgt tttatcccgt acagtttgta 1740

cactatgagg aaggcctgca atacgattat tcctacctgc agcacggtcc gcaattacag 1800

atatcgagct acggtgccgt atttattacc ggggtactga aacttgccaa ttacgttagg 1860

gatacccctt atgctttaag taccgagaaa ctggctatat tttcaaagta ttaccgcgac 1920

agttatctga aagctatccg tggaagttat atggatttta acgtagaagg ccgcggagta 1980

agccggccag acattctaaa taaaaaggca gaaaaaaaga ggttgctggt ggcgaagatg 2040

atcgatctta agcatactga agaatgggct gatgcgatag ccaggacaga tagcacagtt 2100

gcggccggct ataagattga gccctatcac catcagttct ggaatggtga ttatgtgcaa 2160

catttaagac ctgcctattc ttttaatgtt cgtatggtga gtaagcggac ccgacgcagt 2220

gaatccggca ataaagaaaa cctgctgggc aggtatttat ctgatggggc tactaacata 2280

caattgcgcg gaccagaata ctataacatt atgccggtat gggaatggga caagattcct 2340

ggcataacca gccgtgatta tttaaccgac agacctttga cgaagctttg gggagagcag 2400

gggagcaatg actttgcagg aggggtgtct gatggtgtat acggggccag tgcctacgca 2460

ttggattacg atagcttaca ggcaaagaaa gcctggttct tttttgacaa agagattgta 2520

tgtcttggtg ccggtatcaa cagcaatgcc cctgaaaaca ttaccactac ccttaaccag 2580

agctggttaa atggcccggt tataagtact gcaggtaaaa ccggccgggg taaaataaca 2640

acgtttaaag cacagggaca gttctggttg ttgcacgatg cgattggtta ttactttcct 2700

gaaggggcca accttagtct gagtacccag tcgcaaaaag gcaattggtt ccacatcaac 2760

aattcacatt caaaagatga agtttctggt gatgtattta agctttggat caaccatggt 2820

gccaggccag aaaatgcgca gtatgcttat atcgttttgc cgggaataaa caagccggaa 2880

gaaattaaaa aatataatgg aacggcaccg aaagtccttg ccaataccaa ccagctgcag 2940

gcagtttatc atcagcagtt agatatggta caggctatct tctatacagc tggaaaatta 3000

agcgtagcgg gcatagaaat tgaaacagat aagccatgtg cagtgctgat caagcacatc 3060

aatggcaagc aggtaatttg ggctgccgat ccattgcaaa aagaaaagac tgcagtgttg 3120

agcatcaggg atttaaaaac aggaaaaaca aatcgggtaa aaattgattt tccgcaacag 3180

gaatttgcag gtgcaacggt tgaactgaaa tagtaa 3216

<210> 8

<211> 2268

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 8

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 tggtggtggc ggcagcggtg gcggcggtag cggcggtggt 1140

ggatccggtg gcggtggttc tggtggtggt ggcagccagc aaaaaaaatc cggtaacatc 1200

ccttaccggg taaatgtgca ggccgacagt gctaagcaga gcgagattat tgacaacaaa 1260

tgggtggcag taggcatcaa taaaccttat gcattacaat atgacgataa actgcgcttt 1320

aatggaaaac catcctatcg ctttgagctt aaagccgaag acaattcgct tgaaggttat 1380

gctgcaggag aaacaaaggg ccgtatagaa ttgtcgtaca gctatgcaac caccaatgat 1440

tttaagaaat ttcccccaag cgtataccaa aatgcgcaaa agctaaaaac cgtttatcat 1500

tacggcaaag ggatttgtga acaggggagc tcccgcagct ataccttttc agtgtacata 1560

ccctcctcct tccccgacaa tgcgactact atttttgccc aatggcatgg tgcacccagc 1620

agaacgcttg tagctacacc agagggagaa attaaaacac tgagcataga agagtttttg 1680

gccttatacg accgcatgat cttcaaaaaa aatatcgccc atgataaagt tgaaaaaaaa 1740

gataaggacg gaaaaattac ttatgtagcc ggaaagccaa atggctggaa ggtagaacaa 1800

ggtggttatc caccgctggc ctttggtttt tctaaagggt atttttacat caaggcaaac 1860

tccgaccggc agtggcttac cgacaaagcc gaccgtaaca atgccaatcc cgagaatagt 1920

gaagtaatga agccctattc ctcggaatac aaaacttcta ccattgccta taaaatgccc 1980

tttgcccagt tccctaaaga ttgctggatt acttttgatg tcgccataga ctggacgaaa 2040

tatggaaaag aggccaatac aattttgaaa cccggtaagc tggatgtgat gatgacttat 2100

accaagaata agaaaccaca aaaagcgcat atcgtaaacc agcaggaaat cctgatcgga 2160

cgtaacgatg acgatggcta ttacttcaaa tttggaattt acagggtcgg taacagcacg 2220

gtcccggtta cttataacct gagcgggtac agcgaaactg ccagatag 2268

<210> 9

<211> 3420

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 9

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 tggtggtggc ggcagcggtg gcggcggtag cggcggtggt 1140

ggatccggtg gcggtggttc tggtggtggt ggcagccaaa cgaaagcgga cgtcgtctgg 1200

aaagatgtcg acggcgtgag catgcctatt ccgccgaaaa cgcacccgcg tttgtatctg 1260

cgtgaacagc aagtgccgga cctgaagaat cgcatgaacg atccgaagct gaaaaaggtt 1320

tgggccgaca tgatcaaaat gcaggaagat tggaaaccgg cggacattcc ggaagttaag 1380

gattttcgct tttacttcaa tcagaaaggt ctgacggtcc gtgttgagtt gatggcactg 1440

aattatctga tgaccaagga cccgaaagtg ggtcgtgagg cgattacgag catcatcgat 1500

accctggaaa ccgcgacctt taagccagcg ggtgatatct ctcgtggcat tggtctgttt 1560

atggttacgg gcgcaatcgt ttacgactgg tgctacgacc agctgaagcc ggaagagaaa 1620

acccgtttcg tgaaagcttt tgtccgcctg gcgaagatgt tggagtgcgg ttacccaccg 1680

gttaaggata agtcgatcgt cggtcacgcg tccgagtgga tgatcatgcg tgatttgctg 1740

agcgtgggta ttgcaattta cgatgagttc ccggagatgt acaatctggc agcgggtcgc 1800

ttcttcaaag aacacctggt tgcccgtaat tggttttatc cgtcccataa ctaccaccag 1860

ggtatgagct acctgaatgt ccgttttacg aacgatttgt tcgcgctgtg gattctggac 1920

cgtatgggtg cgggtaatgt gtttaacccg ggtcaacagt tcattttgta tgacgcgatt 1980

tacaagcgtc gcccggatgg ccagattctg gcgggtggtg acgtggacta tagccgcaag 2040

aaaccgaagt actataccat gccggccttg ctggcgggta gctactataa agacgaatac 2100

ctgaactacg agtttctgaa agacccgaat gtggaaccgc attgtaagct gttcgagttc 2160

ctgtggcgcg acacccaact gggctcccgc aaacctgacg atctgccact gtcccgttat 2220

tctggcagcc cgtttggctg gatgatcgcg cgtactggct ggggtccgga atcggtgatc 2280

gcagaaatga aagtcaacga atacagcttc ctgaatcacc agcatcaaga cgctggcgct 2340

ttccagatct actacaaggg tccgctggcc attgacgccg gcagctatac gggtagcagc 2400

ggtggttata actccccgca taacaaaaac tttttcaagc gtaccattgc gcacaatagc 2460

ctgctgattt acgaccctaa agaaaccttt agcagcagcg gctacggtgg tagcgaccac 2520

acggattttg ccgcgaacga cggtggccaa cgcctgccgg gtaagggctg gattgcccct 2580

cgtgatctga aagaaatgct ggccggtgac tttcgtacgg gcaagatcct ggcgcagggc 2640

tttggtccgg ataaccaaac cccggactat acttatctga aaggcgatat taccgcagca 2700

tatagcgcaa aagtgaaaga ggtcaaacgt agctttctgt tcctgaacct gaaagacgct 2760

aaggttccgg cagcgatgat cgttttcgat aaggtggtgg catctaatcc ggacttcaag 2820

aagttctggc tgttgcatag catcgagcaa ccggagatca aaggcaacca gattactatc 2880

aaacgtacca aaaacggtga cagcggtatg ctggttaaca ccgctctgct gccagacgct 2940

gccaatagca acatcacctc tattggtggt aagggcaaag atttctgggt gtttggcacc 3000

aattacacga atgacccgaa accgggcact gatgaggcgc tggagcgtgg cgagtggcgc 3060

gtggaaatca ccccgaagaa ggcagcggca gaggactact atttgaacgt aatccaaatt 3120

gctgataata cgcagcagaa actgcacgag gtgaagcgca ttgatggtga taaagttgtc 3180

ggtgttcaac tggcggaccg catcgtcacc ttcagcaaga ccagcgaaac ggttgatcgt 3240

ccgtttggtt ttagcgtggt tggtaaaggc accttcaagt tcgtcatgac cgatctgctg 3300

ccgggcacgt ggcaagttct gaaggatggt aagatcctgt acccggcact gagcgcgaag 3360

ggcgacgatg gtccgttgta cttcgagggt accgaaggca cctatcgttt cctgcgctaa 3420

<210> 10

<211> 3084

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 10

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 tggtggtggc ggcagcggtg gcggcggtag cggcggtggt 1140

ggatccggtg gcggtggttc tggtggtggt ggcagccaat ccagcagcat tactcgtaaa 1200

gacttcgacc acatcaacct ggaatatagc ggcttggaga aagtgaacaa ggcggtggcg 1260

gctggcaatt atgacgatgc cgcgaaagca ttgctggcgt actaccgcga gaaaagcaaa 1320

gcccgcgagc ctgactttag caatgctgag aagccggcag atattcgtca gccgattgat 1380

aaagtaaccc gcgaaatggc cgataaagcc ctggtccacc agttccagcc gcataaaggt 1440

tatggttatt tcgattatgg caaggatatc aactggcaga tgtggccggt caaggataac 1500

gaggttcgct ggcaactgca ccgtgtgaaa tggtggcagg caatggccct ggtctaccat 1560

gcgacgggtg acgagaaata cgctcgtgag tgggtttacc agtacagcga ctgggcgcgt 1620

aagaatccgc tgggtctgag ccaagacaat gacaagtttg tgtggcgtcc tctggaagtg 1680

agcgaccgtg tccaatctct gccgccgacg ttctccctgt tcgtcaactc cccggcattc 1740

acgccagcat tcctgatgga attcctgaac tcttaccacc agcaagcaga ctacctgagc 1800

acccattatg cggaacaagg taaccaccgt ttgttcgagg cgcaacgtaa tctgtttgcg 1860

ggcgtgagct tcccggagtt taaagacagc ccgcgttggc gtcaaaccgg catcagcgtg 1920

ctgaacaccg aaatcaagaa acaggtttat gcggatggta tgcaattcga actgagcccg 1980

atctaccatg ttgccgcgat cgacattttc ctgaaggcgt atggcagcgc caaacgtgtt 2040

aacctggaaa aagaatttcc gcagagctac gttcaaacgg ttgagaatat gatcatggct 2100

ctgatctcta ttagcctgcc ggactataac accccgatgt ttggtgactc gtggattacg 2160

gacaaaaact ttcgcatggc gcagtttgca agctgggcac gcgtctttcc ggcaaaccaa 2220

gcgatcaagt acttcgcgac cgatggtaag caaggtaaag caccgaactt cctgagcaag 2280

gcgctgagca atgcgggctt ctacaccttt cgtagcggct gggataagaa cgccactgtc 2340

atggtcctga aagcgagccc accgggtgaa ttccacgcgc agccggacaa cggtacgttc 2400

gagttgttta tcaaaggtcg caatttcact ccagatgcgg gtgtttttgt gtacagcggc 2460

gacgaggcca tcatgaagct gcgtaactgg tatcgccaga cgcgtattca ctcgaccctg 2520

accctggata atcagaatat ggtgattacc aaggcacgcc agaataagtg ggaaacgggt 2580

aacaacctgg acgtcctgac ctatacgaat ccgagctacc cgaatttgga tcaccagcgt 2640

tccgttctgt tcatcaataa gaaatacttc ttggtcattg accgtgcaat cggtgaggcg 2700

accggtaacc tgggtgttca ttggcagctg aaggaagata gcaatccggt ttttgataaa 2760

accaaaaacc gtgtttacac cacctaccgt gacggcaata acctgatgat tcagagcttg 2820

aatgctgatc gcaccagcct gaacgaggaa gagggcaaag tgtcttatgt atacaataag 2880

gagctgaagc gtccggcgtt cgtttttgag aagccaaaga agaatgcagg tacccaaaac 2940

tttgtgagca ttgtgtaccc gtatgatggt caaaaagctc cggagatttc catccgcgaa 3000

aacaaaggta acgattttga gaaaggcaag ctgaatttga ccctgacgat taatggcaag 3060

caacagttgg tcctggtgcc gtaa 3084

<210> 11

<211> 2622

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 11

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 tggtggtggc ggcagcggtg gcggcggtag cggcggtggt 1140

ggatccggtg gcggtggttc tggtggtggt ggcagccagg ttgttgcttc aaatgaaact 1200

ttataccagg ttgtaaagga ggtaaaaccc ggtggtctgg tacagattgc cgatgggact 1260

tataaagatg ttcagctgat tgtcagcaat tcaggaaaat ctggtttgcc catcactatt 1320

aaagccctga acccgggtaa ggtttttttt accggagatg ctaaagtaga gctgaggggc 1380

gagcacctga tactggaagg catctggttt aaagacggga acagagctat tcaggcatgg 1440

aaatcacatg gacccggatt ggtggctata tatggtagct ataaccgcat taccgcatgt 1500

gtatttgatt gttttgatga agccaattct gcttacatta ctacttcgct taccgaagac 1560

ggaaaggtac ctcaacattg ccgcatagac cattgcagtt ttaccgataa gatcactttt 1620

gaccaggtaa ttaacctgaa caatacagcc agagctatta aagacggttc ggtgggagga 1680

ccggggatgt accatcgtgt tgatcactgt tttttttcca atccgcaaaa accgggtaat 1740

gccggagggg gaatcaggat tggctattac cgtaatgata taggccgttg tctggtagac 1800

tctaacctgt ttatgcgtca ggattcggaa gcagagatca tcaccagcaa atcgcaggaa 1860

aatgtttatt atggtaatac ttacctgaat tgccagggca ccatgaactt tcgtcacggt 1920

gatcatcagg tggccattaa caatttttat ataggcaatg accagcgatt tggatacggg 1980

ggaatgtttg tttggggaag caggcatgtc atagcctgta attattttga gctgtccgaa 2040

accataaagt cgagggggaa cgccgcattg tatttaaacc ccggtgctat ggcttcggag 2100

catgctcttg ctttcgatat gttgatagcc aacaacgctt tcatcaatgt aaatgggtat 2160

gccatccatt ttaatccatt ggatgagcgc agaaaagaat attgtgcagc caataggctt 2220

aagttcgaaa ccccgcacca gctaatgtta aaaggcaatc ttttctttaa ggataaacct 2280

tatgtttacc cattttttaa agatgattat tttatagcag ggaaaaatag ctggactggt 2340

aatgtagcct taggtgtgga aaagggaatc cctgttaaca tttcggccaa taggtctgcc 2400

tataagccgg taaaaattaa agatatccag cccatagaag gaatcgctct tgatctcaat 2460

gcgctgatca gcaaaggcat tacaggaaag ccccttagct gggatgaagt aaggccctac 2520

tggttaaaag aaatgcccgg gacgtatgct ttaacggcca ggctttctgc agatagggct 2580

gcaaagttta aagccgtaat taaaagaaat aaagagcact ga 2622

<210> 12

<211> 3216

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 12

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 tggtggtggc ggcagcggtg gcggcggtag cggcggtggt 1140

ggatccggtg gcggtggttc tggtggtggt ggcagccagc agaccggtac tgcagaactg 1200

attatgaagc gggtgatgct ggaccttaaa aagcctttgc gcaatatgga taaggtggcg 1260

gaaaaggacc tgaatacgct gcagcctgac ggtagctgga aggatgtgcc ttataaagat 1320

gatgccatga ccaattggtt gccaaacaac cacctgctac aattggaaac tattatacag 1380

gcttatattg aaaaagatag tcactattat ggcgacgata aagtgtttga ccagatttcc 1440

aaagctttta agtattggta tgacagcgac ccgaaaagcc gcaactggtg gcacaatgaa 1500

attgccactc cgcaggccct tggtgaaatg ctgatcctga tgcgttacgg taaaaagccg 1560

cttgatgaag cattggtgca taaattgacc gaaagaatga agcggggcga accggagaag 1620

aaaacggggg ccaacaaaac agatatcgcc ctgcattact tttatcgtgc tttgttaacg 1680

tctgatgagg ctttgctttc cttcgccgta aaagaattgt tttatcccgt acagtttgta 1740

cactatgagg aaggcctgca atacgattat tcctacctgc agcacggtcc gcaattacag 1800

atatcgagct acggtgccgt atttattacc ggggtactga aacttgccaa ttacgttagg 1860

gatacccctt atgctttaag taccgagaaa ctggctatat tttcaaagta ttaccgcgac 1920

agttatctga aagctatccg tggaagttat atggatttta acgtagaagg ccgcggagta 1980

agccggccag acattctaaa taaaaaggca gaaaaaaaga ggttgctggt ggcgaagatg 2040

atcgatctta agcatactga agaatgggct gatgcgatag ccaggacaga tagcacagtt 2100

gcggccggct ataagattga gccctatcac catcagttct ggaatggtga ttatgtgcaa 2160

catttaagac ctgcctattc ttttaatgtt cgtatggtga gtaagcggac ccgacgcagt 2220

gaatccggca ataaagaaaa cctgctgggc aggtatttat ctgatggggc tactaacata 2280

caattgcgcg gaccagaata ctataacatt atgccggtat gggaatggga caagattcct 2340

ggcataacca gccgtgatta tttaaccgac agacctttga cgaagctttg gggagagcag 2400

gggagcaatg actttgcagg aggggtgtct gatggtgtat acggggccag tgcctacgca 2460

ttggattacg atagcttaca ggcaaagaaa gcctggttct tttttgacaa agagattgta 2520

tgtcttggtg ccggtatcaa cagcaatgcc cctgaaaaca ttaccactac ccttaaccag 2580

agctggttaa atggcccggt tataagtact gcaggtaaaa ccggccgggg taaaataaca 2640

acgtttaaag cacagggaca gttctggttg ttgcacgatg cgattggtta ttactttcct 2700

gaaggggcca accttagtct gagtacccag tcgcaaaaag gcaattggtt ccacatcaac 2760

aattcacatt caaaagatga agtttctggt gatgtattta agctttggat caaccatggt 2820

gccaggccag aaaatgcgca gtatgcttat atcgttttgc cgggaataaa caagccggaa 2880

gaaattaaaa aatataatgg aacggcaccg aaagtccttg ccaataccaa ccagctgcag 2940

gcagtttatc atcagcagtt agatatggta caggctatct tctatacagc tggaaaatta 3000

agcgtagcgg gcatagaaat tgaaacagat aagccatgtg cagtgctgat caagcacatc 3060

aatggcaagc aggtaatttg ggctgccgat ccattgcaaa aagaaaagac tgcagtgttg 3120

agcatcaggg atttaaaaac aggaaaaaca aatcgggtaa aaattgattt tccgcaacag 3180

gaatttgcag gtgcaacggt tgaactgaaa tagtaa 3216

Claims (4)

1. a kind of MBP fusion heparinase containing link peptide SEQ ID NO.1 merges heparin selected from MBP fusion heparinase I, MBP Enzyme II, MBP fusion heparinase III, nucleotide sequence are respectively SEQ ID NO.3, SEQ ID NO.4, SEQ ID in sequence table NO.5。

2. a kind of MBP containing link peptide SEQ ID NO.1 merges chondrosulphatase, chondrosulphatase is merged selected from MBP B, MBP merges chondrosulphatase AC, and nucleotide sequence is respectively SEQ ID NO.6 in sequence table, SEQ ID NO.7.

3. a kind of MBP fusion heparinase containing link peptide SEQ ID NO.2 merges heparin selected from MBP fusion heparinase I, MBP Enzyme II, MBP fusion heparinase III, nucleotide sequence are respectively SEQ ID NO.8, SEQ ID NO.9, SEQ ID in sequence table NO.10。

4. a kind of MBP containing link peptide SEQ ID NO.2 merges chondrosulphatase, chondrosulphatase is merged selected from MBP B, MBP merges chondrosulphatase AC, and nucleotide sequence is respectively SEQ ID NO.11 in sequence table, SEQ ID NO.12.