CN114836461A - Recombinant plasmid and yeast strain for expressing collagenase, fermentation medium and fermentation culture method thereof - Google Patents
Recombinant plasmid and yeast strain for expressing collagenase, fermentation medium and fermentation culture method thereof Download PDFInfo
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- CN114836461A CN114836461A CN202210605225.4A CN202210605225A CN114836461A CN 114836461 A CN114836461 A CN 114836461A CN 202210605225 A CN202210605225 A CN 202210605225A CN 114836461 A CN114836461 A CN 114836461A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C12N1/14—Fungi; Culture media therefor
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- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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Abstract
The invention discloses a recombinant plasmid for expressing collagenase, a yeast strain, a fermentation culture medium, a fermentation culture method and application thereof, wherein the recombinant plasmid is formed by inserting different modules of an expression box into an expression plasmid, and comprises a signal peptide alpha factor, a ColG or ColH leader peptide and a functional region; and then transforming the recombinant plasmid into a starting yeast strain to obtain the saccharomyces cerevisiae for secreting collagenase. The invention constructs the saccharomyces cerevisiae engineering bacteria for secreting and producing the collagenase, optimizes the signal peptide element in the collagenase sequence by optimizing the components of the yeast culture medium, and improves the capability of the saccharomyces cerevisiae for secreting the collagenase. The obtained recombinant collagenase can effectively degrade gelatin and collagen, and has important practical significance for the application of collagenase and the effective utilization of collagen resources.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant plasmid and a yeast strain for expressing collagenase, a fermentation medium, a fermentation culture method and application thereof.
Background
In the processing production of livestock, poultry and aquatic products, the by-product yield is huge, and the animal horn, hoof, skin, bone and scale contain abundant collagen resources.
The enzymatic extraction is an important means for improving the processing utilization rate of the byproducts, and compared with other non-biological processes such as acid and alkali treatment, the method can better obtain the high-quality bioactive extract and is more environment-friendly. Because the special triple-helix structure of collagen has higher strength, the collagen is difficult to be directly hydrolyzed by common protease, and a chemical method or high-temperature auxiliary hydrolysis is usually needed.
Enzymes commonly used for preparing collagen oligopeptides in industrial production at present mainly comprise papain, alkaline protease, trypsin and the like, but have poor specificity to collagen.
The collagenase is a specific metalloprotein hydrolase aiming at collagen, and can hydrolyze the collagen under physiological conditions, so that collagen peptide with bioactivity can be obtained more easily, and the hydrolysis degree and quality of the collagen under mild treatment conditions can be effectively improved.
The collagenase derived from Clostridium histolyticum is the most widely studied, is the standard enzyme which is generally accepted at present for identifying and analyzing novel collagenase, and is a commercially available microbial collagenase. However, the Clostridium histolyticum-derived collagenase is mainly obtained by purifying fermentation supernatant of Clostridium histolyticum, and the fermentation supernatant has complex components, complicated protein purification steps and large activity difference of collagenase preparations among batches. In addition, Clostridium histolyticum is a pathogenic bacterium, and the application of collagenase secreted by Clostridium histolyticum in the fields of food and the like with higher requirements on safety is limited to a certain extent.
Disclosure of Invention
The invention aims to provide a recombinant plasmid and a yeast strain for expressing collagenase, a fermentation culture medium, a fermentation culture method and application thereof, and aims to overcome the defect that the source of the existing collagenase needs to be expanded. The secretory expression of the collagenase in a food-grade safe host is realized by introducing the collagenase gene into a saccharomyces cerevisiae cell body and optimizing the components of a yeast fermentation culture medium. And further, by optimizing a signal peptide element in a collagenase sequence, the secretion level of the collagenase is improved. The invention obtains the saccharomyces cerevisiae strain capable of efficiently secreting and expressing the collagenase by optimizing the culture medium and the signal peptide element, the obtained recombinant collagenase has the capability of hydrolyzing gelatin and collagen under physiological conditions, and the fermentation supernatant containing the recombinant collagenase can be directly used for processing the substrate.
The purpose of the invention is realized by the following technical scheme:
a recombinant plasmid for expressing collagenase is characterized in that a leader sequence (leader) of alpha factor, a leader peptide sequence (pro peptide) and an enzyme functional region sequence (Chain) of ColG of Clostridium histolyticum collagenase are inserted into the expression plasmid, or the leader sequence (leader) and a spacer sequence (spacer) of alpha factor, the leader peptide sequence (pro peptide) and the enzyme functional region sequence (Chain) of ColH of Clostridium histolyticum collagenase are inserted into the expression plasmid;
the leader sequence (leader) of the alpha factor is shown as SEQ ID No. 1;
the leader peptide sequence and the enzyme functional region sequence (pro peptide-Chain) of the ColG are shown in SEQ ID No. 2;
the leader-spacer of the alpha factor is shown as SEQ ID No. 3;
the leader peptide sequence and the enzyme functional region sequence (pro peptide-Chain) of the ColH are shown in SEQ ID No. 4;
the constructed collagenase expression plasmid is not limited to the collagenase secretion mediated by using only the original signal peptide of the collagenase, but the optimized module combination is obtained by comparing the secretion yield of the collagenase under the combination of different modules (signal peptide, leader peptide and functional region) of an expression cassette, and the efficient secretion in saccharomyces cerevisiae cells is realized.
The expression plasmid is CPOTud, p426GPD, p426TEF, p426ADH, p426CYC, p416GPD, p4216TEF, p4126ADH, p416CYC, p425GPD, p425TEF, p425ADH, p425CYC, p415GPD, p4215TEF, p4125ADH, p415CYC, p424GPD, p424TEF, p424ADH, p424CYC, p414GPD, p4214TEF, p4124ADH, p414CYC, p423GPD, p423TEF, p ADH 423ADH, p423CYC, p413GPD, p4213TEF, p4123ADH, p413CYC, pSP-GM1, pRS303, pRS304, pRS305, pRS306, pRS413, pRS414, pRS415, pRS423, pRS424 or pRS424 pRS 426.
The saccharomyces cerevisiae secreting the collagenase is obtained by converting the recombinant plasmid into a starting yeast strain;
the starting yeast strain is Saccharomyces cerevisiae B184M, CEN.PK 530.1C, CEN.PK 113.5D, BY4742, BY4741, CEN.PK2-1D, CEN.PK2-1C or IMX 581.
A fermentation medium suitable for the above Saccharomyces cerevisiae is prepared by adding Ca into YPD medium 2+ And/or Zn 2+ ；
The Ca 2+ At a concentration of 10-25mM, at which collagenase is secreted in a high amount; considering the potential for higher metal ion concentrations to potentially cause precipitation of the fermentation broth, 10mM is preferred;
said Zn 2+ Is 0.4-1mM, preferably 0.6 mM.
The fermentation culture method of the saccharomyces cerevisiae is to inoculate the saccharomyces cerevisiae into the fermentation culture medium for culture;
the recombinant collagenase can be collected by culturing for at least 96 hours at the temperature of 30-37 ℃.
The recombinant collagenase which is the fermentation product of the saccharomyces cerevisiae can effectively degrade gelatin and collagen, so the recombinant plasmid and the saccharomyces cerevisiae can be applied to producing the collagenase and degrading the gelatin and the collagen.
Compared with the prior art, the invention has the following advantages and effects:
the invention constructs the saccharomyces cerevisiae engineering bacteria for secreting and producing the collagenase, optimizes the signal peptide element in the collagenase sequence by optimizing the components of the yeast culture medium, and improves the capability of the saccharomyces cerevisiae for secreting the collagenase. The obtained recombinant collagenase can effectively degrade gelatin and collagen, and has important practical significance for the application of collagenase and the effective utilization of collagen resources.
Drawings
FIG. 1 is a map of collagenase expression plasmids pCP _ G01 and pCP _ H01.
FIG. 2 shows different Ca 2+ Enzyme activity and OD at ion concentration 600 The value is obtained.
FIG. 3 shows different Zn 2+ Enzyme activity and OD at ion concentration 600 The value is obtained.
FIG. 4 shows different Zn 2+ Enzyme activity and OD at ion concentration 600 The value is obtained.
FIG. 5 is a plasmid construction framework diagram.
FIG. 6 shows the enzyme activities and OD of different strains 600 The value is obtained.
FIG. 7 shows the results of plate hydrolysis experiments with different strains.
FIG. 8 shows the enzyme activity and OD of different strains 600 The value is obtained.
FIG. 9 shows the results of plate hydrolysis experiments with different strains.
FIG. 10 shows the effect of collagenase on gelatin degradation.
FIG. 11 shows the effect of collagenase on collagen degradation.
FIG. 12 is the morphology of collagen 24h after collagenase treatment.
FIG. 13 shows the production of secreted and expressed collagenase by the strains.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified;
the experimental procedures under the conditions not specified in the examples below were generally carried out under the conventional conditions as described in molecular cloning, A laboratory Manual (Beijing: scientific Press, 2017) and Yeast genetics methods, an laboratory Manual (Beijing: scientific Press, 2016).
In order to better understand the content of the invention, Saccharomyces cerevisiae B184M (PNAS,2015,112: E4689-E4696) is used as a starting strain, and plasmid CPOTud (Biotechnology and Bioengineering,2012,109(5):1259-1268) is used as an expression plasmid, and specific examples are further explained.
The media involved in the following examples are as follows:
LB culture medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl;
LB/Amp Medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, sterilized, cooled to about 50 ℃, added 100. mu.g/mL ampicillin (filter sterilized);
YPD medium: 20g/L peptone, 10g/L yeast extract, 20g/L glucose (added after separate sterilization);
modified YPD medium: 20g/L peptone, 10g/L yeast extract, 20g/L glucose (added after separately sterilized), 10mM CaCl 2 And 0.6mM ZnCl 2 ；
YPE medium: 20g/L peptone, 10g/L yeast extract, 0.5g/L glucose (added after separate sterilization), 10ml absolute ethanol/L (filter sterilized);
the solid culture medium is added with 20g/L agar powder on the basis of the corresponding liquid culture medium.
In the following examples, the activity of collagenase in relation to the enzyme activity was measured as follows:
(1) yeast strains (BG01, BH01) containing collagenase expression plasmids were activated on YPD plates and single colonies were picked with 1. mu.L inoculating loop and transferred to 2.5mL medium at 30 ℃ and cultured at 200rpm for 96 h.
(2) And (4) measuring biomass. Diluting the bacterial liquid by a proper multiple, and measuring the light absorption value at 600nm wavelength to obtain OD 600 The value is obtained.
(3) Collagenase enzyme activity was determined by the indetrione method (Anal Biochem,2013,437: 46-48). And centrifuging the cultured bacterial liquid to obtain fermentation supernatant. mu.L of the fermentation supernatant was collected and 480. mu.L of the reaction buffer (50mM Tris. Cl, 5mM CaCl) was added 2 、1μM ZnCl 2 pH 7.5) was reacted at 37 ℃ for 30min, and 500 μ L of stop solution (12% (w/v) PEG6000, 25mM EDTA) was added to stop the reaction. Adding 90 μ L deionized water into 10 μ L reaction solution, adding 500 μ L ninhydrin color development solution (containing 0.16% SnCl) 2 200mM citric acid buffer solution with pH 5.0 and 50g/L ninhydrin-DMSO solution are mixed in equal volume and prepared at present), heated in water bath at 80 deg.C for 10min, cooled, added with 600 μ L deionized water, mixed well, and measured for absorbance at 570 nm. Hydrolysis of collagen per 100 μ L of enzyme solution per minute at 37 c and pH 7.5 produced 1 enzyme activity unit U equivalent to 1 μ g glycine.
(4) The plate transparent ring qualitatively characterizes the enzyme activity. The solid medium was supplemented with 1% skim milk powder, and the strain was cultured on a plate at 30 ℃ for 4 days, then transferred to 37 ℃ for 2-3 days, examined for the size of the hydrolysis ring formed around the colony, and photographed.
The primer sequences involved in the following examples are shown in Table 1:
table 1: primer sequences
# underlined marker restriction sites
Example 1 acquisition of a Gene encoding Clostridium histolyticum collagenase
Based on the amino acid sequences of Clostridium histolyticum collagenase ColG (Q9X721) and ColH (Q46085) disclosed in the UniProt database, DNA coding sequences were synthesized by King of King-Shi Biotech, Inc., south Beijing after codon optimization for Saccharomyces cerevisiae to obtain plasmids pUC57-Mini _ G (SEQ ID No.5) and pUC57-Mini _ H (SEQ ID No.6) (containing pre-peptide-pro-peptide-Chain).
Example 2 construction of engineered Saccharomyces cerevisiae expressing collagenase ColG and ColH
(1) Carrying out enzyme digestion on CPOTud by Kpn I and Nhe I, and then recovering to obtain a carrier;
(2) amplifying a signal peptide (alpha factor leader + spacer) with G, H homology arms by using primer pairs AF/ARproG and AF/ARproH and pAlphaAmyCPOT (Biotechnology and Bioengineering,2012,109:1259-1268) as a template (SEQ ID No. 3); using primer pairs proGF/GR, proHF/HR, pUC57-Mini _ G and pUC57-Mini _ H as templates to amplify ColG and ColH fragments (SEQ ID No.2 and SEQ ID No. 4); the corresponding fragments (ColG or ColH and the corresponding signal peptide) were fused by PCR amplification using the corresponding primers AF/GR, AF/HR to give inserts G01, H01.
(3) The fused fragment was digested with Kpn I and Nhe I, and then ligated with a vector using T4 DNA Ligase to obtain plasmids pCP _ G01(SEQ ID No.7) and pCP _ H01(SEQ ID No. 8).
(4) The ligation products were transformed into E.coli competent cells using conventional procedures. After overnight incubation of the coated plates at 37 ℃ colonies were picked to PCR systems containing the identifying primers G1/G2 or H1/H2 for colony PCR identification.
(5) The correct clone was identified and inoculated into LB/Amp medium, cultured at 37 ℃ and 250rpm for 12-16H, and plasmid pCP _ G01(SEQ ID No.7) and pCP _ H01(SEQ ID No.8) were identified and sequenced successfully. Plasmid pCP _ G01 contains the collagenase ColG expression cassette: alpha factor leader + spacer-colG pro peptide-colG chain; plasmid pCP _ H01 contains the collagenase ColH expression cassette: alpha factor leader + spacer-colH pro peptide-colH chain (map is shown in FIG. 1).
(6) The resulting plasmids pCP _ G01, pCP _ H01 and the control empty plasmid CPOTud were transformed into Saccharomyces cerevisiae B184M using a conventional lithium acetate transformation method, resulting in strains BG01, BH01 and B0.
EXAMPLE 3 optimization of culture conditions for collagenase-producing bacteria
Since ColG and ColH are metalloproteinases, Ca is required 2+ Zn of heel 2+ As a metal cofactor.
The medium was optimized as represented by strain BG 01. Conventional YPD medium was supplemented with Ca at concentrations of 2.5mM, 5mM, 10mM, 15mM, 20mM, and 25mM, respectively 2+ (CaCl 2 ) And Zn at a concentration of 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1mM 2+ (ZnCl 2 ) And performing after-fermentation for 96h, and measuring the enzyme activity. The results are shown in FIGS. 2 and 3, and the single factor experiment shows that Ca is added 2+ Or Zn 2+ So that the yield of the collagenase is obviously improved. Wherein, Ca 2+ The enzyme activity can reach higher between 10mM and 25 mM. To avoid higher concentrations of Ca 2+ Potential precipitating effects, preferably 10mM Ca 2+ As the added concentration of the metal ion. For Zn 2+ 0.6mM Zn is selected 2+ As the metal ion addition concentration.
Based on the optimization results of ColG, we also added 10mM Ca to YPD medium 2+ Zn was supplemented at 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1mM 2+ (ZnCl 2 ) Inoculating strain BH01, fermenting for 96h, and determining enzyme activity. The results are shown in FIG. 4, 0.6mM Zn 2+ The enzyme activity is highest under the concentration.
Taken together, 10mM Ca 2+ And 0.6mM Zn 2+ The concentration of metal ions added is optimized.
Example 4 optimization of element Adaptation of Saccharomyces cerevisiae expression cassettes
The construction framework of the element adaptation assembly optimization series plasmid is shown in FIG. 5, the construction process is the same as that of example 2, and different fragments are obtained by amplification and fusion of corresponding primer pairs.
Amplifying a signal peptide (alpha factor leader) with a G homologous arm by using a primer pair AF/ARWproG and using pAlphaAmyCPOT as a template (SEQ ID No. 1); amplifying a ColG fragment by using a primer pair WproGF/GR and pUC57-Mini _ G as a template; the corresponding fragment (ColG and corresponding signal peptide) was fused by PCR amplification using the corresponding primer AF/GR to give insert G02.
Using the primer pair PproG/GR, a ColG with an alpha factor pre homology arm was amplified using pUC57-Mini _ G as a template, and the resulting fragment was amplified using the primer pair AP/GR to obtain the insert G03.
Amplifying a signal peptide with a G homologous arm (alpha factor leader + spacer) by using a primer pair AF/ARG and using pAlphaAmyCPOT as a template; a ColG fragment is amplified by using a primer pair AGF/GR and pUC57-Mini _ G as a template; the corresponding fragment (ColG and corresponding signal peptide) was fused by PCR amplification using the corresponding primer AF/GR to give insert G04.
Amplifying a signal peptide with a G homologous arm (alpha factor leader + spacer) by using a primer pair AF/ARWG and using pAlphaAmyCPOT as a template; a ColG fragment is amplified by using a primer pair WGF/GR and pUC57-Mini _ G as a template; the corresponding fragment (ColG and corresponding signal peptide) was fused by PCR amplification using the corresponding primer AF/GR to give insert G05.
Using the primer pair PG/GR, a ColG with an alpha factor pre homology arm was amplified using pUC57-Mini _ G as a template, and the resulting fragment was amplified using the primer pair AP/GR to obtain an insert G06.
The insert G07 was amplified using the primer set GF/GR, pUC57-Mini _ G as a template.
Amplifying a signal peptide (alpha factor leader) with an H homologous arm by using a primer pair AF/ARWproH and using pAlphaAmyCPOT as a template; amplifying a ColH fragment by using a primer pair WproHF/HR and pUC57-Mini _ H as a template; the corresponding fragment (ColH and corresponding signal peptide) was fused by PCR amplification using the corresponding primer AF/HR to give insert H02.
Amplifying a signal peptide with an H homologous arm (alpha factor leader + spacer) by using a primer pair AF/ARH and using pAlphaAmyCPOT as a template; a ColH fragment is amplified by using a primer pair AHF/HR and pUC57-Mini _ H as a template; the corresponding fragment (ColH and corresponding signal peptide) was fused by PCR amplification using the corresponding primer AF/HR to give insert H03.
Amplifying a signal peptide with an H homologous arm (alpha factor leader + spacer) by using a primer pair AF/ARWH and using pAlphaAmyCPOT as a template; a ColH fragment is amplified by using a primer pair WHF/HR and pUC57-Mini _ H as a template; the corresponding fragment (ColH and corresponding signal peptide) was fused by PCR amplification using the corresponding primer AF/HR to give insert H04.
The insert H05 was amplified using pUC57-Mini _ H as template using the primer pair HF/HR.
The above fragments G02, G03, G04, G05, G06, G07, H02, H03, H04 and H05 are inserted into the plasmid CPOTud by enzymatic ligation, and a part of the fragments are ligated to the plasmid using a Seamless Cloning master mix (see the Specification of the Shilian research kit B632219); expression plasmids pCP _ G02, pCP _ G03, pCP _ G04, pCP _ G05, pCP _ G06, pCP _ G07, pCP _ H02, pCP _ H03, pCP _ H04 and pCP _ H05 were constructed accordingly.
The resulting plasmids and the expression cassettes containing them were:
pCP _ G01 (containing alpha factor leader + spacer-colG pro peptide-colG chain)
pCP _ G02 (containing alpha factor leader-colG pro peptide-colG chain)
pCP _ G03 (containing alpha factor pre-colG pro peptide-colG chain)
pCP _ G04 (containing alpha factor leader + space-colG chain)
pCP _ G05 (containing alpha factor leader-colG chain)
pCP _ G06 (containing alpha factor pre-colG chain)
pCP _ G07 (containing colG pre peptide-colG pro peptide-colG Chain)
pCP _ H01 (containing alpha factor leader + spacer-colH pro peptide-colH chain)
pCP _ H02 (containing alpha factor leader-colH pro peptide-colH chain)
pCP _ H03 (containing alpha factor leader + spacer-colH chain)
pCP _ H04 (containing alpha factor leader-colH chain)
pCP _ H05 (containing colH pre-peptide-colH pro peptide-colH chain)
After sequencing and verification, the strain is transferred into B184M to obtain corresponding strains BG01, BG02, BG03, BG04, BG05, BG06, BG07, BH01, BH02, BH03, BH04 and BH 05.
After the constructed strain is cultured, enzyme activity detection is carried out, and as can be seen from figures 6 and 7, BG02 has the highest enzyme activity for expressing collagenase ColG, and the hydrolysis loop of the flat plate is the most obvious. As can be seen from FIGS. 8 and 9, BH01 has the highest enzyme activity for expressing collagenase ColH, and the plate hydrolysis loop is the most obvious. The strain BG02 carries a plasmid pCP _ G02(SEQ ID No.9), and contains an expression frame of alpha factor leader-colG pro peptide-colG chain; the strain BH01 carries a plasmid pCP _ H01(SEQ ID No.8), and contains an expression frame of alpha factor leader + spacer-colH pro peptide-colH chain.
Subsequent experiments were performed using strain BG02 and strain BH01 as described above.
Example 5 degradation of various substrates by recombinant collagenase
(1) Degrading the gelatin. 1% gelatin was added additionally to the modified YPD medium. Strains B0, BG02 and BH01 were inoculated to 2.5mL of modified YPD + gelatin medium, respectively, and fermented at 30 ℃ and 200rpm for 96 h. After the fermentation was completed, 500. mu.L of the fermentation broth was centrifuged to remove the bacterial cells. And then 20 mu L of fermentation supernatant is taken, 5 mu L of 5 xSDS-PAGE protein loading buffer solution is added, the mixture is boiled at 100 ℃ for 10min, and then the loading electrophoresis is carried out until bromophenol blue reaches the bottom of the precast gel. After the electrophoresis is finished, the albumin glue is placed in a staining solution for staining for 30min, and after the excessive staining solution is eluted, a picture is taken by a gel imager. As shown in FIG. 10, the gelatin of the lanes corresponding to the blank control and the control strain B0 was not decomposed, and the whole lane was stained; the gelatin of the lanes corresponding to the strains BG02 and BH01 is obviously hydrolyzed by secreted collagenase.
(2) Degrading insoluble bovine bone collagen. Strains B0, BG02 and BH01 were inoculated into 40mL of modified YPD medium and fermented at 200rpm at 30 ℃ for 96 h. After the fermentation was completed, 20mL of the fermentation broth was centrifuged to remove the bacterial cells. The supernatant from the fermentation was concentrated using a 50kDa ultrafiltration centrifuge tube and diluted to the appropriate concentration with 50mM Tris. Cl. 2mg of insoluble bovine bone collagen was added thereto, allowed to stand at 37 ℃, and the dissolution of the collagen was observed and recorded by photographing. Wherein the G + H group means BG02 mixed with BH01 at a ratio of 1: 1. As can be seen from fig. 11, the collagen fibers were significantly swollen after the treatment with the recombinant collagenase, and the two recombinant collagenases showed a synergistic effect, compared to the control group. Over time, collagen fibers are enzymatically hydrolyzed and thus gradually disappear. Naturally air-drying the collagen fiber treated for 24h on a silicon wafer, adhering the collagen fiber on a conductive adhesive, spraying a gold plating film, and observing the shape and the structure by using an ultra-high resolution cold field emission scanning electron microscope (SU8220, Hitachi). As a result, as shown in FIG. 12, the fibers of the sample treated with the recombinant collagenase ColG were in a loose state, and the surfaces of the fibers of the sample treated with the recombinant collagenase ColH were also significantly changed, compared to the compact and intact fiber structure of the control group. These observations demonstrate, from a microscopic structure, that the recombinant collagenase has a very good collagen hydrolyzing activity.
Example 6 production of recombinant collagenase by shake flask fermentation of engineered Strain
After streaking and activating strains B0, BG02 and BH01 on YPD plates, single colonies were picked with 1. mu.L inoculating loop and transferred to 2.5mL of modified YPD medium for overnight culture to obtain seed solutions. Transfer the appropriate seed solution to 40mL fresh modified YPD Medium, initiate OD 600 Adjusting to about 0.005, performing shake flask fermentation at 30 deg.C and 200rpm under 10% liquid loading condition, sampling at proper time intervals, and detecting enzyme activity. The yield of recombinant collagenase obtained by shake flask fermentation of the engineering strain is shown in FIG. 13, and the yields of collagenase ColG and ColH reach 6.8 × 10 respectively 4 U/L and 5.5X 10 4 U/L. The result shows that the saccharomyces cerevisiae engineering strain constructed by the invention can efficiently secrete and express the recombinant collagenase, can be used for processing and utilizing collagen resources, and has wide industrial application prospect.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of southern China's science
<120> recombinant plasmid and yeast strain for expressing collagenase, and fermentation medium and fermentation culture method thereof
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gaattattca gacacgaata cacccattac ttgcaaggta gatacgctgt cccaggtcaa 1320
tggggtcgta ccaagttgta tgataacgac agattgacct ggtacgaaga aggtggtgct 1380
gaactgtttg ctggttctac tagaacttcc ggcatcttgc caagaaagtc tattgtctct 1440
aacattcaca acaccactag aaacaacaga tacaagctgt ctgacactgt tcattcgaag 1500
tacggtgcct ctttcgaatt ttacaactac gcttgtatgt tcatggacta catgtacaat 1560
aaggacatgg gtattttgaa caagttgaat gatttggcta agaacaacga cgttgacggt 1620
tacgacaatt acatcagaga tttgtcatct aattacgctt tgaacgataa gtaccaagat 1680
cacatgcaag aacgtattga caactatgaa aacttgaccg ttccattcgt tgctgacgac 1740
tacttagtta gacacgctta caagaaccca aacgaaattt actccgaaat ctccgaagtt 1800
gccaagttga aggatgctaa gtctgaagtt aagaaatctc aatacttctc aactttcact 1860
ttgcgtggta gttacaccgg aggtgcttcc aagggtaagc tggaagatca aaaagctatg 1920
aacaagttca ttgacgactc tttgaagaag ttggacacct attcctggtc tggttacaag 1980
actcttactg cttattttac caactacaag gtcgattctt ctaacagagt tacttacgat 2040
gtcgttttcc atggttactt gccaaacgaa ggtgattcca agaacagctt gccatacggt 2100
aagattaacg gtacttacaa gggtactgag aaggaaaaga ttaagttctc ctccgaaggt 2160
tctttcgacc cagacggaaa gatagtttcc tacgaatggg acttcggtga tggtaacaag 2220
tccaacgaag agaaccctga acacagttac gataaggtcg gtacttacac tgtcaagttg 2280
aaggttaccg atgataaggg tgaaagctct gtttctacta ccactgctga aatcaaagat 2340
ttgtctgaaa acaagttgcc agtcatctat atgcacgttc ctaaatcggg tgctttaaac 2400
caaaaggttg tcttctacgg taagggtact tacgacccag acggttctat cgctggctac 2460
caatgggatt tcggtgacgg ttctgacttt tcctctgaac agaacccatc tcacgtctac 2520
accaagaaag gtgaatacac tgtgaccttg agagtcatgg attcttccgg tcaaatgtct 2580
gaaaagacca tgaaaatcaa gatcactgat ccagtatacc ctattggtac tgaaaaggaa 2640
ccaaacaact ctaaggaaac agcctccggt ccaatcgttc cgggtattcc agtctccggt 2700
actatcgaaa acacctctga tcaagactat ttctacttcg atgttattac tccaggtgaa 2760
gtcaagatcg acatcaacaa gctaggttac ggtggtgcta catgggttgt ttacgatgaa 2820
aacaataacg ctgtctccta cgctaccgat gacggtcaaa atttgtctgg taaattcaag 2880
gctgacaagc ctgggagata ctacattcat ttgtacatgt tcaatggttc ttacatgcca 2940
tacagaatta acattgaagg ttctgttggt aga 2973
<210> 5
<211> 5216
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ccaatgataa aggtaccaac aaaatgaaga agaacatctt gaagattttg atggactctt 60
actccaagga atccaaaatc caaactgtta gaagagttac ttctgtttct ttgttggctg 120
tctacttgac tatgaacacc tcaagtctag tgttggctaa gccaatcgaa aacactaacg 180
acaccagcat caagaacgtt gaaaagttga gaaacgcccc aaacgaagaa aactctaaga 240
aagttgaaga ctctaagaat gataaggttg aacacgttaa gaacattgaa gaagctaagg 300
tcgaacaagt tgctccagaa gtcaagtcta aaagtacctt gagatctgct tctatagcta 360
atacaaattc cgaaaagtac gacttcgaat atcttaatgg tttgagttac accgaattga 420
ctaacttgat caagaacatc aagtggaacc aaatcaacgg tttattcaac tactccaccg 480
gttcgcaaaa atttttcggt gacaagaaca gagtccaagc catcatcaac gctctacaag 540
aatctggtag aacttacact gccaatgaca tgaagggtat tgaaactttc accgaagttt 600
tgagagccgg tttttacttg ggctattaca acgatggtct ctcctacctg aacgaccgta 660
atttccaaga caagtgtatc ccagccatga ttgctattca aaagaaccca aacttcaagt 720
taggtactgc tgttcaagac gaagttatca catctttggg taagttgatt ggtaacgcct 780
ctgctaacgc tgaagttgtt aataactgtg tcccagtttt gaagcaattc agagaaaact 840
taaaccaata cgctccagac tacgtcaaag gtactgccgt aaatgaattg atcaagggta 900
ttgaatttga cttttccggt gctgcttacg aaaaggacgt caagaccatg ccatggtacg 960
gtaagattga cccattcatc aatgaattaa aggccttggg tctctacggt aacatcactt 1020
ctgccactga atgggcttca gatgttggta tctactactt gtctaagttc ggtttgtact 1080
caaccaatcg taacgacatt gttcaatcct tagaaaaggc tgttgatatg tacaaatatg 1140
gtaagatcgc tttcgttgct atggaaagaa tcacctggga ttacgatggt atcggttcca 1200
acgggaagaa ggtcgaccac gataagttct tggacgatgc agaaaagcat tatttgccaa 1260
agacctatac tttcgacaac ggaaccttca tcatcagagc tggagataag gtttctgaag 1320
aaaagattaa gagattgtac tgggcttccc gtgaagttaa gtctcaattc cacagagttg 1380
ttggtaacga taaggctttg gaagtgggta acgctgacga tgtcttgacc atgaaaatct 1440
tcaactctcc agaagaatac aagttcaaca ccaacattaa cggtgtctcc actgataacg 1500
gtggtttgta catcgaacca agaggtactt tctacactta cgaaagaacc ccacaacaat 1560
ctattttctc cttggaagaa ctgttcagac acgaatacac ccactacttg caagctagat 1620
acttggttga cggtttgtgg ggtcaaggtc ctttctacga aaaaaacaga ttgacttggt 1680
tcgatgaagg tactgctgaa tttttcgctg gttccaccag aacttctggt gtgttgccaa 1740
gaaagtccat cttgggttac cttgccaagg ataaggtcga tcatagatac tccttgaaga 1800
aaactttgaa ctctggttac gatgactctg actggatgtt ctacaactac ggttttgctg 1860
ttgctcacta cttatacgaa aaggacatgc caacttttat taagatgaac aaggccatct 1920
tgaacactga tgttaagtct tacgacgaaa tcattaaaaa actgtctgac gacgctaaca 1980
agaacactga ataccaaaac cacatccaag aactcgctga taagtaccaa ggtgccggta 2040
taccattggt gtctgatgac tacctgaagg accatggtta caagaaggct tccgaagttt 2100
actcagaaat ctccaaggct gcgtctttaa ctaacacttc tgttaccgct gaaaagtctc 2160
aatacttcaa cacattcact ttgaggggta cttacactgg tgaaacctct aagggtgaat 2220
ttaaggactg ggacgaaatg tctaagaagt tggacggtac tttggagtct ttagctaaga 2280
actcttggtc tggttacaag actttgaccg cttacttcac caactacaga gtcacttctg 2340
ataacaaagt ccaatatgat gttgtcttcc acggtgtttt gactgacaac gccgatattt 2400
ctaacaacaa ggctcctatt gctaaggtca ccggtccatc tactggtgct gttggtagaa 2460
acattgaatt ttctggtaag gattccaagg acgaagatgg caagattgtc tcttatgact 2520
gggatttcgg tgatggtgct acctccagag gtaagaactc tgtccacgct tacaagaagg 2580
ctggtactta caacgtcact ttgaaggtca ccgatgacaa gggtgctacg gctactgaaa 2640
gtttcactat tgaaattaaa aacgaagaca ctaccacccc aatcaccaag gaaatggaac 2700
caaacgatga cattaaggaa gccaacggcc cgattgtcga aggcgtcacg gtcaaaggtg 2760
acttgaacgg ttctgatgac gctgacactt tctactttga tgtcaaggaa gacggtgacg 2820
ttaccatcga attgccatac tctggttcct ctaactttac ctggttagtc tacaaggaag 2880
gtgacgacca aaaccacatc gcctccggta tcgataaaaa caactccaag gttggtactt 2940
tcaagtccac taagggtcgt cattacgttt tcatctacaa gcacgactct gcttccaaca 3000
tttcatactc cttgaacatc aagggtttgg gtaacgaaaa gttgaaagag aaggaaaaca 3060
atgactcctc tgacaaggca accgttattc caaatttcaa taccaccatg caaggttctc 3120
tattagggga cgattctaga gactactact ccttcgaagt caaggaagaa ggtgaagtta 3180
acattgaatt ggataaaaag gatgaatttg gtgttacttg gaccttgcac ccagaaagca 3240
acatcaacga cagaattacc tacggtcaag tcgatggtaa caaggtatcc aacaaggtca 3300
aattgagacc aggtaagtac tacttgttgg tttacaagta cagtggttcc ggtaactacg 3360
aattgcgggt taacaagtaa gctagcttta tcggaaagaa catgtgagca aaaggccagc 3420
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 3480
ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 3540
aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 3600
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 3660
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 3720
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 3780
cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 3840
ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 3900
gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 3960
gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4020
agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 4080
acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 4140
tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 4200
agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 4260
gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 4320
agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 4380
cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 4440
ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 4500
cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 4560
cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 4620
ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 4680
tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 4740
catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 4800
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 4860
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 4920
tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 4980
catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5040
aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 5100
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 5160
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc 5216
<210> 6
<211> 4925
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ccaatgataa aggtaccaac aaaatgaaga gaaaatgttt gtctaagaga ttgatgttgg 60
ctatcaccat ggctaccatc ttcaccgtca attctacatt gccaatttac gctgctgttg 120
acaagaacaa tgccactgct gccgtccaaa acgaatccaa gcgttacact gtttcttact 180
taaagacctt gaactattac gacttggtcg atctattggt taagaccgaa atcgaaaact 240
taccagactt gtttcaatac tcttccgacg ctaaggaatt ttacggtaac aagactagaa 300
tgtcctttat catggacgaa attggtcgta gagctccaca atacactgaa attgaccaca 360
agggtatacc aaccctcgtt gaagtcgtca gagccgggtt ctacttgggt tttcacaaca 420
aagaattgaa tgaaatcaac aagagatctt tcaaagaaag agttatcccg tctatcttgg 480
ctatccaaaa gaacccaaac ttcaaattag gtactgaagt tcaagacaag attgtctctg 540
ctaccggtct attggctggt aacgaaaccg ctccaccaga agttgttaac aacttcaccc 600
caatcttgca agattgtatc aagaacatcg acagatacgc cttggacgac ttgaaaagta 660
aagcattgtt caacgttttg gccgccccaa cctacgacat tactgaatac ctcagagcta 720
ccaaggaaaa gccagaaaac actccatggt acggtaagat cgatggtttc atcaacgaat 780
tgaagaagct cgctttgtac ggtaaaatta acgacaacaa ctcttggatt atcgataacg 840
gtatttacca cattgctcca ttgggtaaat tgcactctaa caacaaaatc ggtatcgaaa 900
ctctaactga agttatgaag gtctacccat acttgtccat gcaacacttg caatccgctg 960
accaaatcaa gagacactac gactctaagg atgccgaagg taacaagatc ccattagata 1020
agttcaagaa ggaaggcaag gaaaagtact gcccaaagac ctacactttt gatgatggta 1080
aggttatcat caaggccggt gctcgtgtcg aagaagaaaa ggtcaaaaga ttatactggg 1140
cttccaagga agtcaacagt caattcttca gagtttatgg tattgacaag ccattggaag 1200
aaggtaaccc agacgacatc cttaccatgg ttatttacaa ctctccagaa gaatacaaac 1260
ttaactccgt cttgtacggt tacgatacca acaacggtgg tatgtacatt gaaccagaag 1320
gtactttttt cacttacgaa agagaagctc aagaatctac gtacactttg gaagaattat 1380
tcagacacga atacacccat tacttgcaag gtagatacgc tgtcccaggt caatggggtc 1440
gtaccaagtt gtatgataac gacagattga cctggtacga agaaggtggt gctgaactgt 1500
ttgctggttc tactagaact tccggcatct tgccaagaaa gtctattgtc tctaacattc 1560
acaacaccac tagaaacaac agatacaagc tgtctgacac tgttcattcg aagtacggtg 1620
cctctttcga attttacaac tacgcttgta tgttcatgga ctacatgtac aataaggaca 1680
tgggtatttt gaacaagttg aatgatttgg ctaagaacaa cgacgttgac ggttacgaca 1740
attacatcag agatttgtca tctaattacg ctttgaacga taagtaccaa gatcacatgc 1800
aagaacgtat tgacaactat gaaaacttga ccgttccatt cgttgctgac gactacttag 1860
ttagacacgc ttacaagaac ccaaacgaaa tttactccga aatctccgaa gttgccaagt 1920
tgaaggatgc taagtctgaa gttaagaaat ctcaatactt ctcaactttc actttgcgtg 1980
gtagttacac cggaggtgct tccaagggta agctggaaga tcaaaaagct atgaacaagt 2040
tcattgacga ctctttgaag aagttggaca cctattcctg gtctggttac aagactctta 2100
ctgcttattt taccaactac aaggtcgatt cttctaacag agttacttac gatgtcgttt 2160
tccatggtta cttgccaaac gaaggtgatt ccaagaacag cttgccatac ggtaagatta 2220
acggtactta caagggtact gagaaggaaa agattaagtt ctcctccgaa ggttctttcg 2280
acccagacgg aaagatagtt tcctacgaat gggacttcgg tgatggtaac aagtccaacg 2340
aagagaaccc tgaacacagt tacgataagg tcggtactta cactgtcaag ttgaaggtta 2400
ccgatgataa gggtgaaagc tctgtttcta ctaccactgc tgaaatcaaa gatttgtctg 2460
aaaacaagtt gccagtcatc tatatgcacg ttcctaaatc gggtgcttta aaccaaaagg 2520
ttgtcttcta cggtaagggt acttacgacc cagacggttc tatcgctggc taccaatggg 2580
atttcggtga cggttctgac ttttcctctg aacagaaccc atctcacgtc tacaccaaga 2640
aaggtgaata cactgtgacc ttgagagtca tggattcttc cggtcaaatg tctgaaaaga 2700
ccatgaaaat caagatcact gatccagtat accctattgg tactgaaaag gaaccaaaca 2760
actctaagga aacagcctcc ggtccaatcg ttccgggtat tccagtctcc ggtactatcg 2820
aaaacacctc tgatcaagac tatttctact tcgatgttat tactccaggt gaagtcaaga 2880
tcgacatcaa caagctaggt tacggtggtg ctacatgggt tgtttacgat gaaaacaata 2940
acgctgtctc ctacgctacc gatgacggtc aaaatttgtc tggtaaattc aaggctgaca 3000
agcctgggag atactacatt catttgtaca tgttcaatgg ttcttacatg ccatacagaa 3060
ttaacattga aggttctgtt ggtagataag ctagctttat cggaaagaac atgtgagcaa 3120
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 3180
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 3240
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3300
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3360
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3420
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3480
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3540
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3600
acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3660
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3720
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3780
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3840
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 3900
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 3960
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4020
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 4080
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 4140
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 4200
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 4260
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4320
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4380
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4440
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4500
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 4560
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4620
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4680
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4740
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4800
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4860
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 4920
acgtc 4925
<210> 7
<211> 11468
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240
ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300
agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360
cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatctaaa 420
aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat 480
tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 540
cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 600
tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 660
agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 720
tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 780
catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 840
aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 900
ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 960
taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag 1020
gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgga 1080
taaaggcgcg ccgtcgacaa tatagaaatt tcttagtttt aatgattgaa tgaatccatt 1140
tcgaacacgt caatgtactt ggacaagctt tgtagaacat gaagtacttg agcaatggac 1200
gcccaactgg aatttacttt agcgacacta ttccaagacc agcaactcaa agaagtaaat 1260
tctttttttc acaatagttg tttttttgat gatgtacgtc ccagcagcac ctaatggtat 1320
ttttatgagt attggcagtt tgccttatta attaagcaaa ccggtcatat attttaaaaa 1380
ataggaactt tgtagtcctt attaatgctg aataaaaaaa taggaagaga aatcagcaag 1440
tataaacagt taaatgtttt ttaactataa tggttgcatt caagaggaca tgtaaattgc 1500
gtactcggta caagttggta aaaacgttaa agcactccta cgagatttag tcagtcctgg 1560
cattaataaa caaaaagacc tgtatctata tttcatgaac tgggtaaaat caccacacaa 1620
aaaagaataa acaaatataa tgaactactt ttggtcttgg actcgcagtt tatagtttgt 1680
taaaattgac aaacacacat gttctgatta acttacttcc gcttcaaagt actgaaaaca 1740
tcatacgaac tggtataaga aggaagatag caaataaaaa taaatcaaat attgcttgta 1800
aaaaattagg atataaaaaa atctctattc attaaagaca aaaagctaaa ggaataaaaa 1860
agtcattcgc tttacgccaa ataaaacgtc tatgcacgtt tttaaaggct gtgaacatta 1920
acaatgttgt ggaattcagg cttgagagaa gcaccgccaa ccaagaaacc gtcaatatcg 1980
tggaacttga ggaactcctt gcagttacca ccgttaacgg aaccaccgta gatgacacgg 2040
agaccctcgg caacagatgc accaagcttg ttggtagccc acttgcggat ctcagcgtga 2100
acctcttgag cttgctcagg ggtggcagtc ttaccagtac caatggccca gacaggctca 2160
taagcaatga caatcttgga ccagttctgg accttgtcag cgatggcgtt caattgacga 2220
acaacaacgt tgatggtctc gttagcctca cgctcggcca aagtctcacc aatgcaggca 2280
acgacagtaa gaccttgttc aagggcaaac ttggtcttgt cggcaacgaa ctcgtcagac 2340
tccttgaaga tggtacgacg ctcggagtga ccagtcaaag tgtaggtaat accagcatca 2400
atcaaagatt gagcactgtt ctcaccagtg taggcaccgt tcttcttgtc gaagacgttt 2460
tgggcaccaa cgccaatatc cttcttgact tgttggcggg tggtgatgag gtacatgttt 2520
tgagggaaga tgacagtttc gacatcacca acgttaagct tggtggtgtt caaaccctca 2580
ataatagtct tcatggactc caaagagcca ttcatcttaa agttaccacc gacaaagaat 2640
ttacgtgcca ttttgatcct agttctacta ttgaaatgta gttggggaga gacgagagtt 2700
ggcccgtccg ctcattatat ataacgtagc ggacagtcac acagttaagg ggaattaccg 2760
agcttcggca atttaccccg tcgatagcaa ccgttggcat ggatccggcc ggccagatct 2820
acgtatggtc atttcttctt cagattccct catggagaaa gtgcggcaga tgtatatgac 2880
agagtcgcca gtttccaaga gactttattc aggcacttcc atgataggca agagagaaga 2940
cccagagatg ttgttgtcct agttacacat ggtatttatt ccagagtatt cctgatgaaa 3000
tggtttagat ggacatacga agagtttgaa tcgtttacca atgttcctaa cgggagcgta 3060
atggtgatgg aactggacga atccatcaat agatacgtcc tgaggaccgt gctacccaaa 3120
tggactgatt gtgagggaga cctaactaca tagtgtttaa agattacgga tatttaactt 3180
acttagaata atgccatttt tttgagttat aataatccta cgttagtgtg agcgggattt 3240
aaactgtgag gaccttaata cattcagaca cttctgcggt atcaccctac ttattccctt 3300
cgagattata tctaggaacc catcaggttg gtggaagatt acccgttcta agacttttca 3360
gcttcctcta ttgatgttac acctggacac cccttttctg gcatccagtt tttaatcttc 3420
agtggcatgt gagattctcc gaaattaatt aaagcaatca cacaattctc tcggatacca 3480
cctcggttga aactgacagg tggtttgtta cgcatgctaa tgcaaaggag cctatatacc 3540
tttggctcgg ctgctgtaac agggaatata aagggcagca taatttagga gtttagtgaa 3600
cttgcaacat ttactatttt cccttcttac gtaaatattt ttctttttaa ttctaaatca 3660
atctttttca attttttgtt tgtattcttt tcttgcttaa atctataact acaaaaaaca 3720
catacataaa ctaaaaggta ccaacaaaat gagatttcca tctattttta ctgctgtttt 3780
gtttgctgct tcttctgctt tggctgctcc agttaatact actactgaag atgaaactgc 3840
tcaaattcca gctgaagctg ttattggtta ttctgatttg gagggtgact ttgatgttgc 3900
tgttttgcca ttttctaact ctactaacaa cggtttgcta ttcatcaaca ctactatcgc 3960
ttctatcgct gctaaagaag aaggtgtttc tttggataaa agagaagaag gtgaaccaaa 4020
aaagccaatc gaaaacacta acgacaccag catcaagaac gttgaaaagt tgagaaacgc 4080
cccaaacgaa gaaaactcta agaaagttga agactctaag aatgataagg ttgaacacgt 4140
taagaacatt gaagaagcta aggtcgaaca agttgctcca gaagtcaagt ctaaaagtac 4200
cttgagatct gcttctatag ctaatacaaa ttccgaaaag tacgacttcg aatatcttaa 4260
tggtttgagt tacaccgaat tgactaactt gatcaagaac atcaagtgga accaaatcaa 4320
cggtttattc aactactcca ccggttcgca aaaatttttc ggtgacaaga acagagtcca 4380
agccatcatc aacgctctac aagaatctgg tagaacttac actgccaatg acatgaaggg 4440
tattgaaact ttcaccgaag ttttgagagc cggtttttac ttgggctatt acaacgatgg 4500
tctctcctac ctgaacgacc gtaatttcca agacaagtgt atcccagcca tgattgctat 4560
tcaaaagaac ccaaacttca agttaggtac tgctgttcaa gacgaagtta tcacatcttt 4620
gggtaagttg attggtaacg cctctgctaa cgctgaagtt gttaataact gtgtcccagt 4680
tttgaagcaa ttcagagaaa acttaaacca atacgctcca gactacgtca aaggtactgc 4740
cgtaaatgaa ttgatcaagg gtattgaatt tgacttttcc ggtgctgctt acgaaaagga 4800
cgtcaagacc atgccatggt acggtaagat tgacccattc atcaatgaat taaaggcctt 4860
gggtctctac ggtaacatca cttctgccac tgaatgggct tcagatgttg gtatctacta 4920
cttgtctaag ttcggtttgt actcaaccaa tcgtaacgac attgttcaat ccttagaaaa 4980
ggctgttgat atgtacaaat atggtaagat cgctttcgtt gctatggaaa gaatcacctg 5040
ggattacgat ggtatcggtt ccaacgggaa gaaggtcgac cacgataagt tcttggacga 5100
tgcagaaaag cattatttgc caaagaccta tactttcgac aacggaacct tcatcatcag 5160
agctggagat aaggtttctg aagaaaagat taagagattg tactgggctt cccgtgaagt 5220
taagtctcaa ttccacagag ttgttggtaa cgataaggct ttggaagtgg gtaacgctga 5280
cgatgtcttg accatgaaaa tcttcaactc tccagaagaa tacaagttca acaccaacat 5340
taacggtgtc tccactgata acggtggttt gtacatcgaa ccaagaggta ctttctacac 5400
ttacgaaaga accccacaac aatctatttt ctccttggaa gaactgttca gacacgaata 5460
cacccactac ttgcaagcta gatacttggt tgacggtttg tggggtcaag gtcctttcta 5520
cgaaaaaaac agattgactt ggttcgatga aggtactgct gaatttttcg ctggttccac 5580
cagaacttct ggtgtgttgc caagaaagtc catcttgggt taccttgcca aggataaggt 5640
cgatcataga tactccttga agaaaacttt gaactctggt tacgatgact ctgactggat 5700
gttctacaac tacggttttg ctgttgctca ctacttatac gaaaaggaca tgccaacttt 5760
tattaagatg aacaaggcca tcttgaacac tgatgttaag tcttacgacg aaatcattaa 5820
aaaactgtct gacgacgcta acaagaacac tgaataccaa aaccacatcc aagaactcgc 5880
tgataagtac caaggtgccg gtataccatt ggtgtctgat gactacctga aggaccatgg 5940
ttacaagaag gcttccgaag tttactcaga aatctccaag gctgcgtctt taactaacac 6000
ttctgttacc gctgaaaagt ctcaatactt caacacattc actttgaggg gtacttacac 6060
tggtgaaacc tctaagggtg aatttaagga ctgggacgaa atgtctaaga agttggacgg 6120
tactttggag tctttagcta agaactcttg gtctggttac aagactttga ccgcttactt 6180
caccaactac agagtcactt ctgataacaa agtccaatat gatgttgtct tccacggtgt 6240
tttgactgac aacgccgata tttctaacaa caaggctcct attgctaagg tcaccggtcc 6300
atctactggt gctgttggta gaaacattga attttctggt aaggattcca aggacgaaga 6360
tggcaagatt gtctcttatg actgggattt cggtgatggt gctacctcca gaggtaagaa 6420
ctctgtccac gcttacaaga aggctggtac ttacaacgtc actttgaagg tcaccgatga 6480
caagggtgct acggctactg aaagtttcac tattgaaatt aaaaacgaag acactaccac 6540
cccaatcacc aaggaaatgg aaccaaacga tgacattaag gaagccaacg gcccgattgt 6600
cgaaggcgtc acggtcaaag gtgacttgaa cggttctgat gacgctgaca ctttctactt 6660
tgatgtcaag gaagacggtg acgttaccat cgaattgcca tactctggtt cctctaactt 6720
tacctggtta gtctacaagg aaggtgacga ccaaaaccac atcgcctccg gtatcgataa 6780
aaacaactcc aaggttggta ctttcaagtc cactaagggt cgtcattacg ttttcatcta 6840
caagcacgac tctgcttcca acatttcata ctccttgaac atcaagggtt tgggtaacga 6900
aaagttgaaa gagaaggaaa acaatgactc ctctgacaag gcaaccgtta ttccaaattt 6960
caataccacc atgcaaggtt ctctattagg ggacgattct agagactact actccttcga 7020
agtcaaggaa gaaggtgaag ttaacattga attggataaa aaggatgaat ttggtgttac 7080
ttggaccttg cacccagaaa gcaacatcaa cgacagaatt acctacggtc aagtcgatgg 7140
taacaaggta tccaacaagg tcaaattgag accaggtaag tactacttgt tggtttacaa 7200
gtacagtggt tccggtaact acgaattgcg ggttaacaag caccaccacc accaccacta 7260
agctagcctc gagtctagaa actaagatta atataattat ataaaaatat tatcttcttt 7320
tctttatatc tagtgttatg taaaataaat tgatgactac ggaaagcttt tttatattgt 7380
ttctttttca ttctgagcca cttaaatttc gtgaatgttc ttataaggga cggtagattt 7440
acaagtgata caacaaaaag caaggcgctt tttctaataa aaagaagaaa agcatttaac 7500
aattgaacac ctctatatca acgaagaata ttactttgtc tctaaatcct tgtaaaatgt 7560
gtacgatctc tatatgggtt actcagaagt gtaccgaaga ctgcattgaa agtttatgtt 7620
ttttcactgc aagcgtcatt ttcgctttga gaagatgttc ttattcaaat ttcaactgtt 7680
atatagaaga gcaaaaaatt gccaaaaaaa acaacattta ttcatttaaa atataaaatt 7740
tgggcttcta tattttaata ttgcttttca attactgtta ttaaatgtaa gtactgcgtc 7800
tatgaaaata tatgcaaatg ctaagaaaaa tcctaaaaat ttgaatatga gatattcctc 7860
agtatttctt tttcatcctt tcttctgcgg ctctagccct ttgttctctc atcaatctgc 7920
gtctctgttc atcggtcaaa gaattcaaat tttgttgctg aattgaagga ataacgcgtg 7980
tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaagatc 8040
ctccggatcg tttcgccggc gtttatccag ctgcattaat gaatcggcca acgcgcgggg 8100
agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 8160
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 8220
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 8280
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 8340
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 8400
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 8460
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 8520
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 8580
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 8640
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 8700
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 8760
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 8820
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 8880
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 8940
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 9000
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 9060
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 9120
tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 9180
ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 9240
ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 9300
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 9360
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 9420
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 9480
aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 9540
tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 9600
ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 9660
agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 9720
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 9780
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 9840
accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 9900
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 9960
cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 10020
ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt 10080
tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat 10140
ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc 10200
atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc 10260
tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat 10320
acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct 10380
tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca 10440
ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa 10500
aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt 10560
caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa 10620
cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta 10680
ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac 10740
tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata 10800
aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt 10860
atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga 10920
agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa 10980
agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag 11040
agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa 11100
tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg 11160
cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac 11220
ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc 11280
attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg 11340
ccactcctca attggattag tctcatcctt caatgctatc atttcctttg atattggatc 11400
atactaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 11460
ctttcgtc 11468
<210> 8
<211> 11222
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240
ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300
agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360
cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatctaaa 420
aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat 480
tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 540
cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 600
tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 660
agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 720
tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 780
catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 840
aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 900
ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 960
taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag 1020
gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgga 1080
taaaggcgcg ccgtcgacaa tatagaaatt tcttagtttt aatgattgaa tgaatccatt 1140
tcgaacacgt caatgtactt ggacaagctt tgtagaacat gaagtacttg agcaatggac 1200
gcccaactgg aatttacttt agcgacacta ttccaagacc agcaactcaa agaagtaaat 1260
tctttttttc acaatagttg tttttttgat gatgtacgtc ccagcagcac ctaatggtat 1320
ttttatgagt attggcagtt tgccttatta attaagcaaa ccggtcatat attttaaaaa 1380
ataggaactt tgtagtcctt attaatgctg aataaaaaaa taggaagaga aatcagcaag 1440
tataaacagt taaatgtttt ttaactataa tggttgcatt caagaggaca tgtaaattgc 1500
gtactcggta caagttggta aaaacgttaa agcactccta cgagatttag tcagtcctgg 1560
cattaataaa caaaaagacc tgtatctata tttcatgaac tgggtaaaat caccacacaa 1620
aaaagaataa acaaatataa tgaactactt ttggtcttgg actcgcagtt tatagtttgt 1680
taaaattgac aaacacacat gttctgatta acttacttcc gcttcaaagt actgaaaaca 1740
tcatacgaac tggtataaga aggaagatag caaataaaaa taaatcaaat attgcttgta 1800
aaaaattagg atataaaaaa atctctattc attaaagaca aaaagctaaa ggaataaaaa 1860
agtcattcgc tttacgccaa ataaaacgtc tatgcacgtt tttaaaggct gtgaacatta 1920
acaatgttgt ggaattcagg cttgagagaa gcaccgccaa ccaagaaacc gtcaatatcg 1980
tggaacttga ggaactcctt gcagttacca ccgttaacgg aaccaccgta gatgacacgg 2040
agaccctcgg caacagatgc accaagcttg ttggtagccc acttgcggat ctcagcgtga 2100
acctcttgag cttgctcagg ggtggcagtc ttaccagtac caatggccca gacaggctca 2160
taagcaatga caatcttgga ccagttctgg accttgtcag cgatggcgtt caattgacga 2220
acaacaacgt tgatggtctc gttagcctca cgctcggcca aagtctcacc aatgcaggca 2280
acgacagtaa gaccttgttc aagggcaaac ttggtcttgt cggcaacgaa ctcgtcagac 2340
tccttgaaga tggtacgacg ctcggagtga ccagtcaaag tgtaggtaat accagcatca 2400
atcaaagatt gagcactgtt ctcaccagtg taggcaccgt tcttcttgtc gaagacgttt 2460
tgggcaccaa cgccaatatc cttcttgact tgttggcggg tggtgatgag gtacatgttt 2520
tgagggaaga tgacagtttc gacatcacca acgttaagct tggtggtgtt caaaccctca 2580
ataatagtct tcatggactc caaagagcca ttcatcttaa agttaccacc gacaaagaat 2640
ttacgtgcca ttttgatcct agttctacta ttgaaatgta gttggggaga gacgagagtt 2700
ggcccgtccg ctcattatat ataacgtagc ggacagtcac acagttaagg ggaattaccg 2760
agcttcggca atttaccccg tcgatagcaa ccgttggcat ggatccggcc ggccagatct 2820
acgtatggtc atttcttctt cagattccct catggagaaa gtgcggcaga tgtatatgac 2880
agagtcgcca gtttccaaga gactttattc aggcacttcc atgataggca agagagaaga 2940
cccagagatg ttgttgtcct agttacacat ggtatttatt ccagagtatt cctgatgaaa 3000
tggtttagat ggacatacga agagtttgaa tcgtttacca atgttcctaa cgggagcgta 3060
atggtgatgg aactggacga atccatcaat agatacgtcc tgaggaccgt gctacccaaa 3120
tggactgatt gtgagggaga cctaactaca tagtgtttaa agattacgga tatttaactt 3180
acttagaata atgccatttt tttgagttat aataatccta cgttagtgtg agcgggattt 3240
aaactgtgag gaccttaata cattcagaca cttctgcggt atcaccctac ttattccctt 3300
cgagattata tctaggaacc catcaggttg gtggaagatt acccgttcta agacttttca 3360
gcttcctcta ttgatgttac acctggacac cccttttctg gcatccagtt tttaatcttc 3420
agtggcatgt gagattctcc gaaattaatt aaagcaatca cacaattctc tcggatacca 3480
cctcggttga aactgacagg tggtttgtta cgcatgctaa tgcaaaggag cctatatacc 3540
tttggctcgg ctgctgtaac agggaatata aagggcagca taatttagga gtttagtgaa 3600
cttgcaacat ttactatttt cccttcttac gtaaatattt ttctttttaa ttctaaatca 3660
atctttttca attttttgtt tgtattcttt tcttgcttaa atctataact acaaaaaaca 3720
catacataaa ctaaaaggta ccaacaaaat gagatttcca tctattttta ctgctgtttt 3780
gtttgctgct tcttctgctt tggctgctcc agttaatact actactgaag atgaaactgc 3840
tcaaattcca gctgaagctg ttattggtta ttctgatttg gagggtgact ttgatgttgc 3900
tgttttgcca ttttctaact ctactaacaa cggtttgcta ttcatcaaca ctactatcgc 3960
ttctatcgct gctaaagaag aaggtgtttc tttggataaa agagaagaag gtgaaccaaa 4020
agctgttgac aagaacaatg ccactgctgc cgtccaaaac gaatccaagc gttacactgt 4080
ttcttactta aagaccttga actattacga cttggtcgat ctattggtta agaccgaaat 4140
cgaaaactta ccagacttgt ttcaatactc ttccgacgct aaggaatttt acggtaacaa 4200
gactagaatg tcctttatca tggacgaaat tggtcgtaga gctccacaat acactgaaat 4260
tgaccacaag ggtataccaa ccctcgttga agtcgtcaga gccgggttct acttgggttt 4320
tcacaacaaa gaattgaatg aaatcaacaa gagatctttc aaagaaagag ttatcccgtc 4380
tatcttggct atccaaaaga acccaaactt caaattaggt actgaagttc aagacaagat 4440
tgtctctgct accggtctat tggctggtaa cgaaaccgct ccaccagaag ttgttaacaa 4500
cttcacccca atcttgcaag attgtatcaa gaacatcgac agatacgcct tggacgactt 4560
gaaaagtaaa gcattgttca acgttttggc cgccccaacc tacgacatta ctgaatacct 4620
cagagctacc aaggaaaagc cagaaaacac tccatggtac ggtaagatcg atggtttcat 4680
caacgaattg aagaagctcg ctttgtacgg taaaattaac gacaacaact cttggattat 4740
cgataacggt atttaccaca ttgctccatt gggtaaattg cactctaaca acaaaatcgg 4800
tatcgaaact ctaactgaag ttatgaaggt ctacccatac ttgtccatgc aacacttgca 4860
atccgctgac caaatcaaga gacactacga ctctaaggat gccgaaggta acaagatccc 4920
attagataag ttcaagaagg aaggcaagga aaagtactgc ccaaagacct acacttttga 4980
tgatggtaag gttatcatca aggccggtgc tcgtgtcgaa gaagaaaagg tcaaaagatt 5040
atactgggct tccaaggaag tcaacagtca attcttcaga gtttatggta ttgacaagcc 5100
attggaagaa ggtaacccag acgacatcct taccatggtt atttacaact ctccagaaga 5160
atacaaactt aactccgtct tgtacggtta cgataccaac aacggtggta tgtacattga 5220
accagaaggt acttttttca cttacgaaag agaagctcaa gaatctacgt acactttgga 5280
agaattattc agacacgaat acacccatta cttgcaaggt agatacgctg tcccaggtca 5340
atggggtcgt accaagttgt atgataacga cagattgacc tggtacgaag aaggtggtgc 5400
tgaactgttt gctggttcta ctagaacttc cggcatcttg ccaagaaagt ctattgtctc 5460
taacattcac aacaccacta gaaacaacag atacaagctg tctgacactg ttcattcgaa 5520
gtacggtgcc tctttcgaat tttacaacta cgcttgtatg ttcatggact acatgtacaa 5580
taaggacatg ggtattttga acaagttgaa tgatttggct aagaacaacg acgttgacgg 5640
ttacgacaat tacatcagag atttgtcatc taattacgct ttgaacgata agtaccaaga 5700
tcacatgcaa gaacgtattg acaactatga aaacttgacc gttccattcg ttgctgacga 5760
ctacttagtt agacacgctt acaagaaccc aaacgaaatt tactccgaaa tctccgaagt 5820
tgccaagttg aaggatgcta agtctgaagt taagaaatct caatacttct caactttcac 5880
tttgcgtggt agttacaccg gaggtgcttc caagggtaag ctggaagatc aaaaagctat 5940
gaacaagttc attgacgact ctttgaagaa gttggacacc tattcctggt ctggttacaa 6000
gactcttact gcttatttta ccaactacaa ggtcgattct tctaacagag ttacttacga 6060
tgtcgttttc catggttact tgccaaacga aggtgattcc aagaacagct tgccatacgg 6120
taagattaac ggtacttaca agggtactga gaaggaaaag attaagttct cctccgaagg 6180
ttctttcgac ccagacggaa agatagtttc ctacgaatgg gacttcggtg atggtaacaa 6240
gtccaacgaa gagaaccctg aacacagtta cgataaggtc ggtacttaca ctgtcaagtt 6300
gaaggttacc gatgataagg gtgaaagctc tgtttctact accactgctg aaatcaaaga 6360
tttgtctgaa aacaagttgc cagtcatcta tatgcacgtt cctaaatcgg gtgctttaaa 6420
ccaaaaggtt gtcttctacg gtaagggtac ttacgaccca gacggttcta tcgctggcta 6480
ccaatgggat ttcggtgacg gttctgactt ttcctctgaa cagaacccat ctcacgtcta 6540
caccaagaaa ggtgaataca ctgtgacctt gagagtcatg gattcttccg gtcaaatgtc 6600
tgaaaagacc atgaaaatca agatcactga tccagtatac cctattggta ctgaaaagga 6660
accaaacaac tctaaggaaa cagcctccgg tccaatcgtt ccgggtattc cagtctccgg 6720
tactatcgaa aacacctctg atcaagacta tttctacttc gatgttatta ctccaggtga 6780
agtcaagatc gacatcaaca agctaggtta cggtggtgct acatgggttg tttacgatga 6840
aaacaataac gctgtctcct acgctaccga tgacggtcaa aatttgtctg gtaaattcaa 6900
ggctgacaag cctgggagat actacattca tttgtacatg ttcaatggtt cttacatgcc 6960
atacagaatt aacattgaag gttctgttgg tagacaccac caccaccacc actaagctag 7020
cctcgagtct agaaactaag attaatataa ttatataaaa atattatctt cttttcttta 7080
tatctagtgt tatgtaaaat aaattgatga ctacggaaag cttttttata ttgtttcttt 7140
ttcattctga gccacttaaa tttcgtgaat gttcttataa gggacggtag atttacaagt 7200
gatacaacaa aaagcaaggc gctttttcta ataaaaagaa gaaaagcatt taacaattga 7260
acacctctat atcaacgaag aatattactt tgtctctaaa tccttgtaaa atgtgtacga 7320
tctctatatg ggttactcag aagtgtaccg aagactgcat tgaaagttta tgttttttca 7380
ctgcaagcgt cattttcgct ttgagaagat gttcttattc aaatttcaac tgttatatag 7440
aagagcaaaa aattgccaaa aaaaacaaca tttattcatt taaaatataa aatttgggct 7500
tctatatttt aatattgctt ttcaattact gttattaaat gtaagtactg cgtctatgaa 7560
aatatatgca aatgctaaga aaaatcctaa aaatttgaat atgagatatt cctcagtatt 7620
tctttttcat cctttcttct gcggctctag ccctttgttc tctcatcaat ctgcgtctct 7680
gttcatcggt caaagaattc aaattttgtt gctgaattga aggaataacg cgtgtacgca 7740
tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa gatcctccgg 7800
atcgtttcgc cggcgtttat ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 7860
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 7920
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 7980
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 8040
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 8100
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 8160
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 8220
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 8280
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 8340
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 8400
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 8460
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 8520
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 8580
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 8640
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 8700
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 8760
aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 8820
taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 8880
gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 8940
agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 9000
cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 9060
tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 9120
gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 9180
agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 9240
gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 9300
atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 9360
gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 9420
tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 9480
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 9540
agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 9600
gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 9660
cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 9720
tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 9780
ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc attttgtaga 9840
acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac 9900
agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt 9960
gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt 10020
tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct 10080
tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt 10140
actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag 10200
gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc 10260
tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat 10320
aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa 10380
gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt 10440
ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg 10500
aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat 10560
gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag 10620
ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt 10680
attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg 10740
tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata 10800
ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac 10860
gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta 10920
tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat 10980
gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca 11040
tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc 11100
ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatacta 11160
agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 11220
tc 11222
<210> 9
<211> 11462
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240
ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300
agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360
cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatctaaa 420
aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct tcaatttaat 480
tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 540
cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 600
tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 660
agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 720
tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 780
catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 840
aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 900
ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 960
taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag 1020
gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctgga 1080
taaaggcgcg ccgtcgacaa tatagaaatt tcttagtttt aatgattgaa tgaatccatt 1140
tcgaacacgt caatgtactt ggacaagctt tgtagaacat gaagtacttg agcaatggac 1200
gcccaactgg aatttacttt agcgacacta ttccaagacc agcaactcaa agaagtaaat 1260
tctttttttc acaatagttg tttttttgat gatgtacgtc ccagcagcac ctaatggtat 1320
ttttatgagt attggcagtt tgccttatta attaagcaaa ccggtcatat attttaaaaa 1380
ataggaactt tgtagtcctt attaatgctg aataaaaaaa taggaagaga aatcagcaag 1440
tataaacagt taaatgtttt ttaactataa tggttgcatt caagaggaca tgtaaattgc 1500
gtactcggta caagttggta aaaacgttaa agcactccta cgagatttag tcagtcctgg 1560
cattaataaa caaaaagacc tgtatctata tttcatgaac tgggtaaaat caccacacaa 1620
aaaagaataa acaaatataa tgaactactt ttggtcttgg actcgcagtt tatagtttgt 1680
taaaattgac aaacacacat gttctgatta acttacttcc gcttcaaagt actgaaaaca 1740
tcatacgaac tggtataaga aggaagatag caaataaaaa taaatcaaat attgcttgta 1800
aaaaattagg atataaaaaa atctctattc attaaagaca aaaagctaaa ggaataaaaa 1860
agtcattcgc tttacgccaa ataaaacgtc tatgcacgtt tttaaaggct gtgaacatta 1920
acaatgttgt ggaattcagg cttgagagaa gcaccgccaa ccaagaaacc gtcaatatcg 1980
tggaacttga ggaactcctt gcagttacca ccgttaacgg aaccaccgta gatgacacgg 2040
agaccctcgg caacagatgc accaagcttg ttggtagccc acttgcggat ctcagcgtga 2100
acctcttgag cttgctcagg ggtggcagtc ttaccagtac caatggccca gacaggctca 2160
taagcaatga caatcttgga ccagttctgg accttgtcag cgatggcgtt caattgacga 2220
acaacaacgt tgatggtctc gttagcctca cgctcggcca aagtctcacc aatgcaggca 2280
acgacagtaa gaccttgttc aagggcaaac ttggtcttgt cggcaacgaa ctcgtcagac 2340
tccttgaaga tggtacgacg ctcggagtga ccagtcaaag tgtaggtaat accagcatca 2400
atcaaagatt gagcactgtt ctcaccagtg taggcaccgt tcttcttgtc gaagacgttt 2460
tgggcaccaa cgccaatatc cttcttgact tgttggcggg tggtgatgag gtacatgttt 2520
tgagggaaga tgacagtttc gacatcacca acgttaagct tggtggtgtt caaaccctca 2580
ataatagtct tcatggactc caaagagcca ttcatcttaa agttaccacc gacaaagaat 2640
ttacgtgcca ttttgatcct agttctacta ttgaaatgta gttggggaga gacgagagtt 2700
ggcccgtccg ctcattatat ataacgtagc ggacagtcac acagttaagg ggaattaccg 2760
agcttcggca atttaccccg tcgatagcaa ccgttggcat ggatccggcc ggccagatct 2820
acgtatggtc atttcttctt cagattccct catggagaaa gtgcggcaga tgtatatgac 2880
agagtcgcca gtttccaaga gactttattc aggcacttcc atgataggca agagagaaga 2940
cccagagatg ttgttgtcct agttacacat ggtatttatt ccagagtatt cctgatgaaa 3000
tggtttagat ggacatacga agagtttgaa tcgtttacca atgttcctaa cgggagcgta 3060
atggtgatgg aactggacga atccatcaat agatacgtcc tgaggaccgt gctacccaaa 3120
tggactgatt gtgagggaga cctaactaca tagtgtttaa agattacgga tatttaactt 3180
acttagaata atgccatttt tttgagttat aataatccta cgttagtgtg agcgggattt 3240
aaactgtgag gaccttaata cattcagaca cttctgcggt atcaccctac ttattccctt 3300
cgagattata tctaggaacc catcaggttg gtggaagatt acccgttcta agacttttca 3360
gcttcctcta ttgatgttac acctggacac cccttttctg gcatccagtt tttaatcttc 3420
agtggcatgt gagattctcc gaaattaatt aaagcaatca cacaattctc tcggatacca 3480
cctcggttga aactgacagg tggtttgtta cgcatgctaa tgcaaaggag cctatatacc 3540
tttggctcgg ctgctgtaac agggaatata aagggcagca taatttagga gtttagtgaa 3600
cttgcaacat ttactatttt cccttcttac gtaaatattt ttctttttaa ttctaaatca 3660
atctttttca attttttgtt tgtattcttt tcttgcttaa atctataact acaaaaaaca 3720
catacataaa ctaaaaggta ccaacaaaat gagatttcca tctattttta ctgctgtttt 3780
gtttgctgct tcttctgctt tggctgctcc agttaatact actactgaag atgaaactgc 3840
tcaaattcca gctgaagctg ttattggtta ttctgatttg gagggtgact ttgatgttgc 3900
tgttttgcca ttttctaact ctactaacaa cggtttgcta ttcatcaaca ctactatcgc 3960
ttctatcgct gctaaagaag aaggtgtttc tttggataaa agagaggctg aagctaagcc 4020
aatcgaaaac actaacgaca ccagcatcaa gaacgttgaa aagttgagaa acgccccaaa 4080
cgaagaaaac tctaagaaag ttgaagactc taagaatgat aaggttgaac acgttaagaa 4140
cattgaagaa gctaaggtcg aacaagttgc tccagaagtc aagtctaaaa gtaccttgag 4200
atctgcttct atagctaata caaattccga aaagtacgac ttcgaatatc ttaatggttt 4260
gagttacacc gaattgacta acttgatcaa gaacatcaag tggaaccaaa tcaacggttt 4320
attcaactac tccaccggtt cgcaaaaatt tttcggtgac aagaacagag tccaagccat 4380
catcaacgct ctacaagaat ctggtagaac ttacactgcc aatgacatga agggtattga 4440
aactttcacc gaagttttga gagccggttt ttacttgggc tattacaacg atggtctctc 4500
ctacctgaac gaccgtaatt tccaagacaa gtgtatccca gccatgattg ctattcaaaa 4560
gaacccaaac ttcaagttag gtactgctgt tcaagacgaa gttatcacat ctttgggtaa 4620
gttgattggt aacgcctctg ctaacgctga agttgttaat aactgtgtcc cagttttgaa 4680
gcaattcaga gaaaacttaa accaatacgc tccagactac gtcaaaggta ctgccgtaaa 4740
tgaattgatc aagggtattg aatttgactt ttccggtgct gcttacgaaa aggacgtcaa 4800
gaccatgcca tggtacggta agattgaccc attcatcaat gaattaaagg ccttgggtct 4860
ctacggtaac atcacttctg ccactgaatg ggcttcagat gttggtatct actacttgtc 4920
taagttcggt ttgtactcaa ccaatcgtaa cgacattgtt caatccttag aaaaggctgt 4980
tgatatgtac aaatatggta agatcgcttt cgttgctatg gaaagaatca cctgggatta 5040
cgatggtatc ggttccaacg ggaagaaggt cgaccacgat aagttcttgg acgatgcaga 5100
aaagcattat ttgccaaaga cctatacttt cgacaacgga accttcatca tcagagctgg 5160
agataaggtt tctgaagaaa agattaagag attgtactgg gcttcccgtg aagttaagtc 5220
tcaattccac agagttgttg gtaacgataa ggctttggaa gtgggtaacg ctgacgatgt 5280
cttgaccatg aaaatcttca actctccaga agaatacaag ttcaacacca acattaacgg 5340
tgtctccact gataacggtg gtttgtacat cgaaccaaga ggtactttct acacttacga 5400
aagaacccca caacaatcta ttttctcctt ggaagaactg ttcagacacg aatacaccca 5460
ctacttgcaa gctagatact tggttgacgg tttgtggggt caaggtcctt tctacgaaaa 5520
aaacagattg acttggttcg atgaaggtac tgctgaattt ttcgctggtt ccaccagaac 5580
ttctggtgtg ttgccaagaa agtccatctt gggttacctt gccaaggata aggtcgatca 5640
tagatactcc ttgaagaaaa ctttgaactc tggttacgat gactctgact ggatgttcta 5700
caactacggt tttgctgttg ctcactactt atacgaaaag gacatgccaa cttttattaa 5760
gatgaacaag gccatcttga acactgatgt taagtcttac gacgaaatca ttaaaaaact 5820
gtctgacgac gctaacaaga acactgaata ccaaaaccac atccaagaac tcgctgataa 5880
gtaccaaggt gccggtatac cattggtgtc tgatgactac ctgaaggacc atggttacaa 5940
gaaggcttcc gaagtttact cagaaatctc caaggctgcg tctttaacta acacttctgt 6000
taccgctgaa aagtctcaat acttcaacac attcactttg aggggtactt acactggtga 6060
aacctctaag ggtgaattta aggactggga cgaaatgtct aagaagttgg acggtacttt 6120
ggagtcttta gctaagaact cttggtctgg ttacaagact ttgaccgctt acttcaccaa 6180
ctacagagtc acttctgata acaaagtcca atatgatgtt gtcttccacg gtgttttgac 6240
tgacaacgcc gatatttcta acaacaaggc tcctattgct aaggtcaccg gtccatctac 6300
tggtgctgtt ggtagaaaca ttgaattttc tggtaaggat tccaaggacg aagatggcaa 6360
gattgtctct tatgactggg atttcggtga tggtgctacc tccagaggta agaactctgt 6420
ccacgcttac aagaaggctg gtacttacaa cgtcactttg aaggtcaccg atgacaaggg 6480
tgctacggct actgaaagtt tcactattga aattaaaaac gaagacacta ccaccccaat 6540
caccaaggaa atggaaccaa acgatgacat taaggaagcc aacggcccga ttgtcgaagg 6600
cgtcacggtc aaaggtgact tgaacggttc tgatgacgct gacactttct actttgatgt 6660
caaggaagac ggtgacgtta ccatcgaatt gccatactct ggttcctcta actttacctg 6720
gttagtctac aaggaaggtg acgaccaaaa ccacatcgcc tccggtatcg ataaaaacaa 6780
ctccaaggtt ggtactttca agtccactaa gggtcgtcat tacgttttca tctacaagca 6840
cgactctgct tccaacattt catactcctt gaacatcaag ggtttgggta acgaaaagtt 6900
gaaagagaag gaaaacaatg actcctctga caaggcaacc gttattccaa atttcaatac 6960
caccatgcaa ggttctctat taggggacga ttctagagac tactactcct tcgaagtcaa 7020
ggaagaaggt gaagttaaca ttgaattgga taaaaaggat gaatttggtg ttacttggac 7080
cttgcaccca gaaagcaaca tcaacgacag aattacctac ggtcaagtcg atggtaacaa 7140
ggtatccaac aaggtcaaat tgagaccagg taagtactac ttgttggttt acaagtacag 7200
tggttccggt aactacgaat tgcgggttaa caagcaccac caccaccacc actaagctag 7260
cctcgagtct agaaactaag attaatataa ttatataaaa atattatctt cttttcttta 7320
tatctagtgt tatgtaaaat aaattgatga ctacggaaag cttttttata ttgtttcttt 7380
ttcattctga gccacttaaa tttcgtgaat gttcttataa gggacggtag atttacaagt 7440
gatacaacaa aaagcaaggc gctttttcta ataaaaagaa gaaaagcatt taacaattga 7500
acacctctat atcaacgaag aatattactt tgtctctaaa tccttgtaaa atgtgtacga 7560
tctctatatg ggttactcag aagtgtaccg aagactgcat tgaaagttta tgttttttca 7620
ctgcaagcgt cattttcgct ttgagaagat gttcttattc aaatttcaac tgttatatag 7680
aagagcaaaa aattgccaaa aaaaacaaca tttattcatt taaaatataa aatttgggct 7740
tctatatttt aatattgctt ttcaattact gttattaaat gtaagtactg cgtctatgaa 7800
aatatatgca aatgctaaga aaaatcctaa aaatttgaat atgagatatt cctcagtatt 7860
tctttttcat cctttcttct gcggctctag ccctttgttc tctcatcaat ctgcgtctct 7920
gttcatcggt caaagaattc aaattttgtt gctgaattga aggaataacg cgtgtacgca 7980
tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa gatcctccgg 8040
atcgtttcgc cggcgtttat ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 8100
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 8160
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 8220
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 8280
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 8340
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 8400
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 8460
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 8520
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 8580
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 8640
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 8700
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 8760
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 8820
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 8880
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 8940
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 9000
aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 9060
taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 9120
gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 9180
agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 9240
cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 9300
tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 9360
gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 9420
agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 9480
gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 9540
atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 9600
gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 9660
tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 9720
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 9780
agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 9840
gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 9900
cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 9960
tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 10020
ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc attttgtaga 10080
acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac 10140
agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt 10200
gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt 10260
tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct 10320
tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt 10380
actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag 10440
gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc 10500
tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat 10560
aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa 10620
gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt 10680
ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg 10740
aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat 10800
gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag 10860
ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt 10920
attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg 10980
tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata 11040
ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac 11100
gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta 11160
tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat 11220
gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca 11280
tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc 11340
ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatacta 11400
agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 11460
tc 11462
Claims (10)
1. A recombinant plasmid expressing collagenase, characterized in that: the leader sequence (leader) of alpha factor, the leader peptide sequence (pro peptide) and the enzyme functional region sequence (Chain) of the collagenase ColG of the clostridium histolyticum are inserted into an expression plasmid, or the leader sequence (leader) and the spacer sequence (spacer) of the alpha factor, the leader peptide sequence (pro peptide) and the enzyme functional region sequence (Chain) of the collagenase ColH of the clostridium histolyticum are inserted into the expression plasmid;
the leader sequence (leader) of the alpha factor is shown as SEQ ID No. 1;
the leader peptide sequence and the enzyme functional region sequence (pro peptide-Chain) of the ColG are shown in SEQ ID No. 2;
the leader-spacer sequence of the alpha factor is shown as SEQ ID No. 3;
the leader peptide sequence and the enzyme functional region sequence (pro peptide-Chain) of the ColH are shown in SEQ ID No. 4.
2. The recombinant plasmid according to claim 1, characterized in that: the expression plasmid is CPOTud, p426GPD, p426TEF, p426ADH, p426CYC, p416GPD, p4216TEF, p4126ADH, p416CYC, p425GPD, p425TEF, p425ADH, p425CYC, p415GPD, p4215TEF, p4125ADH, p415CYC, p424GPD, p424TEF, p424ADH, p424CYC, p414GPD, p4214TEF, p4124ADH, p414CYC, p GPD, p423TEF, p ADH 423ADH, p CY423C, p413GPD, p4213TEF, p4123ADH, p413CYC, pSP-1, pRS303, pRS304, pRS305, pRS306, pRS413, pRS414, pRS415, pRS416, pRS424 or pRS 424.
3. Saccharomyces cerevisiae secreting collagenase is characterized by: is obtained by transforming the recombinant plasmid of claim 1 or 2 into a starting yeast strain.
4. The Saccharomyces cerevisiae according to claim 3, wherein: the starting yeast strain is Saccharomyces cerevisiae B184M, CEN.PK 530.1C, CEN.PK 113.5D, BY4742, BY4741, CEN.PK2-1D, CEN.PK2-1C or IMX 581.
5. A fermentation medium suitable for the Saccharomyces cerevisiae as claimed in claim 3 or 4, characterized in that: ca is added into YPD medium 2+ And/or Zn 2+ ；
The Ca 2+ In a concentration of 10-25mM, said Zn 2+ Is in a concentration of 0.4-1 mM.
6. The fermentation medium of claim 5, wherein: the Ca 2+ In a concentration of 10mM, said Zn 2+ Is 0.6 mM.
7. The method for fermentation culture of saccharomyces cerevisiae according to claim 3 or 4, wherein: inoculating the saccharomyces cerevisiae into the fermentation medium of claim 5 or 6 for culture.
8. The fermentation culture method according to claim 7, wherein: the culture is carried out at 30-37 ℃ for at least 96h, and then the recombinant collagenase can be collected.
9. Use of the recombinant plasmid of claim 1 or 2, the saccharomyces cerevisiae of claim 3 or 4 for the production of collagenase.
10. Use of the recombinant plasmid of claim 1 or 2, the saccharomyces cerevisiae of claim 3 or 4 for degrading gelatin and collagen.
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