1. A5-12-5 tricyclic sesterterpene skeleton compound Schultriene is characterized in that: the molecular formula is C₂₅H₄₀The structural formula is as follows:

2. the 5-12-5 tricyclic sesterterpene skeleton compound synthase CcCS according to claim 1, wherein the amino acid sequence of the synthase is shown in SEQ ID NO.3, and the N-terminal and the C-terminal of the synthase are respectively responsible for terpene cyclization and prenyl transfer functions.

3. The synthetase according to claim 2, characterized in that the synthetase comprises two conserved domains: the terpene cyclase domain contains two recognition domains for Mg²⁺And the characteristic conserved motifs of the substrates DDACE and NDYWSWPRE, the E-IPPS domain also contains two characteristic conserved motifs with similar functions DDIED and DDYMN.

4. A gene encoding the synthetase according to claim 2, characterized in that: the polynucleotide sequence of the strain CS12565(Cytospora schulzeri12565) is shown in SEQ ID NO.1, the strain CS12565 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 21944.

5. The gene of claim 4, wherein the gene comprises 3 introns, the cDNA size is 2280bp, and the sequence is shown as SEQ ID NO. 2.

6. A recombinant expression vector of 5-12-5 tricyclic sesterterpene skeleton compound, wherein the recombinant expression vector is a eukaryotic or prokaryotic expression vector carrying the synthetase of claim 2 or 3 or carrying the gene of claim 3 or 4.

7. A recombinant expression host cell of a 5-12-5 tricyclic sesterterpene skeleton compound, characterized in that: comprising the recombinant expression vector of claim 6.

8. Use of the synthetase of claim 2 or 3 or the gene of claim 4 or 5 for the synthesis of 5-12-5 tricyclic sesterterpene skeleton compounds.

9. A method for the heterologous expression of a 5-12-5 tricyclic sesterterpene skeleton compound comprising the steps of:

A. construction of heterologous expression vector for CcCS Gene

By PCR technology, a gene sequence containing CcCS is obtained by amplification by taking a CS12565 genome as a template, and primer sequences used for amplification are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

connecting the amplified fragment with a pUARA2 vector through homologous recombination to construct pUARA2-CcCS expression plasmid, transforming the ligation product into Escherichia coli DH10B, screening positive transformants, extracting plasmid PCR verification after culture to obtain pUARA2-CcCS plasmid,

B. protoplast transformation

Culturing Aspergillus oryzae NSAR1, collecting protoplast, mixing with pUARA2-CcCS plasmid, culturing, performing PCR verification on the grown transformant, wherein the positive transformant is CcCS heterologous expression strain AO-CcCS,

C. culture of heterologous expression strain AO-CcCS and product separation

Inoculating a heterologous expression strain AO-CcCS, screening by a pUARA2 plasmid screening liquid culture medium, performing fermentation culture, separating and purifying the obtained fermentation crude extract by adopting a forward silica gel column chromatography method, detecting each flow part rapidly by TLC, combining the same flow parts with spots, concentrating under reduced pressure, performing rotary evaporation to dryness, transferring into a weighed sample bottle, weighing a sample and recording the weight.

Background

Terpenoids are a generic term for all isoprene polymers and their derivatives. The terpenoid plays an important role in the medical, food and cosmetic industries as the most abundant type of small molecular natural products^[1]. The sesterterpene is a terpenoid synthesized by taking GFPP as a precursor through catalysis, only occupies a very small part (less than 2%) of the terpenoid, and the sesterterpene usually has a complex cyclization structure and has high medicament forming potential^[2]. Such as: the obtained opsiobolina has cytotoxic and antimalarial activities, calmodulin activity and anti-glioma activity^[3-5]. Sesterterpenes that are catalytically synthesized by bifunctional terpene synthases (BFTSs) are often favored by their novel catalytic mechanisms.

The traditional chemical synthesis of sesterterpene has the problems of complicated synthesis steps, poor catalytic specificity, limited new framework acquisition capacity and the like, and the technical situation of low yield and high cost makes the sesterterpene compounds of a novel framework difficult to directly acquire from nature. The rapid development of genomic technology and bioinformatics has led to the realization that microorganisms also have considerable terpenoid synthesis potential. Through the combination of microorganism whole genome analysis and genome mining strategies, a plurality of bifunctional terpene synthases with brand-new catalytic mechanisms can be mined from microorganisms, and a large amount of new skeleton terpene products can be obtained by combining a high-efficiency terpene biosynthesis gene cluster heterologous expression system, so that the method becomes an important method for finding new drug lead compounds.

Cytospora schulzeri belongs to Ascomycota (Ascomycota), Chaetomium (Sordariomycetes), Diapurthales (Diaporthales), and Chaetomium (Valsaceae) of Cytospora, and can cause apple trees to rot^[6]. There has been no report to date on the isolation of natural products from Cytospora schulzeri that give rise to sesterterpene activity. The mining of the secondary metabolic biosynthesis gene cluster of Cytospora schulzeri is helpful for enriching natural product compound libraries and providing valuable lead compounds for new drug discovery.

Disclosure of Invention

The invention is carried out by the research, and provides a 5-12-5 tricyclic sesterterpene skeleton compound, synthetase thereof, a coding gene of the synthetase and a heterologous expression method of the compound.

The idea of the invention is as follows: in the biosynthesis process of the dug strain C.schulzeri12565 metabolite sesterterpene compounds, the correlation between the CcCS gene and terpene synthesis is known through gene function analysis, which indicates that the CcCS gene participates in the biosynthesis of 5-12-5 tricyclic sesterterpene skeleton compounds. Further obtains the CcCS protein through heterogeneously expressing the CcCS gene to carry out in vitro enzymatic reaction, and verifies that the CcCS gene is a synthetic gene of a 5-12-5 tricyclic diterpene skeleton compound Schultriene.

The invention aims at providing the structure of 5-12-5 tricyclic sesterterpene skeleton compounds; the second purpose is to provide an enzyme and a gene for synthesizing the compound; the third objective is to provide a method for heterologous expression of 5-12-5 tricyclic sesterterpene skeleton compounds.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided a 5-12-5 tricyclic sesterterpene skeleton compound Schultriene of formula C₂₅H₄₀The structural formula is as follows:

in a second aspect of the invention, the synthetase CcCS of the 5-12-5 tricyclic sesterterpene skeleton compound is provided, the amino acid sequence of the synthetase is shown in SEQ ID No.3, and the N end and the C end of the synthetase are respectively responsible for terpene cyclization and isopentenyl group transfer functions.

Further, the synthetase contains two conserved domains: the terpene cyclase domain contains two recognition domains for Mg²⁺And the characteristic conserved motifs of the substrates DDACE and NDYWSWPRE, the E-IPPS domain also contains two characteristic conserved motifs with similar functions DDIED and DDYMN.

In a second aspect of the present invention, there is provided a gene encoding the above-mentioned synthetase cloned from the genome of the strain CS12565 (C. schulzeri12565), the polynucleotide sequence of which is shown in SEQ ID NO. 1. The CS12565 strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 21944. The gene contains 3 introns, the cDNA size is 2280bp, and the sequence is shown in SEQ ID NO. 2.

In a third aspect of the present invention, there is provided a recombinant expression vector of 5-12-5 tricyclic sesterterpene skeleton compound, wherein the recombinant expression vector is a eukaryotic or prokaryotic expression vector carrying the above-mentioned synthetase or gene, such as E.coli, yeast system and filamentous fungi.

In the fourth aspect of the invention, the invention provides a recombinant expression host cell of 5-12-5 tricyclic sesterterpene skeleton compounds, which contains the recombinant expression vector to realize the heterologous expression of the compounds.

In a fifth aspect of the invention, the application of the above-mentioned synthetase or gene in the synthesis of terpenoid, specifically in the synthesis of 5-12-5 tricyclic sesterterpene skeleton compounds is provided.

In a sixth aspect of the present invention, there is provided a method for the heterologous expression of a 5-12-5 tricyclic sesterterpene skeleton compound, comprising the steps of:

A. construction of heterologous expression vector for CcCS Gene

By using a PCR technology, a gene sequence containing CcCS is obtained by amplification by using a C.schulzeri12565 genome as a template, and primer sequences used for amplification are respectively shown as SEQ ID NO.4 and SEQ ID NO. 5;

primer sequences used for amplification:

CcCS-F：cgGAATTCGAGCTCGATGCTTGAAGCAAACGAGCT；

CcCS-R：tactacaGATCCCCGGTCAAACTCGCAAGGTTTCCACCAG。

connecting the amplified fragment with a pUARA2 vector through homologous recombination to construct pUARA2-CcCS expression plasmid, transforming the ligation product into Escherichia coli DH10B, screening positive transformants, extracting plasmid PCR verification after culture to obtain pUARA2-CcCS plasmid,

B. protoplast transformation

Culturing Aspergillus oryzae NSAR1, collecting protoplast, mixing with pUARA2-CcCS plasmid, culturing, performing PCR verification on the grown transformant, wherein the positive transformant is CcCS heterologous expression strain AO-CcCS,

C. culture of heterologous expression strain AO-CcCS and product separation

Inoculating a heterologous expression strain AO-CcCS, screening by a pUARA2 plasmid screening liquid culture medium, performing fermentation culture, separating and purifying the obtained fermentation crude extract by adopting a forward silica gel column chromatography method, detecting each flow part rapidly by TLC, combining the same flow parts with spots, concentrating under reduced pressure, performing rotary evaporation to dryness, transferring into a weighed sample bottle, weighing a sample and recording the weight.

The invention has the following beneficial effects:

the invention discovers the CcCS gene for synthesizing a 5-12-5 tricyclic diterpene skeleton compound Schultriene, and the coded CcCS protein can assist the synthesis of the mother nucleus of the Schultriene compound. The invention provides a new resource for the biosynthesis of 5-12-5 tricyclic sesterterpene compounds and provides a choice for the synthesis of the compounds.

The CcCS gene discovered by the invention catalyzes the generation of a new sesterterpene skeleton compound, and provides a valuable lead compound resource for enriching a natural product compound library and discovering new antibiotics.

Drawings

FIG. 1 is a HR-EI-MS spectrum of the compound Schultriene of the present invention.

FIG. 2 shows the dissolution of the compound Schultriene in Benzene-d₆In (1)¹H-NMR spectrum.

FIG. 3 shows the dissolution of the compound Schultriene in Benzene-d₆In (1)¹³C-NMR spectrum.

FIG. 4 shows the dissolution of the compound Schultriene in Benzene-d₆In¹³C-DEPT 135 Spectrum.

FIG. 5 shows the dissolution of the compound Schultriene in Benzene-d₆In (1)¹H-¹H COSY spectra.

FIG. 6 shows the dissolution of the compound Schultriene in Benzene-d₆HSQC spectrum in (1).

FIG. 7 shows the dissolution of the compound Schultriene in Benzene-d₆HMBC spectrum in (1).

FIG. 8 shows the dissolution of the compound Schultriene in Benzene-d₆NOESY spectrum of (1).

FIG. 9 is a diagram showing the alignment of the amino acid sequences of the protein encoded by the CsSS gene in Cytospora schulzeri12565 and the reported proteins.

Strain preservation information: CS12565(Cytospora schulzeni 12565) is preserved in the China general microbiological culture Collection center, the preservation address is No.3 of West Lu No.1 of the Chaoyang district in Beijing, the preservation date is No. 02 of 04.2021 years, and the preservation number is CGMCC No. 21944.

Detailed Description

The following embodiments are implemented on the premise of the technical scheme of the present invention, and give detailed implementation modes and specific operation procedures, but the protection scope of the present invention is not limited to the following embodiments.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The 5-12-5 tricyclic sesterterpene skeleton compound Schultriene synthetic gene provided by the invention is cloned from Cytospora schulzeri12565 and is named as CcCS gene, and the gene sequence of the CcCS gene is shown in SEQ ID NO. 1. The CcCS gene contains 3 introns, the cDNA size is 2280bp, and the sequence is shown in SEQ ID NO. 2. The protein coded by the CcCS gene is named as CcCS protein, and the amino acid sequence of the protein is shown in SEQ ID NO. 3.

CcCS protein belongs to chimeric terpene synthetase, and the N end and the C end of the CcCS protein are respectively responsible for terpene cyclization and isopentenyl transfer functions. The CcCS protein contains two conserved domains, wherein the terpene cyclase domain contains two domains for recognizing Mg²⁺And the characteristic conserved motifs of the substrates DDACE and NDYWSWPRE, the E-IPPS domain also contains two characteristic conserved motifs with similar functions DDIED and DDYMN (fig. 9).

Example 1: heterologous expression and structural identification of 5-12-5 tricyclic diterpene skeleton compound Schultriene synthetic gene

By using a heterologous expression method, the CcCS gene in the Cytospora schulzeri12565 thallus is transferred into a host Aspergillus oryzae by constructing an expression plasmid, and the production condition of a heterologous expression strain product is detected. The medium formulation used is shown in Table 1.

TABLE 1 culture Medium formulation used in the examples

Construction of CcCS Gene Source expression vector

(1) The gene sequence containing the CcCS was amplified by PCR using the c.schulzeri12565 genome as a template. Primer sequences used for amplification:

CcCS-F：cgGAATTCGAGCTCGATGCTTGAAGCAAACGAGCT(SEQ ID NO.4)；

CcCS-R：tactacaGATCCCCGGTCAAACTCGCAAGGTTTCCACCAG(SEQ ID NO.5)。

(2) and connecting the amplified fragment with a pUARA2 vector through homologous recombination to construct a pUARA2-CcCS expression plasmid, wherein the two sides of the vector CcCS have homologous sequences which are consistent with those of the pUARA2 vector.

(3) The ligation product was transformed into E.coli DH10B and positive transformants were selected by ampicillin screening. And (3) liquid culturing positive transformants, extracting plasmid PCR verification, and obtaining pUARA2-CcCS plasmid.

2. Transformation of protoplasts

(1) Aspergillus oryzae NSAR1 was spread on PDA plates and cultured at 30 ℃ for 7 days.

(2) Spores were collected in 10mL of 0.1% Tween-80 (1 plate of the brood is typically collected) and counted on a hemocytometer. Inoculation about 10⁷The spores were cultured in 50mL DPY at 30 ℃ and 220rpm for 2-3 days.

(3) 100mg of Yatalase was weighed, dissolved by addition of dissolution 0, 20ml was sterilized by filtration through a 0.22 μm filter and added to a 50ml centrifuge tube.

(4) And collecting the thallus. Pouring 100ml of cultured mycelia into a P250 glass filter, removing a culture medium, washing with sterile water (or 0.8M NaCl) for 3-5 times, squeezing out water with a sterile medicine spoon, and adding the pressed dry mycelia into a Yatalase solution. Culturing at 30 deg.C and 200rpm under shaking for 1-2 hr until the spherical mycelium disappears and the supernatant is clear and dirty.

(5) The digested bacterial solution was filtered through Miracloth, protoplasts were collected and transferred to a new 50ml centrifuge tube, centrifuged at 4 ℃ at 800g for 5 min.

(6) The supernatant was removed, 20ml of 0.8M NaCl was added for resuspension washing, and centrifugation was carried out at 4 ℃ at 800g for 5min (twice washing). The supernatant was removed and 10ml of 0.8M NaCl was added. The number of protoplasts was counted under a microscope using a bacterial counter. Protoplast count-total count/80X 400ml X10⁴X dilution factor.

(7) Adjusting the protoplast concentration to 2X 10⁸cell/ml. (sol 2/sol 3 ═ 4/1), 0.5ml to 2ml of protoplasts were harvested depending on the growth of the cells.

(8) 200. mu.l of the protoplast solution was transferred to a new 50ml centrifuge tube, 10. mu.g of the expression plasmid pUARA2-CcCS was added, and gently mixed. Standing on ice for 20 min. During this time, the sterilized Top agar was incubated in a water bath at 50 ℃.

(9) To the suspension of (8) was added 1ml of sol 3 and gently mixed with a tip. Standing at room temperature for 20 min. Add 10ml of sol 2 and mix gently.

(10) Centrifugation was carried out at 4 ℃ and 800g for 10min to remove the supernatant, 1ml of sol 2 was added, the suspension was gently suspended by a pipette gun, and 200. mu.l of the suspension was added to the center of the pUARA2 plasmid selection solid medium (X3 plate). 5ml of top agar incubated at 50 ℃ was rapidly added around the petri dish and mixed rapidly. After the plate surface was sufficiently dried, it was wound with parafilm, and incubated at 30 ℃ for 3 to 7 days with the lid facing downward.

(11) Each plate was picked 2-3 clones, 8 in total. And carrying out PCR verification on the grown transformant, wherein the positive transformant is the CCCS heterologous expression strain AO-CcCS.

3. Detection of heterologous expression strain AO-CcCS expression product

(1) Inoculating heterologous expression strain AO-CcCS into pUARA2 plasmid to screen liquid culture medium, and culturing at 30 deg.C for 3 d.

(2) Centrifuging at 8000rpm for 10min to collect fermented thallus, adding 100ml of 80% acetone with the same volume, ultrasonicating for 20min, centrifuging at 8000rpm for 10min, and collecting supernatant.

(3) The mixture was extracted 1 time with 2 volumes of ethyl acetate, dried by rotary evaporation and dissolved in 15mL of methanol (chromatographic grade).

(4) Taking 1mL of methanol solution, filtering the solution by a 0.22 mu m filter membrane, and placing the filtered solution in a chromatographic bottle to obtain GC-MS and LC-MS samples.

(5) The samples were subjected to GC-MS detection: an Agilent-HP-5MS chromatographic column is adopted, the initial temperature is 60 ℃, the temperature is increased to 310 ℃ at the speed of 15 ℃/min, then the temperature is increased to 310 ℃ at the speed of 5 ℃/min, and the temperature is kept for 13 min. The GC-MS process parameters were as follows: a Sample module: the needle washing times before and after sample injection are 5 times, the needle washing times of the sample are 2 times, the viscosity compensation time is 0.2s, and the sample injection mode is normal. A GC module: the column temperature was 50 ℃, the sample introduction temperature was 270 ℃, the sample introduction mode was splitless (split), the carrier gas was helium, the flow rate control mode was linear, the total flow rate was 10mL/min, and the column temperature control procedure is as shown in table 3.5. An MS module: the MS ion source temperature is 230 ℃, the interface temperature is 270 ℃, the solvent removal time is 2.5min, the acquisition time is 3min-60min, the acquisition mode is full scanning, the event time is set to be 0.3s, the scanning speed is 2000, and the scanning nuclear-to-mass ratio is 40-600 Da.

(7) Subjecting the sample to LC-MS detection: adopting a Cholester chromatographic column, and adopting mobile phase A-0.1% formic acid water and mobile phase B-acetonitrile with the flow rate of 1mL/min, wherein the proportion of the mobile phase acetonitrile in 30min is increased from 5% to 100%, then maintaining for 6min, then reducing the proportion of the mobile phase acetonitrile in 10s to 5%, and then maintaining for 4min and 50 s.

4. Separation, purification and identification of heterologously expressed recombinant strain AO-CcCS sesterterpene skeleton product

AO-CcCS was co-fermented for 10L to give about 2g of crude extract. The obtained crude fermentation extract is firstly separated and purified by adopting a forward silica gel column chromatography method. Loading the column by adopting a dry method, isocratic eluting by using petroleum ether, collecting one tube of effluent liquid every 10mL, collecting 18 flow parts, quickly detecting each flow part by TLC, combining the flow parts with the same spots, concentrating under reduced pressure, rotary-steaming until the flow parts are dry, transferring the flow parts into a weighed sample bottle, weighing the sample and recording the weight. HPLC is used for analyzing the components of each flow part and accurately positioning the target flow part. Optimizing the preparation conditions, selecting a Cholester semi-preparative chromatographic column to prepare a target compound, and carrying out mobile phase: phase a-0.1% formic acid water; and B phase-acetonitrile with the flow rate of 4mL/min and the isocratic of 95% acetonitrile formic acid, wherein 10 mu L of sample is initially introduced, the sample amount is gradually increased to 80 mu L on the basis of ensuring that the peak type is not changed, the target sesterterpene compound peak appears in about 20min, and when the peak appears, the outflow solution is connected into a conical flask. The purity of the prepared compound was checked by TLC and HPLC.

NMR measurements of the isolated sesterterpene skeleton compounds were carried out using Bruker 600MHz (R) ((R))¹H 600MHz；¹³C150 MHz). The solvent of the sesterterpene skeleton compound is Benzene-d₆The resolution of NMR spectrometer is 600MHz, the first step is¹H NMR and¹³c NMR measurement, and correlation with databaseAnd (4) comparing the data in the step (A), and if the structure is a new structure, complementing HSQC, COSY and HMBC spectrogram resolution to determine the specific structure.

5. Identifying a sesterterpene skeleton compound Schultriene.

Identifying the sesterterpene skeleton compound Schultriene obtained by the method:

(1) appearance: is in the form of transparent grease.

(2) Solubility: is easily dissolved in methanol and hardly dissolved in water.

(3) Nuclear magnetic resonance spectroscopy: FIG. 1 shows the dissolution of the compound Schultriene in Benzene-d₆In (1)¹H-NMR spectrum. FIG. 2 shows the dissolution of the compound Schultriene in Benzene-d₆In (1)¹³C-NMR spectrum. FIG. 3 shows the dissolution of the compound Schultriene in Benzene-d₆In¹³C-DEPT 135 Spectrum. FIG. 4 shows the dissolution of the compound Schultriene in Benzene-d₆In (1)¹H-¹H COSY spectra. FIG. 5 shows the dissolution of the compound Schultriene in Benzene-d₆HSQC spectrum in (1). FIG. 6 shows the dissolution of the compound Schultriene in Benzene-d₆HMBC spectrum in (1). FIG. 7 shows the dissolution of the compound Schultriene in Benzene-d₆NOESY spectrum of (1). The nuclear magnetic resonance spectrum of the compound Schultriene of the invention was studied and the signals of the 1D and 2D spectra were assigned, see Table 2. And finally the structure was determined as follows:

TABLE 2 assignment of peaks in the 1D and 2D spectra of the compound fusaoxyspene A

The references involved in the background of the invention are as follows:

[1]Guan,Z.et al.Metabolic engineering of Bacillus subtilis for terpenoid production.Applied Microbiology andbiotechnology 99,9395-9406(2015).

[2]Chang,M.C.&Keasling,J.D.Production of isoprenoid pharmaceuticals by engineered microbes.Nature chemical biology 2,674-681(2006).

[3]Leung,P.C.,Taylor,W.A.,Wang,J.H.&Tipton,C.L.Role of calmodulin inhibition in the mode ofaction ofophiobolinA.Plant physiology 77,303-308(1985).

[4]Nozoe,S.et al.The structure of ophiobolin,a C25 terpenoid having a novel skeleton.Journal ofthe American Chemical Society 87,4968-4970(1965).

[5]Peters,C.&Mayer,A.Ca 2+/calmodulin signals the completion ofdocking and triggers a late step ofvacuole fusion.Nature 396,575-580(1998).

[6]Jiang,N.,Yang,Q.,Fan,X.-L.&Tian,C.-M.Identification of six Cytospora species on Chinese chestnut in China.MycoKeys 62,1(2020).

while the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Sequence listing

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cgaccagaaa gacttgggct cctctgctac ctgttagatg caggttgttt tcacgatgat 300

gcttgtgagg agatgcccat cgccgcagct caccaagaac acttggactt ggacgcagct 360

atggacgttg aggaccggcg agagttgagt agtgattcac ggtccttgcg gacaaaacaa 420

ctcatctcca acgctgttct tgaatgcatc aaggtcgaca gagtgggcgc tatgcgtatg 480

ctggaagcct acagaaagaa atggctgcgt attatggaga catacaacac cgaggagatc 540

aacactgttg aagactattt ccttgcacga gcaaacaatg ggggaatggg agcgtactac 600

gccatgcttg agttctctct gggcattttg gtcaccgacg aggaatacga gatgatggct 660

gaacccattg cacatgtgga acggtgcatg cttcttacca acgattactg gagctggcca 720

cgtgaacgca agcaagccga gtaccaggag gctggaaagg tcttcaacat cgtgtggttc 780

ctgaagaaaa ttgagctctg caccgaagaa gaggcagtgt caaaagtccg tgatatggtt 840

catgcagaag agcggaactg gactgctgcc aaaactcgcc tgtacagtca gttcggtaac 900

ctgcggcagg acttggtcaa gttcttggag aatttgcaca cggcactggc gggcaacgac 960

tattggagtt cgcagtgtta ccgccacaat gactgggagc acatcccaga tcttcctggc 1020

gaggacgcac caaaacttca cgaacttgcc accctaggtc gacgattatt gctggacgaa 1080

gatctaccgc ccggggcttc gacctatgga caggatacgg atgcggacag cgcgaggttc 1140

actgaatgca agccatccgt gggtaccgtc tctggtgatg aaactcagtc actcccgggc 1200

aggagcacgt ccggcgaaga atcggcttct gggtacatgt actccatctc atcagccagt 1260

gccccaccaa gtccttccaa agagcaccaa tcctcatatc cgagcatccc ctaccagtcg 1320

tctgcgcacg tcgcgggtga tcttgactct ccagtcttga gggaaccgat caagtacatc 1380

aggaacatgc cgtccaagaa ccttcgaaca caactgatcg actgcttcaa catctggcta 1440

aatgcctctg ggcctgcgat ttcggtcatc aaagaagtca ttgactgcct gcaccattcc 1500

tcactgatcc tggatgacat cgaggacggt tcacatctac gacgcgggtt tccagccact 1560

catgtcgtct acggaacatg tcaggccgtc aacagcgcca cgtttctcta cgtccaagca 1620

gtggagagcg tccacgccgc cgcccggaac aaccccgaga tgatggacgt ctttttgaaa 1680

cacctgaggc agctgttcaa cggccaaagc tgggacctgt actggacgta ccaccgccaa 1740

tgccctaccg aggagcaata cctggacatg gtagatcaga aaactggcgc catgttgcaa 1800

ttgttagtgg gcttgatgca gacggcgcag ccgcagcatc cagggaaggt tggcggcgtt 1860

gtccatagtg aggtcctctt caggttcaca cagttgtttg gccgattctt ccaggtccgt 1920

gacgactata tgaacctcac gtcgacagac tacgctcggc agaagggctt tgctgaggac 1980

ctcgacgagc agaagttctc gtacatgata gtacacatgt accagagata ccccgaggcg 2040

aaggacaagg tcgagggtgt cttcagggcg atgcaacagg gtggcatctc acaggttgca 2100

gctgacacga gcaagaggta catcttgtcc atccttgacg agacaggctc aaccgcagct 2160

accaaggcac tgctattaaa gtggcatgac gagattacgg aggagattgg ggctttggag 2220

aggcattttg gggttgataa tgccttactt cgcttgctgg tggaaacctt gcgagtttga 2280

<210> 3

<211> 759

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 3

Met Leu Glu Ala Asn Glu Leu Tyr Pro Tyr Ser Val Ala Val Asp Arg

1 5 10 15

Asp Glu Val Val Gln Ser Gly Ala Leu Thr Ser Leu Pro Val Arg Ile

20 25 30

His Arg Tyr Asn His Leu Ala Asp Ala Gly Ala Leu Cys Leu Thr Asn

35 40 45

Asp Trp Arg Cys Thr Met Lys Asp Gly Gln Asp Arg Lys Ser Asn Gly

50 55 60

Ser Pro Cys Val Val Gly Asn Trp Gly Ser Phe Ile Trp Pro Glu Ser

65 70 75 80

Arg Pro Glu Arg Leu Gly Leu Leu Cys Tyr Leu Leu Asp Ala Gly Cys

85 90 95

Phe His Asp Asp Ala Cys Glu Glu Met Pro Ile Ala Ala Ala His Gln

100 105 110

Glu His Leu Asp Leu Asp Ala Ala Met Asp Val Glu Asp Arg Arg Glu

115 120 125

Leu Ser Ser Asp Ser Arg Ser Leu Arg Thr Lys Gln Leu Ile Ser Asn

130 135 140

Ala Val Leu Glu Cys Ile Lys Val Asp Arg Val Gly Ala Met Arg Met

145 150 155 160

Leu Glu Ala Tyr Arg Lys Lys Trp Leu Arg Ile Met Glu Thr Tyr Asn

165 170 175

Thr Glu Glu Ile Asn Thr Val Glu Asp Tyr Phe Leu Ala Arg Ala Asn

180 185 190

Asn Gly Gly Met Gly Ala Tyr Tyr Ala Met Leu Glu Phe Ser Leu Gly

195 200 205

Ile Leu Val Thr Asp Glu Glu Tyr Glu Met Met Ala Glu Pro Ile Ala

210 215 220

His Val Glu Arg Cys Met Leu Leu Thr Asn Asp Tyr Trp Ser Trp Pro

225 230 235 240

Arg Glu Arg Lys Gln Ala Glu Tyr Gln Glu Ala Gly Lys Val Phe Asn

245 250 255

Ile Val Trp Phe Leu Lys Lys Ile Glu Leu Cys Thr Glu Glu Glu Ala

260 265 270

Val Ser Lys Val Arg Asp Met Val His Ala Glu Glu Arg Asn Trp Thr

275 280 285

Ala Ala Lys Thr Arg Leu Tyr Ser Gln Phe Gly Asn Leu Arg Gln Asp

290 295 300

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

305 310 315 320

Tyr Trp Ser Ser Gln Cys Tyr Arg His Asn Asp Trp Glu His Ile Pro

325 330 335

Asp Leu Pro Gly Glu Asp Ala Pro Lys Leu His Glu Leu Ala Thr Leu

340 345 350

Gly Arg Arg Leu Leu Leu Asp Glu Asp Leu Pro Pro Gly Ala Ser Thr

355 360 365

Tyr Gly Gln Asp Thr Asp Ala Asp Ser Ala Arg Phe Thr Glu Cys Lys

370 375 380

Pro Ser Val Gly Thr Val Ser Gly Asp Glu Thr Gln Ser Leu Pro Gly

385 390 395 400

Arg Ser Thr Ser Gly Glu Glu Ser Ala Ser Gly Tyr Met Tyr Ser Ile

405 410 415

Ser Ser Ala Ser Ala Pro Pro Ser Pro Ser Lys Glu His Gln Ser Ser

420 425 430

Tyr Pro Ser Ile Pro Tyr Gln Ser Ser Ala His Val Ala Gly Asp Leu

435 440 445

Asp Ser Pro Val Leu Arg Glu Pro Ile Lys Tyr Ile Arg Asn Met Pro

450 455 460

Ser Lys Asn Leu Arg Thr Gln Leu Ile Asp Cys Phe Asn Ile Trp Leu

465 470 475 480

Asn Ala Ser Gly Pro Ala Ile Ser Val Ile Lys Glu Val Ile Asp Cys

485 490 495

Leu His His Ser Ser Leu Ile Leu Asp Asp Ile Glu Asp Gly Ser His

500 505 510

Leu Arg Arg Gly Phe Pro Ala Thr His Val Val Tyr Gly Thr Cys Gln

515 520 525

Ala Val Asn Ser Ala Thr Phe Leu Tyr Val Gln Ala Val Glu Ser Val

530 535 540

His Ala Ala Ala Arg Asn Asn Pro Glu Met Met Asp Val Phe Leu Lys

545 550 555 560

His Leu Arg Gln Leu Phe Asn Gly Gln Ser Trp Asp Leu Tyr Trp Thr

565 570 575

Tyr His Arg Gln Cys Pro Thr Glu Glu Gln Tyr Leu Asp Met Val Asp

580 585 590

Gln Lys Thr Gly Ala Met Leu Gln Leu Leu Val Gly Leu Met Gln Thr

595 600 605

Ala Gln Pro Gln His Pro Gly Lys Val Gly Gly Val Val His Ser Glu

610 615 620

Val Leu Phe Arg Phe Thr Gln Leu Phe Gly Arg Phe Phe Gln Val Arg

625 630 635 640

Asp Asp Tyr Met Asn Leu Thr Ser Thr Asp Tyr Ala Arg Gln Lys Gly

645 650 655

Phe Ala Glu Asp Leu Asp Glu Gln Lys Phe Ser Tyr Met Ile Val His

660 665 670

Met Tyr Gln Arg Tyr Pro Glu Ala Lys Asp Lys Val Glu Gly Val Phe

675 680 685

Arg Ala Met Gln Gln Gly Gly Ile Ser Gln Val Ala Ala Asp Thr Ser

690 695 700

Lys Arg Tyr Ile Leu Ser Ile Leu Asp Glu Thr Gly Ser Thr Ala Ala

705 710 715 720

Thr Lys Ala Leu Leu Leu Lys Trp His Asp Glu Ile Thr Glu Glu Ile

725 730 735

Gly Ala Leu Glu Arg His Phe Gly Val Asp Asn Ala Leu Leu Arg Leu

740 745 750

Leu Val Glu Thr Leu Arg Val

755

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

cggaattcga gctcgatgct tgaagcaaac gagct 35

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

tactacagat ccccggtcaa actcgcaagg tttccaccag 40