Marine streptomycete phospholipase D mutant and application

文档序号：1793927 发布日期：2021-11-05 浏览：15次中文

阅读说明：本技术 一种海洋链霉菌磷脂酶d突变体和应用 (Marine streptomycete phospholipase D mutant and application ) 是由王永华王方华胡荣康蓝东明杨博于 2021-07-12 设计创作，主要内容包括：本发明公开了一种海洋链霉菌磷脂酶D突变体和应用,所述的磷脂酶D突变体是在氨基酸序列为SEQ ID NO:1的基础上单点突变380位点氨基酸所得。本发明磷脂酶D突变体酶活力显著提高,实验测定突变体1(S380A)的酶活力是野生型的2.4倍,突变体2(S380F)的酶活力是野生型的1.1倍,突变体3(S380V)的酶活力是野生型的2.6倍,突变体4(S380M)的酶活力是野生型的1.2倍,突变体5(S380L)的酶活力是野生型的1.6倍。同时,本发明突变体的催化效率也显著提高,酶活力和催化性能的改善进一步提升了该酶的工业利用价值。(The invention discloses a streptomyces marinus phospholipase D mutant and application thereof, wherein the phospholipase D mutant is obtained by single point mutation of 380-site amino acid on the basis that the amino acid sequence is SEQ ID NO. 1. The phospholipase D mutant enzyme activity is remarkably improved, and experiments determine that the enzyme activity of the mutant 1(S380A) is 2.4 times of that of a wild type, the enzyme activity of the mutant 2(S380F) is 1.1 times of that of the wild type, the enzyme activity of the mutant 3(S380V) is 2.6 times of that of the wild type, the enzyme activity of the mutant 4(S380M) is 1.2 times of that of the wild type, and the enzyme activity of the mutant 5(S380L) is 1.6 times of that of the wild type. Meanwhile, the catalytic efficiency of the mutant is also obviously improved, and the industrial utilization value of the enzyme is further improved by improving the enzyme activity and the catalytic performance.)

1. The streptomyces marinus phospholipase D mutant is characterized in that the phospholipase D mutant is obtained by single point mutation of 380-site amino acid on the basis that the amino acid sequence is SEQ ID NO. 1.

2. The phospholipase D mutant according to claim 1, wherein the amino acid sequence is SEQ ID NO 2, 3, 4, 5, or 6.

3. A gene encoding the phospholipase D mutant of claim 1, wherein the nucleic acid sequence is SEQ ID NO 8, 9, 10, 11, 12.

4. A recombinant genetically engineered bacterium containing the gene of claim 3.

5. The method for preparing the recombinant genetically engineered bacterium of claim 4, wherein the gene of claim 3 is cloned into an expression vector pET-21a, pET-28a or pET-32a, and Escherichia coli Shuffle T7 competent cells are transformed to obtain the recombinant genetically engineered bacterium.

6. Use of a phospholipase D mutant according to claim 1 or claim 2, wherein the phospholipase D mutant is used to produce phosphatidylserine.

7. The use as claimed in claim 6, wherein L-serine is added to the enzyme solution of the phospholipase D mutant, soybean lecithin is added to the organic solvent, and after mixing, shaking culture is performed at 40 ± 5 ℃ for 12 ± 4 h.

8. The use of claim 7, wherein the volume ratio of the organic solvent to the enzyme solution is 4: 1-4: 4, and the organic solvent is diethyl ether.

9. The use of claim 7 or 8, wherein the phospholipase D mutant is prepared by adding Tris-HCl buffer solution to the phospholipase D mutant at pH 8.

Technical Field

The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to a phospholipase D mutant with remarkably improved catalytic performance obtained by utilizing a molecular biology site-directed mutagenesis technology and a recombinant expression preparation method thereof.

Background

Phospholipids are mixed lipids containing phosphoric acid, which are essential components of biological membranes and constitute essential substances for life. In addition, phospholipids have been widely used in emulsifiers, cosmetic ingredients, pharmaceutical formulations and liposomal formulations due to their unique chemical structures and health-care functions. In industry, natural phospholipids are mainly byproducts of crude oil refining dehydration and degumming. In recent years, much interest has been generated in phospholipids of various molecular properties such as charge, polarity, size, etc. to obtain special functional phospholipids having excellent processability and outstanding physiological and pharmacological functions. Therefore, modification of natural phospholipids has become an important way to realize high-value utilization of phospholipid by-products.

Enzymatic modification is an important direction for preparing functional phospholipids. Phospholipase d (pld), an important member of the phospholipase family, is an important tool enzyme for the synthesis and engineering of phospholipids. Phospholipase D (PLD) (EC 3.1.4.4) can catalyze the transfer reaction of phospholipid polar head groups by transphosphatidylation (transphosphatidylation), thereby synthesizing various rare phospholipids and series of functional phospholipid derivatives. PLD has been used to synthesize less abundant phospholipids, such as Phosphatidylglycerol (PG), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), and Phosphatidylinositol (PI). In addition, a large number of novel phospholipids have been synthesized by PLD, in which choline including Phosphatidylcholine (PC) is substituted with aliphatic alcohol, saccharide, nucleoside, aromatic alcohol or n-heterocyclic alcohol. Therefore, the development of PLD with industrial application value has important strategic significance and development prospect.

At present, the development of PLD enzymatic proteins is still in the beginning stage of the whole world as compared with other types of enzymatic proteins (protease, lipase, etc.), and there is a large development space. There are no more than 10 commercially available PLDs worldwide, and are mainly monopolized by several japanese companies, and no commercially available PLDs are found in China. The phospholipase D has single source, low enzyme activity, high preparation difficulty and high cost, and is the technical bottleneck restricting the development of the enzyme at present. The quality of the PLD is a key factor for determining whether the enzyme can be used for developing commercial enzyme preparations, wherein the low enzyme activity becomes a main restriction factor, and the difference from the requirement of industrial application is large. The main solution to this problem is to modify the enzyme molecules by protein engineering, for example, in the invention patent 201610402557.7 of China, the wild-type phospholipase D gene is subjected to site-directed mutagenesis by recombinant DNA technology to obtain a phospholipase D gene with high activity. After recombinant expression, the specific enzyme activity of the high-activity phospholipase D is improved by 38-140% compared with that of the wild phospholipase D.

The research on the phospholipase D in China starts late, and has great difference with the foreign advanced technology in the aspects of phospholipase D fermentation strains, enzymology properties and the like. At present, no PLD commercial enzyme appears in China, enzyme preparations mainly depend on import, and the high dependence of commercial enzyme sources seriously influences the healthy development of the downstream enzyme method phospholipid modification industry in China. Therefore, there is an urgent need to develop a novel phospholipase D having excellent enzymatic properties and to reduce production costs, thereby promoting the wide application of phospholipase D. The realization of key technical breakthroughs of independent research and development, efficient preparation and the like of new enzymes is an effective way for changing the current situation.

Disclosure of Invention

The invention aims to provide high-activity phospholipase D and a method for preparing phosphatidylserine by using the same.

SEQ ID No.1

DSSATPHLDAVEQTLRQVSPGLEGRVWERTAGNALDAPAGDPAGWLLQTPGCWGDANCAERTGTKRLLARMTENISKATRTVDISTLAPFPNGAFQDAIVAGLKKSVENGNKPKVRVLVGAAPVYHMNVLPSKYRDDLRDKLGKAADGLTLNVASMTTSKTAFSWNHSKLLVVDGQSAITGGINSWKDDYVDTTHPVSDVDLALTGPAAGSAGRYLDQLWTWTCENKSNIASVWFAASPGAGCMPTMEKDANPVPAAATGNVPVIAVGGLGVGIKDSDPSSAFKPELPSAPDTKCVVGLHDNTNADRDYDTVNPEESALRALVGSARSHVEISQQDLNATCPPLPRYDVRLYDALAAKLAAGVKVRIVVSDPENRGAVGSGGYSQIKSLNEISDLLRNRLSLLPGGAQGAKTAMCGNLQLATARSSDSAKWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQDFGYIVESPEAARQLDAELLAPQWKYSQATATFDYARGICQG

The technical scheme of the invention is as follows:

a phospholipase D mutant is obtained by single point mutation of 380 site amino acids into several hydrophobic amino acids (A, F, V, M and L) on the basis of the amino acid sequence of SEQ ID NO: 1. Wherein the amino acid at position 380 is substituted with the following amino acid: position 380: ser380Ala, Ser380Phe, Ser380Val, Ser380Met, Ser380 Leu.

The mutant gene is obtained by taking Streptomyces klenkenii (Streptomyces klenkenkii) with the amino acid sequence of SEQ ID No.1(GenBank: RKN69773.1) as a parent, analyzing a Signal peptide sequence of the mutant gene through Signal peptide online analysis software Signal P on the basis of the parent, carrying out homologous modeling on a protein sequence without the Signal peptide to obtain a three-dimensional structure, analyzing the protein structure, constructing a recombinant vector through enzyme digestion, connection and the like, and then mutating a 380 th site of a wild phospholipase D gene through an overlap PCR technology.

Preferably, the amino acid sequence is SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6.

A gene for coding phospholipase D mutant has the nucleic acid sequence of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.

A preparation method of recombinant genetic engineering bacteria comprises the steps of cloning genes to expression vectors pET-21a, pET-28a or pET-32a, and transforming escherichia coli SHuffle T7 competent cells to obtain the recombinant genetic engineering bacteria.

The phospholipase D mutant is used for producing phosphatidylserine. Preferably, L-serine is added into the enzyme solution of phospholipase D mutant, soybean lecithin is added into the organic solvent, and after mixing, shaking culture is carried out at 40 + -5 ℃ for 12 + -4 h.

Preferably, the volume ratio of the organic solvent to the enzyme solution is 4: 1-4: 4, and the organic solvent is diethyl ether.

Preferably, the enzyme solution of the phospholipase D mutant is prepared by adding a phospholipase D mutant into a Tris-HCl buffer solution, and the pH value is 8.

And (3) determining the enzyme activity and enzyme kinetics of the mutant on soybean phosphatidylcholine by an enzyme-linked colorimetric method.

The following definitions are used in the present invention:

1. nomenclature of amino acid and DNA nucleic acid sequences:

the accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.

2. Identification of phospholipase D mutants

"amino acid substituted by original amino acid position" is used to indicate a mutated amino acid in the phospholipase D mutant. In the case of Ser380Val, the amino acid at position 380 is replaced by Ser in the wild-type phospholipase D for Val. The numbering of positions corresponds to SEQ ID NO:1, amino acid sequence number of phospholipase D. Nucleotide changes are also denoted by "original nucleotide position substituted nucleotide" and the position numbering corresponds to that of SEQ ID NO: 7 nucleotide sequence number of wild-type phospholipase D.

Compared with the prior art, the invention has the following beneficial effects:

(1) the enzyme activity is obviously improved. The enzyme activity of the mutant 1 is 2.4 times of that of the wild type, the enzyme activity of the mutant 2 is 1.1 times of that of the wild type, the enzyme activity of the mutant 3 is 2.6 times of that of the wild type, the enzyme activity of the mutant 4 is 1.2 times of that of the wild type, and the enzyme activity of the mutant 5 is 1.6 times of that of the wild type. The activity of the phospholipase mutant obtained by the invention is obviously improved.

(2) The catalytic efficiency is obviously improved. Catalytic efficiency (k) of mutant 1 of the invention_cat/K_m) 3.5 times that of wild type, catalytic efficiency (k) of mutant 2_cat/K_m) 1.4 times that of wild type, catalytic efficiency (k) of mutant 3_cat/K_m) 4.9 times that of wild type, catalytic efficiency (k) of mutant 4_cat/K_m) 1.4 times that of wild type, catalytic efficiency (k) of mutant 5_cat/K_m) Is 1.9 times of the wild type. Shows that the catalytic efficiency of the phospholipase mutant obtained by the invention is obviously improvedHigh. The improvement of the enzyme activity and the catalytic efficiency further improves the industrial utilization value of the enzyme.

Drawings

FIG. 1 is a SDS-PAGE result of wild type SkPLD and mutant protein purification.

FIG. 2 is a bar graph showing the relative enzyme activities of phospholipase D mutant 1(SkPLD-Ser380Ala), mutant 2(SkPLD-Ser380Phe), mutant 3(SkPLD-Ser380Val), mutant 4(SkPLD-Ser380Met), mutant 5(SkPLD-Ser380Leu) and wild type (SkPLD).

FIG. 3 shows the use of phospholipase D and mutant 3(SkPLD-Ser380Val) for phosphatidylserine production.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.

Example 1: expression vector for phospholipase SkPLD mutant and construction of expression strain

(1) Referring to the entire amino acid sequence (GenBank: RKN69773.1) of wild-type phospholipase D (SkPLD) of Streptomyces klebsiella (Streptomyces klenklenkii), the Signal peptide sequence thereof was analyzed by Signal peptide on-line analysis software Signal P, and amino acids 1 to 33 were deleted from the entire sequence to obtain a phospholipase SkPLD mature peptide coding sequence (SEQ ID NO. 1);

(2) designing a phospholipase SkPLD gene coding sequence according to the amino acid sequence obtained in the step (1) and the base sequence of the phospholipase SkPLD gene coding sequence is shown in SEQ ID NO.7 (wild type) according to the codon bias of the escherichia coli. Introducing ECoR I at the upstream of the sequence, introducing Not I enzyme cutting site at the downstream, and synthesizing the obtained phospholipase SkPLD gene sequence by a biological engineering (Shanghai) GmbH;

(3) the phospholipase SkPLD gene synthesized in (2) was digested with restriction enzymes ECoR I and Not I, respectively, and the purified gene fragment and plasmid pET28a were digested with double restriction enzymes, ligated, and transformed into e.coli DH5 α competent cells. The plates were plated on LB (50. mu.g/mL kanamycin-containing) plates. Selecting positive clones, and performing ECoR I and Not I double enzyme digestion identification and gene sequencing to obtain pET28a-SkPLD recombinant plasmids of wild type SkPLD;

(4) the mutant SEQID NO. 2, SEQID NO. 3, SEQID NO. 4, SEQID NO. 5 and SEQID NO. 6 are constructed by adopting a two-step overlap extension PCR method. Firstly, splicing the full length of the primer, and then amplifying by using a plasmid template containing a target gene. The reaction conditions were as follows:

reaction conditions 1:

wherein the sequences of an upstream primer and a downstream primer used for constructing the mutant 1 are as follows:

an upstream primer: agtaaccgccagcgccaaccgcgcc

A downstream primer: ggcgcggttggcgctggcggttact

The sequences of the upstream primer and the downstream primer used for constructing the mutant 2 are as follows:

an upstream primer: taaccgccaaagccaaccgcgccacgg

A downstream primer: ccgtggcgcggttggctttggcggtta

The sequences of an upstream primer and a downstream primer used for constructing the mutant 3 are as follows:

an upstream primer: gagagtaaccgccaacgccaaccgcgccac

A downstream primer: gtggcgcggttggcgttggcggttactctc

The sequences of the upstream primer and the downstream primer used for constructing the mutant 4 are as follows:

an upstream primer: ctgagagtaaccgcccatgccaaccgcgccacg

A downstream primer: cgtggcgcggttggcatgggcggttactctcag

Mutant 5 construction the sequences of the upstream and downstream primers used were:

an upstream primer: agtaaccgcctaagccaaccgcgccacgg

A downstream primer: the amplification conditions of ccgtggcgcgggttggcttaggcggttactPCR are that the temperature is 98 ℃ and the time is 3 min; 10s at 98 ℃; at 58 ℃ for 15 s; 72 ℃ for 10 s; 20 cycles; 72 ℃ for 2 min. And purifying the amplification product by a DNA purification kit to obtain the full-length primer.

Reaction conditions 2:

PCR amplification conditions were 98 ℃ for 3 min; 10s at 98 ℃; at 58 ℃ for 15 s; at 72 ℃ for 408 s; 31 cycles; 72 ℃ for 2 min. And purifying the PCR product by using a DNA purification kit to obtain the phospholipase mutation gene.

Digesting the template plasmid by using Dpn I, wherein the digestion system of Dpn I is as follows:

and (3) placing the Dpn I enzyme digestion system at 37 ℃ for 2 h. The digest was transformed into e.coli DH5 α competent cells. The plates were plated on LB (50. mu.g/mL kanamycin-containing) plates. The positive clone is picked, and is subjected to ECoR I and Not I double enzyme digestion identification and gene sequencing to obtain pET28 a-SkPLD-mutant plasmid.

(5) And (3) respectively transforming the recombinant plasmids obtained in the step (3) and the step (4) into escherichia coli Shuffle T7 competent cells, selecting positive clones, and performing sequencing verification to obtain the recombinant pET28a-SkPLD wild type and mutant Shuffle T7 escherichia coli expression strains.

Example 2: fermentation of wild SkPLD and its mutant recombinant expression strain and recombinant protein purification

(1) Inoculating the wild type and mutant expression strains of the recombinant escherichia coli SkPLD into a seed culture medium (10 g/L of NaCl, 10g/L of peptone, 5g/L of yeast extract and pH 7.2-7.4) containing kanamycin (50 mu g/mL), and performing shake-flask culture at 37 ℃ and 200r/min until the logarithmic phase to obtain a seed solution;

(2) inoculating the seed liquid in the step (1) into a self-induction liquid fermentation culture medium (10 g/L of enzymatic hydrolysis casein, 5g/L of yeast extract, 0.5g/L of glucose, 2g/L of lactose, 5g/L of glycerol, 3.6g/L of disodium hydrogen phosphate, 3.4g/L of potassium dihydrogen phosphate, 2.7g/L of ammonium chloride, 0.7g/L of sodium sulfate, 1g/L of magnesium sulfate and pH 7.2-7.4) according to the inoculation amount of 5 percent, and performing shake flask culture at 37 ℃ and 200r/min until OD (origin of the product) is within the range of 7.2-7.4₆₀₀Performing induction culture at 20 ℃ and 200r/min for 24h under the condition of 0.6-0.8; (3) centrifuging the fermentation liquor obtained in the step (2) (8000r/min,5min), collecting thalli precipitates, carrying out heavy suspension by using Tris-HCl buffer solution (pH 8.0) and carrying out ultrasonic cell disruption, centrifuging the cell disruption solution (10000r/min,20min), and taking supernatant, namely the prepared crude phospholipase SkPLD;

(4) and (4) carrying out suction filtration on the phospholipase crude enzyme solution obtained in the step (3) by using a filter membrane with the diameter of 0.45 mu m. The filtrate was purified by a nickel column affinity chromatography at a flow rate of 4mL/min, and finally eluted with a gradient of 10-500mM imidazole in Tris-HCl buffer (pH 8.0) to elute the target protein at 250mM imidazole. The eluted target protein was passed through a G-25 desalting column, and then subjected to column chromatography using Q column, followed by elution with Tris-HCl buffer (pH 7.3) containing 700mM NaCl to obtain the target protein (see FIG. 1).

Example 3 phospholipase enzymatic Properties analysis

(1) Method for measuring activity of phosphatidase

The activity of the wild phospholipase SkPLD and the mutant phospholipase is measured by adopting a standard enzyme-linked colorimetric method, and soybean phosphatidylcholine is used as a reaction substrate. And (3) performing activity detection by adopting an enzyme-linked colorimetric method: phospholipase D catalyzes and hydrolyzes L-alpha-lecithin to generate choline, the choline generates hydrogen peroxide under the action of choline oxidase, the hydrogen peroxide generates quinoneimine chromogenic substance with 4-aminoantipyrine and phenol under the action of peroxidase, and the light absorption value is 500 nm. For substrate preparation, 1mmol of soy phosphatidyl choline was dissolved in 5mL of chloroform. After nitrogen blow-drying, SDS and Triton X-100 were added to give a solution containing 1mM soybean phosphatidylcholine, 1.25mM SDS and 4mM Triton X-100. The solution was vortexed and sonicated using a 800W/s probe sonicator for 10 minutes. The reaction mixture (100. mu.L) consisted of 0.4mM soy PC, 50mM Tris-HCl (pH 8.0), 20mM CaCl2, and 10. mu.L of the enzyme sample. After incubation at 40 ℃ for 10min with shaking, 25. mu.L of a solution containing 50mM EDTA and 50mM Tris-HCl (pH 8.0) was added and denatured by heating at 100 ℃ for 5 minutes. After cooling the reaction mixture to room temperature, 25. mu.L of a mixed solution of 50mM Tris-HCl (pH 8.0) containing 42mM phenol, 4-aminoantipyrine 50mM,0.5U horseradish peroxidase and 0.25U choline oxidase was added. After incubation at 37 ℃ for 60min, the absorbance of the reaction mixture was measured at 500 nm.

(2) The kinetics of wild-type and mutant phospholipase enzymes were determined as follows: according to the method for measuring the activity of the phospholipase, under the respective optimal reaction temperature and optimal pH conditions, the enzyme activity is measured by taking the soybean phosphatidylcholine as a substrate under the condition that the substrate concentration is 0-2.8mN, and the result is subjected to nonlinear fitting. The control and sample groups were run in triplicate. As a result, the catalytic efficiency (k) of mutant 1 of the present invention is shown in Table 1_cat/K_m) 3.5 times that of wild type, catalytic efficiency (k) of mutant 2_cat/K_m) 1.4 times that of wild type, catalytic efficiency (k) of mutant 3_cat/K_m) 4.9 times that of wild type, catalytic efficiency (k) of mutant 4_cat/K_m) 1.4 times that of wild type, catalytic efficiency (k) of mutant 5_cat/K_m) Is 1.9 times of the wild type. The catalytic efficiency of the phospholipase mutant obtained by the invention is obviously improved.

TABLE 1 enzyme kinetic parameters of phospholipase SkPLD and its mutants

Example 4 use of the mutant (S380V) for phosphatidylserine synthesis

The reaction is carried out in a biphasic reaction system. The initial transformation conditions were 20mg/mL L-serine added to 500. mu.L of enzyme solution (100. mu.g/mL PLD added to 100mM Tris-HCl buffer, pH 8), soybean lecithin PC (5mg/mL) added to 500. mu.L diethyl ether, and shaking-cultured at 40 ℃ for 12 hours. In order to obtain the optimal conditions for PLD-catalyzed transphosphatidylation, the effect of organic solvent to aqueous phase ratio (4:1,4:2,4:3, and 4:4) on the transphosphatidylation reaction of SkPLD and mutant 3(S380V) was selected. Phospholipid samples were analyzed by Thin Layer Chromatography (TLC). Chloroform-methanol-acetic acid-water (75:40:8:3 by volume) and 0.2% (w/v)2 ', 7' -dichlorofluorescein solution were used as the color developing agent. PS was quantified by HPLC (Waters 1525, USA) and ELSD. The reaction mixture was eluted with a chromatography column (4.6 μm. times.125 mm, Thermo, USA), solvent A (methanol/water/acetic acid/triethylamine, 425:75:2.5:0.25, vol.) and solvent B (n-hexane/isopropanol/solvent A,160:384:256, vol.). The elution profile is 0min, A is 100%; at 9min, B increased to 40%; 13min, B increased to 60%; 17min, B being 100%; the time for 22 minutes decreases to 0%. PS yield (%) is defined as the percentage of PS compared to the initial PC concentration. As shown in FIG. 3, the reaction conversion was 48.58% and the PS cumulative concentration was 2.43mg/mL when the ether was used as the organic phase and shaken at 40 ℃ for 12 hours.

In conclusion, compared with the wild type, the phospholipase mutant obtained by the invention has the advantages that the enzyme activity of the phospholipase mutant is obviously improved, the phospholipase mutant is more suitable for being applied to the industrial fields of food, medicine and the like, and the market space is wide.

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> Streptomyces marinus phospholipase D mutant and application thereof

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Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala

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Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val

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Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn

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Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp

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Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Val Arg Leu Tyr

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Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val

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Val Ser Asp Pro Glu Asn Arg Gly Ala Val Gly Ala Gly Gly Tyr Ser

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

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Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly

405 410 415

Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala

420 425 430

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

435 440 445

Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln

450 455 460

Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp

465 470 475 480

Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr

485 490 495

Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly

500 505

<210> 3

<211> 506

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg

1 5 10 15

Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly

20 25 30

Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln

35 40 45

Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr

50 55 60

Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg

65 70 75 80

Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln

85 90 95

Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys

100 105 110

Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn

115 120 125

Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys

130 135 140

Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys

145 150 155 160

Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln

165 170 175

Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp

180 185 190

Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala

195 200 205

Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys

210 215 220

Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly

225 230 235 240

Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala

245 250 255

Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val

260 265 270

Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro

275 280 285

Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn

290 295 300

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

305 310 315 320

Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp

325 330 335

Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Val Arg Leu Tyr

340 345 350

Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val

355 360 365

Val Ser Asp Pro Glu Asn Arg Gly Ala Val Gly Phe Gly Gly Tyr Ser

370 375 380

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

385 390 395 400

Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly

405 410 415

Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala

420 425 430

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

435 440 445

Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln

450 455 460

Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp

465 470 475 480

Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr

485 490 495

Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly

500 505

<210> 4

<211> 506

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg

1 5 10 15

Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly

20 25 30

Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln

35 40 45

Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr

50 55 60

Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg

65 70 75 80

Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln

85 90 95

Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys

100 105 110

Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn

115 120 125

Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys

130 135 140

Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys

145 150 155 160

Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln

165 170 175

Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp

180 185 190

Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala

195 200 205

Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys

210 215 220

Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly

225 230 235 240

Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala

245 250 255

Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val

260 265 270

Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro

275 280 285

Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn

290 295 300

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

305 310 315 320

Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp

325 330 335

Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Val Arg Leu Tyr

340 345 350

Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val

355 360 365

Val Ser Asp Pro Glu Asn Arg Gly Ala Val Gly Val Gly Gly Tyr Ser

370 375 380

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

385 390 395 400

Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly

405 410 415

Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala

420 425 430

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

435 440 445

Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln

450 455 460

Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp

465 470 475 480

Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr

485 490 495

Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly

500 505

<210> 5

<211> 506

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg

1 5 10 15

Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly

20 25 30

Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln

35 40 45

Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr

50 55 60

Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg

65 70 75 80

Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln

85 90 95

Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys

100 105 110

Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn

115 120 125

Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys

130 135 140

Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys

145 150 155 160

Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln

165 170 175

Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp

180 185 190

Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala

195 200 205

Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys

210 215 220

Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly

225 230 235 240

Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala

245 250 255

Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val

260 265 270

Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro

275 280 285

Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn

290 295 300

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

305 310 315 320

Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp

325 330 335

Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Val Arg Leu Tyr

340 345 350

Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val

355 360 365

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

370 375 380

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

385 390 395 400

Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly

405 410 415

Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala

420 425 430

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

435 440 445

Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln

450 455 460

Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp

465 470 475 480

Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr

485 490 495

Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly

500 505

<210> 6

<211> 506

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg

1 5 10 15

Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly

20 25 30

Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln

35 40 45

Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr

50 55 60

Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg

65 70 75 80

Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln

85 90 95

Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys

100 105 110

Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn

115 120 125

Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys

130 135 140

Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys

145 150 155 160

Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln

165 170 175

Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp

180 185 190

Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala

195 200 205

Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys

210 215 220

Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly

225 230 235 240

Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala

245 250 255

Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val

260 265 270

Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro

275 280 285

Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn

290 295 300

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

305 310 315 320

Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp

325 330 335

Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Val Arg Leu Tyr

340 345 350

Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val

355 360 365

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

370 375 380

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

385 390 395 400

Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly

405 410 415

Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala

420 425 430

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

435 440 445

Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln

450 455 460

Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp

465 470 475 480

Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr

485 490 495

Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly

500 505

<210> 7

<211> 1518

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gatagctctg cgaccccgca cctggatgcg gttgaacaga ccctgcgtca ggttagccct 60

ggtctggaag gccgcgtttg ggaacgtacc gcgggtaacg ctctggatgc gccggcgggt 120

gatccggcgg gttggctgtt gcagacccca ggttgctggg gtgatgcgaa ctgcgcggaa 180

cgtaccggca ccaaacgtct gctggcacgt atgaccgaaa acatctctaa agcgacccgc 240

accgttgaca tctctaccct ggcgccgttc ccgaacggtg cgttccagga tgcgatcgtt 300

gcgggtctga aaaagtctgt tgaaaacggt aacaaaccga aagttcgcgt tctggtgggt 360

gcagcgccgg tttaccacat gaacgttctg ccgtccaaat accgtgatga tctgcgtgat 420

aaactgggca aagccgcgga tggcctgacc ctgaacgttg caagcatgac caccagcaaa 480

accgcattca gctggaacca ctctaaactg ctggttgtgg atggtcagtc tgcgatcacc 540

ggtggcatca acagctggaa agatgattac gttgacacca cccacccggt gagcgacgtt 600

gatctggcgc tgaccggtcc ggcggcgggt agcgcgggtc gttacctgga ccagctgtgg 660

acctggacct gcgaaaacaa atccaacatc gcaagcgttt ggtttgcggc ctctccgggc 720

gctggctgta tgccgacgat ggaaaaagat gctaacccgg ttccggcggc tgcgaccggt 780

aacgttccgg tgatcgcggt gggcggtctg ggtgttggca tcaaagattc cgatccgagc 840

agcgcgttca aaccggaact gccgagcgcc ccggatacca aatgcgttgt tggtctgcac 900

gataacacca acgcggaccg tgattacgat accgttaacc cggaagaaag cgcgctgcgt 960

gctctggtgg gcagcgcgcg ttcccacgtt gaaatctctc agcaggatct gaacgcgacc 1020

tgcccgccgc tgccgcgtta tgacgtgcgc ctgtacgatg cactggcggc gaaactggcg 1080

gctggcgtga aagtgcgtat cgttgtgagc gacccggaaa accgtggcgc ggttggctct 1140

ggcggttact ctcagatcaa atccctgaac gaaatctccg acctgctgcg taaccgtctg 1200

agcctgctgc cgggcggtgc tcagggtgct aaaaccgcta tgtgcggtaa cctgcaactg 1260

gcgaccgcgc gcagcagcga ctctgctaaa tgggctgatg gtaaaccgta cgcgcagcac 1320

cacaaactgg ttagcgttga tgattctgca ttctacatcg gtagcaaaaa cctgtacccg 1380

agctggctgc aagatttcgg ttacatcgtt gaaagcccgg aagcggcgcg tcagctggat 1440

gcggaactgc tggcgccgca gtggaaatac agccaggcga ccgcgacctt cgattacgcg 1500

cgtggcatct gccagggt 1518

<210> 8

<211> 1518

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8