Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The source of the biological material is as follows: pseudomonas carrageenonovora ASY5, the Chinese name of which is Pseudoalteromonas cervicalis, is separated from mansion mangrove soil leaf mold sample and is derived from China center for culture Collection of Industrial microorganisms and strains (CICC) with the preservation number of 23819.
Example 1: acquisition of the XylA Gene with bifunctional Properties
Inoculating Pseudomonas carrageenovora ASY5 into artificial seawater culture medium, and shake culturing at 25 deg.C and 180r/min to OD6001-1.5, taking 1mL of culture solution, and extracting genomic DNA of Pseudomonas carrageenovora ASY5 strain by using a rapid extraction kit (silica gel membrane centrifugation column method) for bacterial genomes of Donghai organisms.
Artificial seawater culture medium: 10g of beef extract, 10g of tryptone, 250mL of distilled water and 750mL of artificial seawater (NaCl 37.51g, KCl 1.03g and CaCl)2 1.61g、MgCl2·6H2O 6.4g、NaHCO3 0.15g、MgSO4·7H2O4.67 g, 1000mL of distilled water). Beef extractDissolving tryptone in distilled water, adjusting pH to 7.8 with NaOH, boiling for 10min, cooling, adjusting pH to 7.3 with NaOH, mixing with artificial seawater, and sterilizing at 121 deg.C for 20 min. The solid medium was supplemented with 20g of agar.
The obtained genome DNA is used as a template to carry out PCR amplification, and the primer sequences are as follows:
forward primer XylA-F (SEQ ID NO: 3):
5’-CCGGAATTCATGAATGCATTTGAATTACGT-3'; underlined is the restriction endonuclease site EcoRI;
reverse primer XylA-R (SEQ ID NO: 4):
5’-CCGCTCGAGTTATTTTGGCCATGCTGTTAG-3'; the restriction enzyme XhoI site is underlined.
The high fidelity DNA polymerase PrimeSTAR HS was purchased from TaKaRa, Daii, China, and the PCR reagents were used according to the product instructions provided by this company.
The PCR reaction system comprises:
the PCR conditions were: 95 ℃ for 5 min; 15s at 94 ℃; 52 ℃, 15s, 72 ℃,1min, 30 cycles, and finally 72 ℃, 10 min.
Sequencing the PCR product, wherein the sequence is shown as SEQ ID NO. 1. Contains 686 amino acids, and the amino acid sequence of the amino acid is shown as SEQ ID NO:2, respectively. Analysis with the protparam tool from ExPASy showed that the protein XylA had a theoretical molecular weight of about 77.88kDa and a theoretical isoelectric point of 5.14. The signal peptide analysis showed no signal peptide. The total number of negative charge residues (Asp + Glu) was 87 and the total number of positive charge residues (Arg + Lys) was 63; molecular formula C3546H5301N917O1023S23(ii) a The half-life period in Escherichia coli is predicted to be more than 10h, the instability coefficient is 32.95, and the protein is classified as stable protein.
The PCR product was the XylA gene.
SEQ ID NO 1 is shown below:
ATGAATGCATTTGAATTACGTGAGCCAGACTGGAACGCAATAGAGCCAGCACGCTTGACTGCAGAGCCTAAGTTACAAGGCCATGTGCTAGTTTTACCGACTCAATTTGGCGAGGTTAGCGTTACAATAAGCCAGTTTGGTTTACGTTTAAATGCAGGCACCACGCATGACGAGACGTTTAAAATATTAACCACCACCCCTTCAAACTTACCGCTATCACTTAATAAATTAGACCAAGGCTTTGCAGCCACCGCCGGTGAGTACCGCTTAGAATTTTACAGCGACCCGTTTTACTTTAAGCTTTATAAAAACGATAAATTAGTACAGCAATCAGCAACCGATGGCCACTTTGTGCGCCAGCACCGTTTACCGCCACTGGCTAAAACCGATAACGGTTGGATTTTAAGCCTAGAGCTTAATTATGACGAAGCCGTATACGGGCTTGGCGAAAAATGGGGCAAGCTTGATAAACGCGGCCAACTTATTCGTTCATACAACCACGATGCGCTAGGCGTCAACGCCGAAAAGTCTTATAAAAACACACCGTATGCATGGAGCCCAGAGGGCTGGAACTTATTTGTACACACACCAGCGCCTGTTACACATGGTGTAGGTTACGCGCTTTGGTCGCAACGTGCTTATGTGTGTTTAGTAGAAGACGACGCGCTAGATGTATTTTTATACCAAGAGCAAACGCCTGCACAAAGTATTAATCGCTACTGCGAGCTAACTGGTTTTGCACCGGTACCACCACAGTGGAGTTTTGGTGTTATTTTATCAAAAGCGTACTACAAAGACGCCAACGAGCTATTAAGTGTCGCCCGCGAAGTACGCGCTAAAAATATGCCGTGCGATGTAATAACTCTTGATGGCCGTGCATGGCAAGACACCGACACCCGCTTTGCCTTTGAGTGGGATCCAACACGTTACGCCGACCCAAAACCTGTGCTTGATGAATTAAAAGCCATGGATTTTAAAATTTGCGTGTGGGAATACCCAATGATCTCGGTAAACAACCCATTATTTGCTAAAGCGGCCGAAAACGGCTGGCTAATAAAAGACAAACGCACAGGTAAAGCGTATCAATACGAGTGGGATTTAAGCCCATTTGGCGAAGTACTTACGCCATTACCGGAGTCGGGTATTTTAGATTTTACTCATCCCGATGCTTACGAATACTGGCTGGAGTCGCACAAGCCATTGTTTGAGTTTGGCGTAGATATGATAAAAGCAGACTTTGGCGAACAGCTAGAAGACGAAAACATGGTATCGCACAGTGGCGACAGCGGCATTAGACTGCATAATGTTTATAGCATGCTGTACAACCGCTGCGTTTATGAAGCCGCAGAAAAGTACTGCAAAACAGGGCCATTTTTATTTAGCCGCTCAGCGTGGACTGGCAGCCAACGTTTCCCTGCACAATGGGGTGGCGACCCACAAGCCGATTGGCAAGGTTTAGCAGCGAGCATTCGCGGTAGCTTAGCGTGGGGCATGTCGGGCGGGCCATTTTTTGCAACCGACATTGGTGGTTTTTACAAAGATACGCGCGATGCAGAGCTTTATGTTCGCTGGGCGCAAGCGTCGGTATTTAGTGCGCACATGCGTTTACATGGCATTGGCCCACGCGAACCGTGGTCATACACCGAGCAAGCAAGTGACGCGGTATTTGCAGCGCTTAAATTACGCTATCAGTTAATTCCTTACTTACAAGAATGTGCAGAGCAAGCGCAGCAAACTGGGCTGCCTATTCAGCGTGCTATGGCACTGGCGTTTCCTGATGACGTACTGGCACATAGTTTCGATCAGCAATTTATGTGCGGCGAAAAACTCCTTGTTGTGCCATGTGTTGTACCAAACGGTAAAGTTAAATTTTATTTACCACAAGGCGAGTGGGTGCGATTCCCTGATGGGCAGGCGTTCCAAGGCGGCAAGTATTACGAAGAAACACTTGAGCTTACACAAATGGCCGTATTTGTTCGTAAAGGCGATACGCTCATGCTAGGCCCAGAGGTTCAACACACAGAGCAAGACATGAGCCAGCTAACAGCATGGCCAAAATAA。
SEQ ID NO 2 is shown below:
MNAFELREPDWNAIEPARLTAEPKLQGHVLVLPTQFGEVSVTISQFGLRLNAGTTHDETFKILTTTPSNLPLSLNKLDQGFAATAGEYRLEFYSDPFYFKLYKNDKLVQQSATDGHFVRQHRLPPLAKTDNGWILSLELNYDEAVYGLGEKWGKLDKRGQLIRSYNHDALGVNAEKSYKNTPYAWSPEGWNLFVHTPAPVTHGVGYALWSQRAYVCLVEDDALDVFLYQEQTPAQSINRYCELTGFAPVPPQWSFGVILSKAYYKDANELLSVAREVRAKNMPCDVITLDGRAWQDTDTRFAFEWDPTRYADPKPVLDELKAMDFKICVWEYPMISVNNPLFAKAAENGWLIKDKRTGKAYQYEWDLSPFGEVLTPLPESGILDFTHPDAYEYWLESHKPLFEFGVDMIKADFGEQLEDENMVSHSGDSGIRLHNVYSMLYNRCVYEAAEKYCKTGPFLFSRSAWTGSQRFPAQWGGDPQADWQGLAASIRGSLAWGMSGGPFFATDIGGFYKDTRDAELYVRWAQASVFSAHMRLHGIGPREPWSYTEQASDAVFAALKLRYQLIPYLQECAEQAQQTGLPIQRAMALAFPDDVLAHSFDQQFMCGEKLLVVPCVVPNGKVKFYLPQGEWVRFPDGQAFQGGKYYEETLELTQMAVFVRKGDTLMLGPEVQHTEQDMSQLTAWPK。
example 2: recombinant expression and purification of the XylA Gene in the E.coli BL21(DE3) Strain
The PCR product obtained in example 1 and the pET-28a plasmid were digested simultaneously with restriction enzymes EcoR I and XhoI, and the product fragment after digestion was collected. Restriction enzymes EcoR I and XhoI were purchased from TaKaRa Bio Inc., Dalian China, and the system, temperature and time for the enzyme to react with the substrate used in the digestion were all operated according to the product instructions provided by the company.
The PCR products which are subjected to double enzyme digestion of EcoR I and XhoI and pET-28a plasmid vectors which are also subjected to double enzyme digestion are connected under the catalysis of T4 DNA ligase, the connection products are transformed into Escherichia coli DH5 alpha strains, the Escherichia coli DH5 alpha strains are coated on an LB solid culture medium containing 0.1mg/mL kanamycin, after inverted culture is carried out at 37 ℃, positive transformants are picked, the positive transformants are inoculated into a liquid LB culture medium containing 0.1mg/mL kanamycin, culture is carried out at 37 ℃ and 180R/min for 12 hours, and bacterial liquid PCR verification is carried out by using forward primers XylA-F and reverse primers XylA-R (namely SEQ ID NO:3 and 4).
Converting a recombinant plasmid with correct PCR verification into escherichia coli BL21(DE3), coating the escherichia coli BL21(DE3) on an LB solid culture medium containing 0.1mg/mL kanamycin, performing inverted culture at 37 ℃ for 16h, selecting a positive transformant, inoculating the positive transformant into a liquid LB culture medium containing 0.1mg/mL kanamycin, performing culture at 37 ℃ for 180R/min for 12h, performing bacterial liquid PCR verification by using a forward primer XylA-F and a reverse primer XylA-R, and obtaining an amplification product with the size of about 2000bp as a result, thereby preliminarily proving that the constructed recombinant plasmid is correct. The recombinant plasmid is sent to Xiamen platinum-Rui Biotechnology Co., Ltd for sequencing, and the result shows that the gene XylA shown in SEQ ID NO.1 is inserted into the EcoR I and XhoI enzyme cutting sites of pET-28a, and the insertion direction is correct, so that the constructed recombinant plasmid is further proved to be correct, and the recombinant plasmid is named as pET-28 a-XylA.
The isopropyl thiogalactoside (IPTG) is used for the induction expression of the recombinant bifunctional enzyme. Adding isopropyl thio-beta-D-galactoside (IPTG) to a final concentration of 0.05mmol/L, inducing at 16 ℃ for 20h, collecting the bacterial liquid into a 200mL centrifuge tube, and centrifuging at 6000rpm to precipitate bacterial cells. Bacterial cells were resuspended in 20mL of lysis buffer (lysis buffer formulation: 0.3mol/L NaCl, 15mmol/L imidazole, 50mmol/L NaH2PO4pH8.0), performing ultrasonic crushing until the bacterial liquid becomes semitransparent (parameters are set to 300w, the ultrasonic time is 5s, the intermittent time is 5s, and the total working time is 15min), centrifuging at 11000rpm for 20min, mixing the supernatant with Ni-NTA Agarose which is balanced by a dissolving buffer in advance, combining the mixture at 4 ℃ for 1h, and performing the purification process according to the instructions of a purification kit (purchased from Qiagen). The purified protein was analyzed by SDS-PAGE and had a molecular weight of about 40kDa, and the concentration of the protein was determined by the Bradford method to give a XylA concentration of about 0.5mg/ml (see FIG. 1 for results). Wherein lane M is a protein Marker, and lane 1 is the target protein XylA (77.88kDa) purified by Ni-NTA. As can be seen, the target protein XylA is induced and expressed, and is purified, so that impurities are effectively removed.
Example 3: analysis of enzymatic Properties of recombinant XylA Gene vectors
1. Determination of the enzyme activity of the recombinant XylA gene vector:
the activities of the alpha-xylosidase and arabinosidase were determined by the amount of p-nitrophenol (pNP) released from the substrates p-nitrophenol-alpha-L-arabinofuranoside (Sigma), p-nitrophenol-alpha-D-xylofuranoside (Sigma), respectivelyAnd (4) determining. The total volume of the system is 800 mu L by spectrophotometry, the reaction system is 200 mu L, and the reaction system contains 10 mu L of 20mmol/L substrate p-nitrophenol-alpha-L-arabinofuranoside or p-nitrophenol-alpha-D-xylofuranoside, 180 mu L of 50mmol/L potassium phosphate buffer solution with pH value of 6.8, and 10 mu L of enzyme solution. Reacting at 60 ℃ for 20min, and then adding 600 mu L of 2mol/L Na2CO3The reaction was stopped and developed and the absorbance was measured at 405 nm. One enzyme unit (U) is defined as the amount of enzyme required to catalyze the production of 1. mu. mol of p-nitrophenol within 1min under the reaction conditions.
2. Effect of temperature on enzyme activity and stability:
the enzyme activity of the recombinant bifunctional enzyme is determined after the reaction at the reaction temperature of 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃, and the highest enzyme activity is 100 percent. When measuring the temperature stability of recombinase XylA, the enzyme solution is incubated for 60min at 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃, the residual enzyme activity is measured at the optimum reaction temperature, and the enzyme activity is taken as 100% when not treated. The results of the optimum temperature measurement are shown in FIG. 2, and when the xylosidase activity is expressed, the optimum temperature is 60 ℃; when the arabinofuranosidase activity is expressed, the maximum enzyme activity is expressed at 55 ℃. The temperature stability results are shown in fig. 3, when the xylosidase activity is expressed, 65% of the residual activity of the xylosidase can be remained after the xylosidase is treated at 50 ℃ for 60 min; when exhibiting arabinofuranosidase activity, 58% of the residual activity was retained after 60min treatment at 50 ℃. It can be seen that the enzyme has better temperature stability no matter which activity is expressed.
3. Effect of pH on enzyme Activity and stability
Preparing a substrate by using buffer systems with different pH values, and measuring the enzyme activity at the optimal reaction temperature, wherein the highest enzyme activity is 100%. The buffers were as follows: 0.2mol/L disodium hydrogen phosphate-citric acid (pH 4.0-8.0); 0.2mol/L glycine-sodium hydroxide buffer (pH8.0-10.0). The study on the pH stability is carried out by mixing the enzyme solution with buffer solutions with different pH values in equal proportion, standing at 4 ℃ for 2h, and then measuring the residual enzyme activity, wherein the enzyme activity is 100% when the enzyme is not treated. The results of pH optima and pH stability are shown in FIG. 4 and FIG. 5, respectively, when xylosidase activity is expressed, the optimum reaction pH is 7, and the enzyme activity can be maintained at 60% or more of the maximum enzyme activity after treatment for 2h under the condition of pH 6-8.5. When the arabinofuranosidase activity is expressed, the optimum pH value is 6.5, the stability is also compared within the range of pH6-8.5, and more than 56% of the maximum enzyme activity can be kept after 2 hours of treatment.
4. Effect of Metal ions on enzyme Activity (with p-nitrophenol-alpha-D-xylofuranoside as substrate)
Adding different metal ions (Mg) with final concentration of 1mmol/L or 10mmol/L to the purified enzyme solution of recombinase XylA2+、Sr2+、Mn2+、Al3+、Ba2+、Cd2+、Fe3+、Ca2+、Cu2+) After standing at 37 ℃ for 1 hour, the residual activity of the enzyme was determined, and the effect of the metal ions on the enzyme activity was investigated with the enzyme activity without the addition of metal ions as 100%, as shown in FIG. 6. As can be seen from FIG. 6, the metal ion Ca2+The recombinant enzyme XylA is slightly promoted at the concentration of 1mmol/L, but the enzyme activity is inhibited when the concentration is increased to 10 mmol/L. Mg (magnesium)2+、Sr2+、Ba2+The recombinant enzyme is slightly inhibited at a concentration of 1 mmol/L. At a concentration of 10mmol/L, Ca2+、Cu2+、Ba2+Has obvious inhibition on enzyme activity. 10mM Mn2+Can almost completely inhibit enzyme activity.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
SEQUENCE LISTING
<110> college university
<120> use of xylA gene with xylosidase and arabinofuranosidase dual functions
<130> JMDXL-19037-CNI
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 2061
<212> DNA
<213> Pseudoalteromonas carrageenovora ASY5
<400> 1
atgaatgcat ttgaattacg tgagccagac tggaacgcaa tagagccagc acgcttgact 60
gcagagccta agttacaagg ccatgtgcta gttttaccga ctcaatttgg cgaggttagc 120
gttacaataa gccagtttgg tttacgttta aatgcaggca ccacgcatga cgagacgttt 180
aaaatattaa ccaccacccc ttcaaactta ccgctatcac ttaataaatt agaccaaggc 240
tttgcagcca ccgccggtga gtaccgctta gaattttaca gcgacccgtt ttactttaag 300
ctttataaaa acgataaatt agtacagcaa tcagcaaccg atggccactt tgtgcgccag 360
caccgtttac cgccactggc taaaaccgat aacggttgga ttttaagcct agagcttaat 420
tatgacgaag ccgtatacgg gcttggcgaa aaatggggca agcttgataa acgcggccaa 480
cttattcgtt catacaacca cgatgcgcta ggcgtcaacg ccgaaaagtc ttataaaaac 540
acaccgtatg catggagccc agagggctgg aacttatttg tacacacacc agcgcctgtt 600
acacatggtg taggttacgc gctttggtcg caacgtgctt atgtgtgttt agtagaagac 660
gacgcgctag atgtattttt ataccaagag caaacgcctg cacaaagtat taatcgctac 720
tgcgagctaa ctggttttgc accggtacca ccacagtgga gttttggtgt tattttatca 780
aaagcgtact acaaagacgc caacgagcta ttaagtgtcg cccgcgaagt acgcgctaaa 840
aatatgccgt gcgatgtaat aactcttgat ggccgtgcat ggcaagacac cgacacccgc 900
tttgcctttg agtgggatcc aacacgttac gccgacccaa aacctgtgct tgatgaatta 960
aaagccatgg attttaaaat ttgcgtgtgg gaatacccaa tgatctcggt aaacaaccca 1020
ttatttgcta aagcggccga aaacggctgg ctaataaaag acaaacgcac aggtaaagcg 1080
tatcaatacg agtgggattt aagcccattt ggcgaagtac ttacgccatt accggagtcg 1140
ggtattttag attttactca tcccgatgct tacgaatact ggctggagtc gcacaagcca 1200
ttgtttgagt ttggcgtaga tatgataaaa gcagactttg gcgaacagct agaagacgaa 1260
aacatggtat cgcacagtgg cgacagcggc attagactgc ataatgttta tagcatgctg 1320
tacaaccgct gcgtttatga agccgcagaa aagtactgca aaacagggcc atttttattt 1380
agccgctcag cgtggactgg cagccaacgt ttccctgcac aatggggtgg cgacccacaa 1440
gccgattggc aaggtttagc agcgagcatt cgcggtagct tagcgtgggg catgtcgggc 1500
gggccatttt ttgcaaccga cattggtggt ttttacaaag atacgcgcga tgcagagctt 1560
tatgttcgct gggcgcaagc gtcggtattt agtgcgcaca tgcgtttaca tggcattggc 1620
ccacgcgaac cgtggtcata caccgagcaa gcaagtgacg cggtatttgc agcgcttaaa 1680
ttacgctatc agttaattcc ttacttacaa gaatgtgcag agcaagcgca gcaaactggg 1740
ctgcctattc agcgtgctat ggcactggcg tttcctgatg acgtactggc acatagtttc 1800
gatcagcaat ttatgtgcgg cgaaaaactc cttgttgtgc catgtgttgt accaaacggt 1860
aaagttaaat tttatttacc acaaggcgag tgggtgcgat tccctgatgg gcaggcgttc 1920
caaggcggca agtattacga agaaacactt gagcttacac aaatggccgt atttgttcgt 1980
aaaggcgata cgctcatgct aggcccagag gttcaacaca cagagcaaga catgagccag 2040
ctaacagcat ggccaaaata a 2061
<210> 2
<211> 686
<212> PRT
<213> Pseudoalteromonas carrageenovora ASY5
<400> 2
Met Asn Ala Phe Glu Leu Arg Glu Pro Asp Trp Asn Ala Ile Glu Pro
Ala Arg Leu Thr Ala Glu Pro Lys Leu Gln Gly His Val Leu Val Leu
Pro Thr Gln Phe Gly Glu Val Ser Val Thr Ile Ser Gln Phe Gly Leu
Arg Leu Asn Ala Gly Thr Thr His Asp Glu Thr Phe Lys Ile Leu Thr
Thr Thr Pro Ser Asn Leu Pro Leu Ser Leu Asn Lys Leu Asp Gln Gly
Phe Ala Ala Thr Ala Gly Glu Tyr Arg Leu Glu Phe Tyr Ser Asp Pro
Phe Tyr Phe Lys Leu Tyr Lys Asn Asp Lys Leu Val Gln Gln Ser Ala
Thr Asp Gly His Phe Val Arg Gln His Arg Leu Pro Pro Leu Ala Lys
Thr Asp Asn Gly Trp Ile Leu Ser Leu Glu Leu Asn Tyr Asp Glu Ala
Val Tyr Gly Leu Gly Glu Lys Trp Gly Lys Leu Asp Lys Arg Gly Gln
Leu Ile Arg Ser Tyr Asn His Asp Ala Leu Gly Val Asn Ala Glu Lys
Ser Tyr Lys Asn Thr Pro Tyr Ala Trp Ser Pro Glu Gly Trp Asn Leu
Phe Val His Thr Pro Ala Pro Val Thr His Gly Val Gly Tyr Ala Leu
Trp Ser Gln Arg Ala Tyr Val Cys Leu Val Glu Asp Asp Ala Leu Asp
Val Phe Leu Tyr Gln Glu Gln Thr Pro Ala Gln Ser Ile Asn Arg Tyr
Cys Glu Leu Thr Gly Phe Ala Pro Val Pro Pro Gln Trp Ser Phe Gly
Val Ile Leu Ser Lys Ala Tyr Tyr Lys Asp Ala Asn Glu Leu Leu Ser
Val Ala Arg Glu Val Arg Ala Lys Asn Met Pro Cys Asp Val Ile Thr
Leu Asp Gly Arg Ala Trp Gln Asp Thr Asp Thr Arg Phe Ala Phe Glu
Trp Asp Pro Thr Arg Tyr Ala Asp Pro Lys Pro Val Leu Asp Glu Leu
Lys Ala Met Asp Phe Lys Ile Cys Val Trp Glu Tyr Pro Met Ile Ser
Val Asn Asn Pro Leu Phe Ala Lys Ala Ala Glu Asn Gly Trp Leu Ile
Lys Asp Lys Arg Thr Gly Lys Ala Tyr Gln Tyr Glu Trp Asp Leu Ser
Pro Phe Gly Glu Val Leu Thr Pro Leu Pro Glu Ser Gly Ile Leu Asp
Phe Thr His Pro Asp Ala Tyr Glu Tyr Trp Leu Glu Ser His Lys Pro
Leu Phe Glu Phe Gly Val Asp Met Ile Lys Ala Asp Phe Gly Glu Gln
Leu Glu Asp Glu Asn Met Val Ser His Ser Gly Asp Ser Gly Ile Arg
Leu His Asn Val Tyr Ser Met Leu Tyr Asn Arg Cys Val Tyr Glu Ala
Ala Glu Lys Tyr Cys Lys Thr Gly Pro Phe Leu Phe Ser Arg Ser Ala
Trp Thr Gly Ser Gln Arg Phe Pro Ala Gln Trp Gly Gly Asp Pro Gln
Ala Asp Trp Gln Gly Leu Ala Ala Ser Ile Arg Gly Ser Leu Ala Trp
Gly Met Ser Gly Gly Pro Phe Phe Ala Thr Asp Ile Gly Gly Phe Tyr
Lys Asp Thr Arg Asp Ala Glu Leu Tyr Val Arg Trp Ala Gln Ala Ser
Val Phe Ser Ala His Met Arg Leu His Gly Ile Gly Pro Arg Glu Pro
Trp Ser Tyr Thr Glu Gln Ala Ser Asp Ala Val Phe Ala Ala Leu Lys
Leu Arg Tyr Gln Leu Ile Pro Tyr Leu Gln Glu Cys Ala Glu Gln Ala
Gln Gln Thr Gly Leu Pro Ile Gln Arg Ala Met Ala Leu Ala Phe Pro
Asp Asp Val Leu Ala His Ser Phe Asp Gln Gln Phe Met Cys Gly Glu
Lys Leu Leu Val Val Pro Cys Val Val Pro Asn Gly Lys Val Lys Phe
Tyr Leu Pro Gln Gly Glu Trp Val Arg Phe Pro Asp Gly Gln Ala Phe
Gln Gly Gly Lys Tyr Tyr Glu Glu Thr Leu Glu Leu Thr Gln Met Ala
Val Phe Val Arg Lys Gly Asp Thr Leu Met Leu Gly Pro Glu Val Gln
His Thr Glu Gln Asp Met Ser Gln Leu Thr Ala Trp Pro Lys
<210> 3
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 3
ccggaattca tgaatgcatt tgaattacgt 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Synthesis
<400> 4
ccgctcgagt tattttggcc atgctgttag 30