The content of the invention
The object of the present invention is to provide a kind of glucose oxidase mutant, heat resistance is significantly improved, is conducive to
It is in the extensive use of field of fodder.
In order to realize foregoing invention purpose, the present invention provides following technical scheme:
The present invention provides a kind of glucose oxidase mutant, have (I), (II) or(Ⅲ)In shown amino acid sequence
Any one:
(I) with the amino acid sequence SEQ ID NO of glucose oxidase:1 has the sequence of at least 95% homology;
(II) there is at least one immune epitope of the glucose oxidase described in (I), and the glucose oxidase
The amino acid sequence that is obtained through modifying, substituting, lacking or adding one or several amino acid of amino acid sequence;
(Ⅲ)By such as SEQ ID NO:When nucleotide sequence or its complementary series shown in 2 or the degeneracy because genetic code with such as
SEQ ID NO:The amino acid sequence of the different sequential coding of the nucleotide sequence of nucleotide sequence or its complementary series shown in 2
Row;
In some embodiments of the invention, it is described to be substituted by 1 amino acid of substitution.
In other embodiments of the present invention, the substitution includes amino acid sequence for SEQ ID NO:1 glucose
The 386th of oxidizing ferment, 387,401,406,408,418,455,466,470,476,478,479,492,493,501,531,
Any one in 532,546,578 amino acids is substituted.
The present invention other embodiments in, it is described substitution include the 386th amino acids from T become A or E or F or
P。
In other embodiments of the present invention, the substitution includes the 387th amino acids and becomes E or P from T.
In other embodiments of the present invention, the substitution includes the 401st amino acids and becomes F from V.
In other embodiments of the present invention, the substitution includes the 406th amino acids and becomes P from A.
In other embodiments of the present invention, the substitution includes the 408th amino acids and becomes A from S.
In other embodiments of the present invention, the substitution includes the 418th amino acids and becomes L from A.
In other embodiments of the present invention, the substitution includes the 455th amino acids and becomes D from E.
In other embodiments of the present invention, the substitution includes the 466th amino acids and becomes K from Q.
In other embodiments of the present invention, the substitution includes the 470th amino acids and becomes E or F or K from N.
In other embodiments of the present invention, the substitution includes the 476th amino acids and becomes P from A.
In other embodiments of the present invention, the substitution includes the 478th amino acids and becomes K from Q.
In other embodiments of the present invention, the substitution includes the 479th amino acids and becomes D from T.
In other embodiments of the present invention, the substitution includes the 492nd amino acids and becomes P from A.
In other embodiments of the present invention, the substitution includes the 493rd amino acids and becomes P from Y.
The present invention other embodiments in, it is described substitution include the 501st amino acids from T become E or K or L or
R, or Y.
In other embodiments of the present invention, the substitution includes the 531st amino acids and becomes F or P from N.
In other embodiments of the present invention, the substitution includes the 532nd amino acids and becomes R from A.
In other embodiments of the present invention, the substitution includes the 546th amino acids and becomes A from G.
The present invention other embodiments in, it is described substitution include the 578th amino acids from S become F or K or R or
Y。
In some embodiments of the invention, the missing is missing 1 or 2 amino acid.
In other embodiments of the present invention, the missing includes the 580th amino acids missing.
In other embodiments of the present invention, the missing includes the 579th and the 580th amino acids lack simultaneously.
The present invention also provides application of the above-mentioned glucose oxidase mutant in feed.
The present invention also provides the recombinant expression carriers with above-mentioned DNA molecular.
The present invention also provides a kind of host cells, include above-mentioned recombinant expression carrier.
In some embodiments of the invention, host cell is Pichia pastoris.
The heat resistance of glucose oxidase mutant provided by the invention is generally higher than wild type, after 60 DEG C handle 10 min
Remnant enzyme activity improves 19.1-63.8%, and remnant enzyme activity improves 27.1-82.6% after 65 DEG C of 5 min of processing.It is above-mentioned so as to illustrate
Mutation causes the heat resistance of glucose oxidase to be increased dramatically, and is more suitable as feed addictive than wild type, is conducive to
Extensive use of the glucose oxidase in feed, thus wide market.
Specific embodiment:
The routine techniques and method that the present invention has used genetic engineering and biology field uses, such as MOLECULAR
CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN
Recorded method in MOLECULAR BIOLOGY (Ausubel, 2003).These general bibliography provide ability
The known definition of field technique personnel and method.But those skilled in the art can be in the technical solution recorded in the present invention
On the basis of, using the other conventional methods in this field, experimental program and reagent, and it is not limited to the limit of the specific embodiment of the invention
It is fixed.
A in the present invention, R, D, C, Q, E, H, I, G, N, L, K, M, F, P, S, T, W, Y, V are alanine Ala respectively, arginine
Arg, aspartic acid Asp, cysteine Cys, glutamine Gln, glutamic acid Glu, histidine, isoleucine Ile, sweet ammonia
Sour Gly, asparagine Asn, leucine Leu, lysine Lys, methionine Met, phenylalanine Phe, proline Pro, silk ammonia
Sour Ser, threonine Thr, tryptophan Trp, tyrosine Tyr, the abbreviation of valine Val.
Experiment material used in the specific embodiment of the invention and reagent are as follows:
Bacterial strain and carrier:Bacillus coli DH 5 alpha, Pichia pastoris GS115, carrier pPIC9K, Amp, G418 are public purchased from Invitrogen
Department.
Enzyme and kit:PCR enzymes and ligase purchase are from Takara companies, and restriction enzyme is purchased from Fermentas public affairs
Department, plasmid extraction kit and glue purification QIAquick Gel Extraction Kit are purchased from Omega companies, the purchase of GeneMorph II Random Mutagenesis Kits
From Beijing Bo Maisi bio tech ltd.
Culture medium prescription:
Escherichia coli culture medium(LB culture mediums):0.5% yeast extract, 1% peptone, 1% NaCl, pH7.0);
LB-AMP culture mediums:LB culture mediums add 100 μ g/mL ampicillins;
Yeast culture medium(YPD culture mediums):1% yeast extract, 2% peptone, 2% glucose;
Yeast screening assay culture medium(MD culture mediums):2% glucose, 2% agarose, 1.34% YNB, 4 × 10-5Biotin;
BMGY culture mediums:2% peptone, 1% yeast extract, 100 mM kaliumphosphate buffers (pH6.0), 1.34% YNB, 4 ×
10-5Biotin, 1% glycerine;
BMMY culture mediums:2% peptone, 1% yeast extract, 100 mM kaliumphosphate buffers (pH6.0), 1.34% YNB, 4 ×
10-5Biotin, 0.5% methanol.
The present invention is described in detail with reference to embodiment.
The acquisition of the heat-resisting mutant of 1 glucose oxidase of embodiment
The amplification of 1.1 glucose oxidase genes
With aspergillus niger(Aspergillus niger)Genome carries out PCR amplification for template, and PCR primer GOD-F1, GOD-R1 is such as
Under:
GOD-F1:GGTATTGAGGCATCTTTGTTGAC
GOD-R1:TTATTGCATAGAAGCGTAATC
Glue recycles PCR product, connects pEASY-T carriers, converts into bacillus coli DH 5 alpha, the correct transformant of picking is surveyed
Sequence.Sequencing result shows that the nucleotides sequence of the genetic fragment expanded is classified as SEQ ID NO:2, the amino acid sequence of coding
It is classified as SEQ ID NO:1.It is compared and found by NCBI BLAST, SEQ ID NO:1 is glycoxidative with the grape from aspergillus niger
Enzyme sequence similitude is up to 100%, so that it is determined that being glucose oxidase gene by the gene that PCR is obtained, is named as GOD.
The amplification and synthesis of glucose oxidase mutant gene
In order to improve the heat resistance of above-mentioned glucose oxidase GOD, applicant has carried out greatly the enzyme by directed evolution technologies
The screening of mutation is measured, design PCR primer GOD-F2, GOD-R2 is as follows:
GOD-F2:GGCGAATTCGGTATTGAGGCATCTTTGTTGAC(Underscore is restriction enzyme EcoRI recognition sites)
GOD-R2:ATAGCGGCCGCTTATTGCATAGAAGCGTAATC(Underscore is restriction enzyme Not I recognition sites)
Using GOD genes as template, with above-mentioned primer GeneMorph II random mutation PCR kits(Stratagene)It carries out
PCR amplification, glue recycling PCR product, EcoRI, Not I connect after carrying out digestion processing with the pET21a carriers after similary digestion
It connects, converts into e. coli bl21 (DE3), be coated on LB+Amp tablets, 37 DEG C are inverted culture, after sub- appearance to be transformed, are used
Toothpick is chosen one by one to 96 orifice plates, adds in the LB+Amp culture mediums that 150 ul contain 0.1mM IPTG in each hole, 37 DEG C 220
Rpm cultivates 6 h or so, and supernatant is abandoned in centrifugation, and thalline is resuspended with buffer solution, multigelation broken wall, and acquisition contains glucose oxidase
Bacillus coli cells lysate.
10 μ L lysates are taken out respectively to two pieces of 96 new orifice plates, one of after 70 DEG C handle 5min, two piece of 96 hole
Plate all adds in 40 μ L substrates, and after 30 DEG C are reacted 30 min, DNS methods measure the reduced sugar of generation, calculate the enzyme solution of high-temperature process
Compared to the opposite enzyme activity of untreated enzyme solution.The experimental results showed that some mutation do not have the heat resistance of glucose oxidase GOD
It influences, some mutation even make its heat resistance or enzyme activity become worse;In addition also some mutation, although grape glycosyloxy can be improved
Change tolerance of the enzyme to temperature, but significant change has occurred in its zymologic property after mutation, and these are undesirable.Finally,
Applicant screens the heat resistance that can significantly improve glucose oxidase GOD and its enzyme activity and original zymology
The mutational site of matter:T386A/E/F/P, T387E/P, V401F, A406P, S408A, A418L, E455D, Q466K, N470E/F/
K, A476P, Q478K, T479D, A492P, Y493P, T501E/K/L/R/Y, N531F/P, A532R, G546A, S578F/K/R/
Y and Q580 missings, M579 and Q580 are lacked simultaneously.
PCR amplification is carried out respectively to above-mentioned mutant with primer GOD-F2, GOD-R2, and primer both ends introduce EcoRI, Not
I site.PCR reaction conditions are:94 DEG C of denaturation 5min;Then 94 DEG C of denaturation 30s, 56 DEG C of renaturation 30s, 72 DEG C of extension 1min, 30
After a cycling, 72 DEG C of heat preservation 10min.Agarose gel electrophoresis is the results show that the mutant gene that amplification obtains is size
The segment of 1800bp or so.
It expands to obtain the genetic fragment of wild type glucose oxidase GOD by above-mentioned same PCR method.
The structure of pichia pastoris engineered strain
The glucose oxidase mutant gene that above-mentioned clone is obtained passes through EcoRI and NotI sites and Expression vector pPIC9K
It is connected, construction of expression vector.
Expression vector is linearized with Sal I, expression vector linearized fragment is converted by electroporation finishes red ferment
Female GS115, screening obtains Pichia pastoris recombinant bacterial strain respectively on MD tablets, then respectively in the Geneticin containing various concentration
The transformant of multicopy is screened on YPD tablets.
The obtained switching of the transformant containing above-mentioned mutant will be screened in BMGY culture mediums, 30 DEG C, 250 rpm vibration trainings
Support 1 d;It is transferred to again in BMMY culture mediums, 30 DEG C, 250 rpm shaken cultivations;The methanol of addition 0.5% daily, 4 d of induced expression;
Centrifugation removal thalline, obtains the fermented supernatant fluid of the mutant containing glucose oxidase;Carried out SDS-PAGE electrophoresis detections point
Analysis.The results show that the molecular size range of glucose oxidase mutant is about 64 kDa in fermented supernatant fluid, with theoretical molecular weight
Size is identical.
It builds to obtain the Pichia yeast engineering of recombination expression wild type glucose oxidase by above-mentioned same method.
The horizontal fermentation of shaking flask, 30 DEG C, 250 rpm shaken cultivations;The methanol of addition 0.5% daily, 4 d of induced expression;Degerming is gone in centrifugation
Body obtains the fermented supernatant fluid of the GOD of glucose oxidase containing wild type.
(1)The definition of glucose oxidase enzyme-activity unit
It is per minute β-D-Glucose of 1 μm of ol to be oxidized to maltonic acid and hydrogen peroxide under the conditions of pH6.0,30 DEG C
Required enzyme amount is defined as 1 enzyme activity unit(IU).
(2)Enzyme activity determination method
Crude enzyme liquid is directly diluted to about 10U/mL with buffer solution.The test tube of 4 150*15 is taken, adds in 2ml buffer solutions, 0.3ml
Glucose, 0.4ml phenol, 0.1ml 4- amino antipyrine, 0.1ml horseradish peroxidases, 30 DEG C of preheating 5min.Xiang Qi
In a pipe add in 0.1ml distilled water, as blank return to zero.Water-bath is placed on to be conveniently operated by spectrophotometer, to sample cell
Middle addition 0.1ml sample solutions, start timing, after vortex mixing immediately at 500nm wavelength with 1cm cuvette colorimetrics at this time.
Absorbance is A0 when reading 0.5 min, then after reacting 1min, reads absorbance A1, draw Δ A500=A1-A0.
Enzyme activity calculation formula:
Enzyme activity X1 in sample(U/mL or U/g)It is calculated according to equation below:
X1=ΔA500×f×B ×1000/(887×t×A×d)=33.82×ΔA500×f
In formula:
F--------------------- enzyme solution extension rates
B-------------------- reaction solution volumes(3 ml)
1000---------------- extinction coefficient unit conversion factors
887----------------- extinction coefficients (Lmol-1cm-1)
The t--------------------- reaction time(min), i.e., the time difference 1min between reading A1 and A0.
A-------------------- adds in sample volume(0.1 ml)
The thickness of d-------------------- cuvettes(cm)
(3)Enzyme activity determination result
The enzyme activity determination of fermented supernatant fluid, the results show are carried out according to the method described above:Recombinantly express wild type glucose oxidase
Pichia pastoris fermented supernatant fluid enzyme activity for 105 U/ml, and recombinantly express the Pichia pastoris hair of glucose oxidase mutant
The enzyme activity of ferment supernatant is about 111-165U/mL.
Fermentation verification
Carry out the fermentation for the Pichia yeast engineering that above-mentioned structure obtains respectively on 10 liters of fermentation tanks, ferment the culture medium used
It is formulated and is:1.1 g/L of calcium sulfate, 5.5 g/L of potassium dihydrogen phosphate, 55 g/L of ammonium dihydrogen phosphate, 20.3 g/L of potassium sulfate, magnesium sulfate
16.4 g/L, 1.65 g/L of potassium hydroxide, antifoaming agent 0.05%.
Zymotechnique:PH value 5.0,30 DEG C of temperature, 300 rpm of stir speed (S.S.), ventilation quantity 1.0-1.5(v/v), dissolved oxygen control
More than 20%.
Entire fermentation process is divided into three phases:First stage be thalline cultivation stage, in 7% ratio access seed, 30 DEG C
24-26 h are cultivated, to have mended glucose as mark;Second stage is the hungry stage, after glucose has been mended, is not flowed plus any
Carbon source terminates, by a definite date about 30-60 min when dissolved oxygen rose to for 80% stage indicated above;Phase III is induced expression rank
Section, stream plus methanol induction, and dissolved oxygen is kept more than 20%, incubation time is between 150-180 h.After fermentation, ferment
Liquid obtains crude enzyme liquid after being handled by flame filter press.
Enzyme activity assay is carried out to crude enzyme liquid using in the embodiment 1 1.3 glucose oxidase enzyme activity determination methods, as a result
It has been shown that, the fermentation enzyme activity that the Pichia pastoris of recombination expression wild type glucose oxidase is final is 3050 U/ml, and is recombinantly expressed
The fermentation enzyme activity that the Pichia pastoris of glucose oxidase mutant is final reaches 3155-3561 U/ml.
Glucose oxidase zymologic property measures
1st, most suitable action pH
It is respectively 2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0 phosphoric acid hydrogen using pH value
Disodium-citrate buffer solution, fermentation crude enzyme liquid described in embodiment 1 1.4 carries out glucose oxidase work under the conditions of 30 DEG C
Power measures, and using highest enzyme activity as 100%, calculates with respect to enzyme activity, the results show wild type glucose oxidase GOD and mutant
Optimun pH is all 6.0, and the horizontal difference of opposite enzyme activity under condition of different pH is little.
2nd, thermal stability analysis
By above-mentioned crude enzyme liquid with after the acetic acid-sodium acetate buffer solution dilution of pH 6.0,10min, 65 DEG C of processing are handled at 60 DEG C
After 5min, enzyme activity is measured respectively, using the enzyme activity of untreated samples as 100%, calculates remnant enzyme activity.The results are shown in table below.
It can be seen that from the data in table compared with wild type, the heat resistance of 35 mutant provided by the invention has
It significantly improves, remnant enzyme activity improves 19.1-63.8% after 60 DEG C of 10 min of processing, and remnant enzyme activity improves after 65 DEG C of 5 min of processing
27.1-82.6%.So as to illustrate that above-mentioned mutation causes the heat resistance of glucose oxidase to be increased dramatically, than wild type more
Feed addictive is suitable as, is conducive to extensive use of the glucose oxidase in feed, wide market.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Qingdao Weilan Biology Group Co., Ltd.
<120>A kind of glucose oxidase mutant
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 581
<212> PRT
<213> 1
<400> 1
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 2
<211> 1746
<212> DNA
<213> 2
<400> 2
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accactgccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acgcttacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttgg ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctcacaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctgct gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccgctgg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746