CN113736753B - Vibrio alginolyticus bacteriophage and application thereof - Google Patents

Abstract

The invention discloses a vibrio alginolyticus bacteriophage and application thereof. The Vibrio alginolyticus phage vB _ ValS _ R12Z has a preservation number of GDMCC No: 61738-B1. The Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z provided by the invention has the characteristics of wide cracking spectrum and short latency, can crack various Vibrio alginolyticus and Vibrio owensis 3186, has the latency of 10 min, and can quickly and effectively kill and prevent and treat corresponding vibrio diseases. In addition, virulence factor genes and drug resistance genes are not detected in the phage genome, which shows that the phage can not enhance the host toxicity strength and can not cause drug resistance pollution of the surrounding water environment, and the safety is high, and the environment is friendly, so the vibrio alginolyticus phage vB _ ValS _ R12Z can be completely used for preventing and treating vibrio alginolyticus diseases of aquatic products.

Description

Vibrio alginolyticus bacteriophage and application thereof

Technical Field

The invention relates to the technical field of microorganism application, in particular to a vibrio alginolyticus phage vB _ ValS _ R12Z and application thereof.

Background

With the rising demand of aquatic products, the aquaculture of ChinaThe industry scale is enlarged year by year, the breeding mode is gradually transformed to high-density intensive breeding, however, infectious diseases can cause greater threat to high-density breeding, the breeding products are reduced in large scale due to disease outbreak, and great economic loss is caused to farmers. Wherein, Vibrio (Vibrio)Vibrio) Caused Vibriosis (Vibriosis) is one of the most common diseases in aquaculture industry causing massive death of fish, shrimp and shellfish, and Vibrio alginolyticus (Vibrio)Vibrio alginolyticus) Is also a common pathogenic bacterium in vibrio, and the infection of vibrio alginolyticus can cause the organs such as digestive tracts, skins and the like of aquatic animals to be diseased and finally cause the death of the organs. When the uncooked aquatic product infected with the vibrio alginolyticus is eaten, infection can be caused to people, and symptoms such as diarrhea, dizziness and the like can be caused to the people. Currently, putting antibiotics in the aquaculture system is the most common and effective means for preventing and treating bacterial aquatic diseases, but because the antibiotic treatment has the problems of drug residues, increased drug resistance, long development period of new antibiotics, high development cost and the like, the aquaculture industry may face the embarrassment of no antibiotics in the near future, and therefore, the search for efficient and environment-friendly alternative prevention and treatment methods is urgent.

As one of the methods for coping with bacterial aquatic diseases, a method for controlling aquatic diseases by using bacteriophage has been attracting attention in recent years. The bacteriophage prevention and control mainly utilizes the lysis effect of virulent bacteriophage on bacteria to kill pathogenic bacteria so as to achieve the effect of treating and preventing bacterial diseases of aquatic animals. The bacteriophage is an organism with the highest abundance and diversity in nature, and has the characteristics of high specificity degree and strong self-multiplication capacity. A bacteriophage will generally only infect a particular host bacterium or class of host bacterium and will not destroy the structure of other bacterial populations in the aquatic product or aquatic environment. After the virulent phage invades the host, a large amount of self-propagation replication is rapidly completed until the host is cracked, and then other host bacteria are infected and cracked, so that pathogenic bacteria can be rapidly and effectively killed. So, comparatively speaking, the phage prevention method has good killing effect on bacteria and is environment-friendly, so the method can be completely used as a substitute for preventing and controlling bacterial diseases and even antibiotic prevention of drug-resistant strains. Therefore, in order to develop a phage control method capable of effectively treating vibrio alginolyticus aquatic diseases, it is necessary to screen and utilize phage which can target vibrio alginolyticus and has strong antibacterial ability or relative superiority in other aspects of antibacterial effect.

Disclosure of Invention

The invention aims to provide a safe and effective bacteriophage which can be used for preventing and treating vibrio alginolyticus infectious aquatic diseases so as to solve the problem of drug resistance of antibiotics in preventing and treating vibrio alginolyticus diseases.

Aiming at the above purpose, the invention is realized by adopting the following technical scheme:

the first purpose of the invention is to provide a Vibrio alginolyticus phage vB _ ValS _ R12Z with the preservation number as follows: GDMCC No. 61738-B1.

The invention obtains a new vibrio alginolyticus virulent phage by screening and separating the eighth market wastewater of Fujian Xiamen, determines the form and classification status of the phage through form observation, whole gene sequencing and phylogenetic analysis, and names the phage as vibrio alginolyticus phageVibrio phagevB _ ValS _ R12Z, deposited at the Guangdong province culture Collection (GDMCC) at 8/4/2021 with the accession number GDMCC No: 61738-B1, accession number: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070.

further, transmission electron microscope observation shows that the Vibrio alginolyticus phage vB _ ValS _ R12Z is a head-tail virus with a head length of 100 nm, a width of 52 nm and a tail length of 145 nm, and belongs to long-tail phage according to the standard of the latest virus classification system of the International Commission on Virus Classification (ICTV).

Further, the Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z to Vibrio alginolyticus ATCC 17749^T（Vibrio alginolyticus ATCC 17749^T）、JL2674（Vibrio alginolyticus JL2674）、BVA2（Vibrio alginolyticusBVA 2) and Vibrio owens 3186: (Vibrioowensii 3186) All have a cracking effect.

Further, the genome of the vibrio alginolyticus phage vB _ ValS _ R12Z is respectively compared and analyzed in a pathogenic bacteria Virulence Factor Database (VFDB) and a comprehensive drug resistance database (CARD), and virulence genes and drug resistance genes are not detected, so that the infection of the vibrio alginolyticus phage vB _ ValS _ R12Z cannot cause the virulence enhancement of host bacteria and the drug resistance pollution of a culture environment and a surrounding water environment.

It is a second object of the present invention to provide a phage composition comprising the Vibrio alginolyticus phage vB _ ValS _ R12Z.

The third object of the present invention is to provide the use of the above-mentioned Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z or the bacteriophage composition for killing and/or preventing microorganisms of Vibrio.

The fourth purpose of the invention is to provide the application of the vibrio alginolyticus phage vB _ ValS _ R12Z or the phage composition in preparing the medicine for resisting the vibrio microorganisms.

The fifth purpose of the invention is to provide the application of the vibrio alginolyticus bacteriophage vB _ ValS _ R12Z or the bacteriophage composition in preparing aquatic feed or feed additive resisting vibrio microorganisms.

Further, the microorganism of Vibrio isVibrio alginolyticus ATCC 17749^T、Vibrio alginolyticus JL2674、Vibrio alginolyticus BVA2、Vibrioowensii 3186 one or more of the above.

It is a sixth object of the present invention to provide a medicament against microorganisms of the genus Vibrio, which contains the above-mentioned Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z or a phage composition.

It is a seventh object of the present invention to provide an aquatic feed or feed additive against Vibrio microorganisms, which contains the above Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z or the bacteriophage composition.

The eighth purpose of the invention is to provide a method for preventing and treating the harmful infection of vibrio alginolyticus, which kills the vibrio alginolyticus by using the lysis effect of the vibrio alginolyticus bacteriophage vB _ ValS _ R12Z or the bacteriophage composition on the vibrio alginolyticus.

The invention has the following beneficial effects:

the vibrio alginolyticus phage vB _ ValS _ R12Z is a lytic long-tail phage, has a wide lysis spectrum, can lyse various vibrio alginolyticus and vibrio erwinii 3186, can effectively kill and prevent related vibrio diseases, does not detect virulence factor genes and drug resistance genes in the phage, has no toxic or side effect, high safety and environmental friendliness, and shows that the vibrio alginolyticus phage vB _ ValS _ R12Z can be completely used as a bactericide for the vibrio alginolyticus diseases of the aquatic products.

Vibrio alginolyticus bacteriophage of the present inventionVibrio phage vB _ ValS _ R12Z was deposited at the guangdong province collection of microorganisms (GDMCC) at 8/4/2021, address: building 59 of first fure middle way 100, large yard, Guangdong province, Guangzhou city, zip code: 510070, accession number GDMCC No: 61738-B1.

Drawings

FIG. 1 shows the plaque morphology of the Vibrio alginolyticus phage vB _ ValS _ R12Z.

FIG. 2 is an electron micrograph of Vibrio alginolyticus phage vB _ ValS _ R12Z.

FIG. 3 phylogenetic tree of the Vibrio alginolyticus phage vB _ ValS _ R12Z and a close canonical phage strain.

FIG. 4 is a one-step growth curve of Vibrio alginolyticus phage vB _ ValS _ R12Z.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof. The methods and techniques used are conventional unless otherwise specified.

Example 1: isolation and purification of bacteriophages of Vibrio alginolyticus

A sample of a wastewater sample collected from the eighth market of Fujian Xiamen was filtered through a 0.22 μm filter membrane and returned to the laboratory in a refrigerated state (stored in a refrigerator at 4 ℃).

In order to increase the abundance of the phage and improve the success rate of phage separation, before the separation experiment, the phage which may exist in the sample is firstly expanded and cultured, specifically as follows: to 10 mL of host bacteria (Vibrio alginolyticus ATCC 17749) in the initial logarithmic growth stage^T) Adding 2 mL of water sample filtered again by 0.22 μm syringe filter into the culture solution, placing in shaking table, culturing for 24 hr, recovering mixed culture solution, and filteringRemoving cells and impurities in the system by a 0.22-micron syringe filter to obtain the mixed phage liquid after propagation, and storing the mixed phage liquid in a refrigerator at 4 ℃.

1mL of the mixed phage liquid was taken, and gradient dilution (dilution gradient: X10) was carried out using SM buffer (SM buffer was prepared by adding 5.844 g NaCl, 2.4647 g MgSO4 & 7H2O, 6.057 g Tris, and 0.100 g Gelatin to 1L of ultrapure water, pH was adjusted to 7.50 after preparation, and autoclaving was carried out at 100 ℃ for 30 min, followed by cooling) to obtain a diluted phage liquid⁰、×10^-2、×10^-4、×10^-6) And the quantity of the phage in the sample is ensured to be suitable. Adding 1mL of host bacteria culture solution at the initial logarithmic growth stage into each diluted gradient phage solution, shaking, standing at room temperature in the dark for 15 min, and allowing the phage to adsorb and infect the host bacteria.

After adsorption, adding 5-6 mL of melted 2216E semisolid culture medium (30 g of 2216E liquid culture medium powder (Haibo organism, Qingdao) is added according to 1L of ultrapure water into the mixed sample, boiling for 1 min to dissolve the powder, cooling, filtering by a 0.45-micron MCE water-based microporous membrane (Tianjin, Temminck laboratories, Inc.), obtaining a filtrate which is an unsterilized liquid culture medium, adding 0.5% of agar powder with final concentration (wt/vol), sterilizing by high-pressure steam at 121 ℃ for 15 min, cooling for later use, obtaining the 2216E semisolid culture medium, heating and melting again before use, controlling the temperature at 50 ℃ in the use process, quickly mixing, pouring onto the prepared single-layer flat-plate 2216E solid culture medium (adding agar powder with final concentration (wt/vol) of 1.5% into the unsterilized liquid culture medium, sterilizing by high-pressure steam at 121 ℃ for 15 min, pouring the culture medium into a culture dish (Biosharp, China) with the diameter of 9 cm before cooling and solidifying, and cooling and solidifying for later use to obtain the solid culture medium). And (3) after the upper flat plate is cooled and solidified, inversely placing the upper flat plate in a constant-temperature incubator at 30 ℃ for culture, and observing whether the plaques appear or not at regular time after 3-4 hours.

After plaques appeared on the double-layer plate, the plaques were carefully observed and recorded by morphological size classification, and individual plaques were picked with a sterilized snip tip and placed in 1mL of SM buffer. The plaques are stored overnight at 4 ℃ in the dark to allow the phage to fillThe fractions were dissolved in SM buffer. The resulting phage extract was diluted in a gradient (dilution X10)^-2、×10^-4、×10^-6、×10^-8) The double-plate experiment was repeated to purify and pick individual plaques (FIG. 1) to obtain a single class of purified phage.

Example 2: expanding culture and enrichment of bacteriophages of vibrio alginolyticus

Adding 1mL of pure phage liquid into 10 mL of host bacteria (Vibrio alginolyticus ATCC 17749) at initial logarithmic growth stage^T) Culturing in a shaking table at 30 deg.C. After the bacterial culture solution has been significantly lysed (i.e., the culture solution has recovered to be transparent and has floccules with cell lysis residues), the culture solution is recovered and filtered through a 0.22 μm syringe filter, and the filtrate is retained. The filtrate was added to 50 mL of host bacteria (Vibrio alginolyticus ATCC 17749) at the early logarithmic growth stage^T) And in the culture solution, continuously culturing until obvious cracking phenomenon appears. The filtration process was repeated, and 200 mL of host bacteria (Vibrio alginolyticus ATCC 17749) at the initial logarithmic growth stage were added to the filtrate^T) The culture is continued in the culture medium. The culture system was further expanded to 1L in this manner.

When the 1L culture system has obvious cracking phenomenon, adding DNase I and RNase A (Takara, Japan) to the final concentration of 1g/L, uniformly mixing, and digesting at room temperature for 30-60 min to degrade free host DNA and RNA in the system. NaCl was added to a final concentration of 1 mol/L, and after sufficient solubilization, the phage was placed at 4 deg.C (or ice bath) for 1 h to allow the phage to fall off the cells or debris into the liquid. Centrifugation (5000 rpm, 4 ℃ C., 5 min) was carried out, and the supernatant was filtered at 0.22 μm to remove cells and a part of debris from the system, thereby obtaining 1L of phage liquid.

Adding 10% (wt/vol) PEG8000 (living creature, Shanghai) into the phage liquid, mixing to dissolve the PEG8000 completely, and processing at 4 deg.C in dark for 1-3 days. The PEG and the biomacromolecule are fully combined and aggregated to form polymer sedimentation. The precipitated sample was centrifuged (12000 Xg, 4 ℃ C., 1 hour) and the supernatant was discarded, and the remaining precipitate was the phage precipitate. To this, SM buffer was added to resuspend the pellet, and the phage was redissolved from the PEG pellet in SM buffer and transferred to a 15 mL centrifuge tube. The phage was placed at 4 ℃ in the dark for more than 12 h to allow the phage to be fully resuspended in SM buffer. If the suspended sample has more impurities, adding equal volume of chloroform (for removing lipids, cell debris and other impurities), slightly shaking to make the two liquids fully contact and mix uniformly, centrifuging at low speed (5000 Xg, 4 ℃, 5 min) for phase separation, and taking the supernatant to obtain phage concentrate.

CsCl powder was dissolved in SM buffer, and the solution density was adjusted to 1.65 g/mL, 1.5 g/mL, 1.4 g/mL, 1.3 g/mL gradient solutions. Add slowly 6 mL of the density gradient solution to the centrifuge tube in sequence, add all of the phage concentrate above the density gradient solution, fill the centrifuge tube with SM buffer and weigh down. The density gradient centrifugation was carried out at 200000 Xg at 4 ℃ for 24 h. After the centrifugation is finished, a blue or white bacteriophage strip can be observed to be the bacteriophage pure particle, and the strip is carefully taken out to be arranged in a centrifuge tube. The removed phage bands were transferred to a 30 kDa ultrafiltration tube, centrifuged (5000 Xg, 4 ℃, 5 min) for dialysis, and washed with SM buffer to remove CsCl. After the completion, the phage solution was transferred to a 1.5 mL centrifuge tube to obtain a high-concentration enriched phage solution sample, which was stored at 4 ℃ in the dark.

Example 3: morphological observation and identification of vibrio alginolyticus phage

And (3) dripping 20 mu L of concentrated phage onto a 200-mesh copper net coated with a carbon film, carrying out dark adsorption for 10-30 min, carrying out negative staining for about 20 min by using 1% phosphotungstic acid, drying for more than 30 min, observing a sample by using a JEM 2100 type transmission electron microscope under the voltage of 120 kV, and collecting images by using an GATAN INC CCD image transmission system.

As shown in FIG. 2, the Vibrio alginolyticus bacteriophage has a head length of 100 nm, a width of 52 nm, and a tail length of 145 nm, and is flexible, and belongs to the long-tailed bacteriophage according to the standard of the latest virus classification system of the International Commission on viral classifications (ICTV). Combining the genome represented by the nucleotide sequence of the phage (as shown in SEQ ID NO. 1) and its position in the phylogenetic tree as shown in FIG. 3, it was named Vibrio alginolyticus phageVibrio phage vB _ ValS _ R12Z (deposited at the Guangdong province Collection of microorganisms at 8/4.2021, accession number GDM)CC No: 61738-B1, accession number: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070).

Example 4: chloroform sensitivity detection of Vibrio alginolyticus phage vB _ ValS _ R12Z

Mixing 0. mu.L, 20. mu.L and 200. mu.L chloroform with the purified phage vB _ ValS _ R12Z solutions with different abundances respectively, and adjusting the phage abundance to 10³、10⁵、10⁷、10⁹ PFU/mL, mixed and shaken for 30 s, then left to stand at 25 ℃ for 30 min. After phase separation by low speed centrifugation (5000 Xg, 45 min), 10. mu.L of supernatant was carefully taken out and dropped on a previously prepared host bacterium (Vibrio alginolyticus ATCC 17749)^T) And (3) performing static culture on the double-layer plate in an incubator at 30 ℃, setting three parallel samples in an experiment, simultaneously replacing phage liquid with SM buffer solution as a negative control, and regularly observing whether the plaques appear. The chloroform sensitivity detection result of the vibrio alginolyticus phage vB _ ValS _ R12Z is shown in Table 1, and the phage after chloroform treatment still has obvious plaques, which shows that the phage is not sensitive to chloroform and has no lipid structure.

TABLE 1 detection results of the sensitivity of Vibrio alginolyticus phage vB _ ValS _ R12Z to chloroform

Note: "+" indicates the presence of plaques by infection and "-" indicates the absence of plaques by infection.

Example 5: one-step growth curve of Vibrio alginolyticus phage

The concentration of pure Vibrio alginolyticus phage vB _ ValS _ R12Z was adjusted to 2X 10 with SM buffer⁶PFU/mL, using RO liquid culture medium (1L seawater after 30 kDa filtration added with 1g yeast extract, 1g peptone and 1g sodium acetate, adjusting pH to 7.4-7.8, sterilizing with 110 deg.C high pressure steam for 30 min, cooling for use, to obtain RO liquid culture medium) to treat host bacteria (Vibrio alginolyticus ATCC 17749)^T) Cultured to OD₆₀₀And = 0.2. Mixing 1000 μ L host bacteria liquid and 100 μ L phage sample, and standing in dark for 5 minPhage-infected bacteria were centrifuged (4 min, 8000 Xg, 4 ℃) and 1mL of supernatant removed, followed by addition of 1mL of RO liquid medium to resuspend the pellet. This step was repeated twice to remove free phage, taking 20 min. Immediately after the second resuspension, 1mL of the mixture was added to 100 mL of RO medium and shaken, 3 replicates were set (30 ℃, 160 rpm), phage samples were collected 1 time every 10 min, and phage abundance was determined by the double-layer plate method.

As shown in FIG. 4, the latency of Vibrio alginolyticus phage vB _ ValS _ R12Z is 10 min, and the lysis amount is 200 PFU/cell, which indicates that the phage is a virulent phage, and the latency is short and the lysis amount is large.

Example 6: host range detection of Vibrio alginolyticus bacteriophage

Marine planktonic bacteria and aquatic animal pathogenic bacteria of various common species of Vibrio are selected (Table 2), includingV. alginolyticus(3 strains),V. parahemolyticus(7 strains),V. owensii(4 strains),V. campbellii(2 strains),V.harveyi(2 strains),V. chagasii、V. cholerae、V.fortis、V. inhibens、VVariabilis. Culturing vibrio to be detected to logarithmic growth initial stage with 2216E liquid culture medium, and mixing with melted semi-solid culture medium to prepare double-layer flat plate; 10 μ L of sterile high concentration (10)⁸ PFU/mL) Vibrio alginolyticus phage vB _ ValS _ R12Z is dripped on the solidified double-layer plate and is statically cultured in an incubator at 30 ℃; and observing whether plaques appear or not at regular time. Three replicates were set up in the experiment with Vibrio alginolyticus ATCC 17749^TThe test strain was replaced with SM buffer as a positive control and the phage liquid as a negative control.

As shown in Table 2, the Vibrio alginolyticus phage vB _ ValS _ R12Z has a smaller host range and can cleave 4 host strains out of the 23 target host strains, respectivelyV. Algynolyticus ATCC 17749^T、V. Algynolyticus JL2674、V. Algynolyticus BVA2、V. owensii 3186。

TABLE 2 host Range results for Vibrio alginolyticus phage vB _ ValS _ R12Z

Note: "+" indicates an infestation, and "-" indicates a failure to infect; and V.alginolyticus ATCC 17749^THost strains for isolation experiments

Example 7: genomic analysis of Vibrio alginolyticus phage

Firstly, extracting DNA by adopting a phenol-chloroform extraction method, and specifically comprising the following steps:

to the suspension of Vibrio alginolyticus phage vB _ ValS _ R12Z, 1.5. mu.L of 100 mg/mL proteinase K solution, 100. mu.L of 10% (wt/vol) SDS solution, and 10. mu.L (0.5 mol/L, pH = 8.0) EDTA solution were added, mixed, and digested in a thermostatic water bath at 55 ℃ for 3 hours.

Adding an equal volume of phenol-chloroform-isoamyl alcohol (25: 24: l, volume ratio) mixed solution into the digested phage suspension, mixing uniformly, centrifuging (12000 Xg, 4 ℃, 5 min), and transferring the upper aqueous phase into a new centrifuge tube; this operation was repeated twice.

To the collected upper aqueous phase solution, an equal volume of chloroform-isoamyl alcohol (24: 1, volume ratio) mixture was added, sufficiently shaken for 30 s, and centrifuged (12000 Xg, 4 ℃, 5 min) to collect the upper aqueous phase solution. Mixing with isopropanol liquid of the same volume, mixing, standing at-20 deg.C for l h, centrifuging again (12000 Xg, 4 deg.C, 5 min), collecting bacteriophage nucleic acid precipitate, washing with pre-cooled 70% ethanol solution twice, and air drying.

The nucleic acid pellet was dissolved in 100. mu.L of TE buffer (10 mmol/L Tris-HCI, 1 mmol/LEDTA, pH = 8.0) and stored at-20 ℃ until use.

The phage vB _ ValS _ R12Z whole genome was sequenced using the Illumina HiSeq4000 platform with an end read size of 150 bp. The raw data were collated with trimmatic v0.32 and phage genomes were assembled with Velvet v1.2.03 software.

Virulence gene detection of bacteriophages in the pathogenic Virulence Factor Database (VFDB) and resistance gene detection in the integrated resistance database (CARD).

The dsDNA of the vibrio alginolyticus phage vB _ ValS _ R12ZR12Z is circularly arranged, the genome size is 32827 bp (the nucleotide sequence of the genome is shown as SEQ ID NO. 1), and virulence genes and drug resistance genes are not found in the phage genes.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Sequence listing

<110> Nanhai ocean institute of Chinese academy of sciences

<120> Vibrio alginolyticus bacteriophage and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 32827

<212> DNA

<213> Vibrio alginolyticus phage vB _ ValS _ R12Z (Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z)

<400> 1

aagccccacc ccgtctgggc tgggactcca tcggagtgcc ggacggattt tgtctgccac 60

gtgttgggga ccacgaccaa cgaccattta ctaatggacc tcgaccacga ccaccgggac 120

catcggacaa tgaccgttag taaatggacc attggaccga aggggtttgt aaattatcat 180

gggggtgtcg tatagttcgt ggaccaattt tttccgggac cagttttgaa gtttttacta 240

aaacggagga ccagatgacc agaccgaaga ggagattgat tgtagtcgag gaccgatgct 300

tccggggaca tgcccccatt cgaattgtgc ggggctctgc gaaggcttct gtatgctatc 360

aatgtcatcg tatgcaaaga aagcacgcac gcatggtagc acgctgccac aacccaaagg 420

acccaaggta caagaactac ggagggcgcg ggatcagcgt atgcccagaa tggcgtaact 480

catttactaa atggtatgag gacttcggct acctcgtaga tgggaatgac ctgactatgg 540

atcgtaggga taacgatggg gattacgagc cggggaatat gcgtgcagcc actacccaag 600

agcaaaaccg caaccaacga agaagtgttt tactaacgta cgatggggag acgctgaatc 660

aacaggtgtg ggcagatagg ttcggctttc ataaatccag tatacagcga cgccttggtg 720

ggaagtaccg gaatgatatg gaatgggtac tctttggcgt acgcactact attactaaaa 780

cggaggtact gattactaag gacaggaagc ggttgaactt gcgtgcttgg tgtagatcgc 840

tgggtttctc actacgcaca gtagaggacc gggtatacaa gttcggctgg agttatgcgc 900

aggcactagg ctttgcctcc cggaagggaa aacttagacc accgcctaca aaggattagt 960

aaataatagt tgcagttagt aacactgtgg catatactct ttatacacca actaactaag 1020

aaggattagt aaaatgactc cagaacagaa acacctagaa gacaagctta taggggtgga 1080

ccgtaactgg cgtggaactg ctaccaactg cctctactca ctagagtcta tgatgcgact 1140

actagataat ggcgtatcag agtacgtcct taagtgtcag gtacacaaca tgatggcttg 1200

gggtctagga cataacgctc agcaggcggt agcacttgcc tactcaatgc tgcacaacga 1260

tggcacctca atcccaatgg tccgggagtg cctagtgcga gcgatctctc agcagaaaca 1320

atacatccat acacttacta aggaggatta gtaaaatgaa ggacttcaat atgagcgagg 1380

agcttactcg cgtacaggct ggctggtatg agcagtctaa ggacgccgtg tacaacttga 1440

aggatgttac taaagcactc acagacggca agtcgctaaa gcattgtaag cgcatggtgg 1500

aagacatccc cggtattggt ctctcccaac gtacgcagga atatttgcag gtggctttga 1560

actgtaaacg catggagaac tttagtaaat gcttgttcaa ggcttatcaa attcaaacgc 1620

gtaatgcagc aagtctgaag aaggactcgt agaatggtta aaccgacaca ccgcacactg 1680

gtagtaactt ctaagggtcg tgtaccctac gatgagaagc attactacca cactggcaaa 1740

aagggagagg atgttacgtt agtaaacgag cgtaacttca aaacggtaca gggacagcga 1800

ttcgaccttg tacttttcgt tggcaacata tccggggaag ttcgtacgaa gttcgctggg 1860

ttggttggag attactaaaa tgaaacgcaa gtacttcgac gaagctttac catcgttctt 1920

tgtgttcggg gagcacgcta acggtacggt tgacctatcg tgtggtgagt ttgatgttgt 1980

aacatactgc cctacagaaa tggcagaagc cgccatagct tactacgaca gcctgctgct 2040

taagctgcat gagtgggtag tcgatcagga gaaaccctcg gaagccctcg acaaactctg 2100

tgacgctttc caagatgttc acgcggggca tcattacatt ggcgacttcg gggctgtaat 2160

gttactaaaa ctgctgtggg aagcgtacac caagatagcg acaatcgtcc atccgagatt 2220

cttacgtagc gtgtacggtg tagagtggac cccatcactg gtgcaggcga acttactaac 2280

gcaacgtggg tacacccctt actgtggtgg tcgtgactgc aagtcggtcc ctcgcacagc 2340

gttccgggag gagcagttcc gctgccctag ctgcggttgg gagtcgaact ttgaccctga 2400

gctcattgct gggtacaagt acgtgtggga cttgccggga gttgactgcg agatgctaga 2460

cgagttccgt gcaatactac cactatcact acacttcgta atggtagacg cggacatgcg 2520

atacgcgggg cgtattgatg atgagtagga agcgtttacc tttagtaaaa cgtgcaagcg 2580

tacattgggc tcagctcacc gggattgtcg taatggaccc ggatggatgg gaccgccaga 2640

acttcgaata cagctggtat gaagagacca ttaccattct ggagttctgg gatcgcgtgt 2700

acaactcaac aacactcatg aaattggaga attagtaaaa tggaaagttc atttggttgg 2760

aaccgtgtat tactcgtagg cgggacaaac gcgggcacgt tcatgggcgt gcttcgtgac 2820

cagcaggtga tacgtatgga gaagaaagac cccaacgtac aacgccttgg aacacacgtc 2880

ctacaacgtg agcaagcccc ggatgaaaca ttcgctaccg aggagtacgt agccacagaa 2940

cacgccttac gagggactac gatctttgta ctgcgaggaa tggctggtgc agacatcccc 3000

tgcgagatgt tgaagatcat cgctaagctg gatagtaaga ttggtgagct aaattctagg 3060

atggagcttg ctaatatagc cgcgatgaac gttgctcact acgtgaagag agaccaataa 3120

tatgcgtatg accattatag cgtgtgcggt taagatacac taccgtgtcc cggtattgcc 3180

ggagatgccg atccttatcg ggaataccag agcattcgtg cagggtcgtg ctgctggtat 3240

tgggtttagt aaccgcgtcc actacgacgt gtccatgccc aagttcgtac gctcagctgc 3300

tacgcaagca ttcggacgcc ttgaccgcga gtttaaggaa gccacaatag accttacgga 3360

ttatcaacat gcttggctgg aggtgagccc caaatgagcg tagaagctgt tttcgagcag 3420

gacatgcagt tagaccctga tatacgcaat agcaacgttg caggggcttt ctgcgtggct 3480

aaagcgaacc tttgcaacgc ctgccagtgg gacggtacta tcgacgctgc tatgatggtc 3540

ctgttgttca tgggcgttac taatttcgac gacgacctga taaagcgttc tgtcgagctg 3600

ggctacttta gcatcaaact catgggtatg gttgatccta tccgtgtaaa ccggggcgat 3660

gtgctcgtcc gatcatctac gagtttcgtg gtggtaacta acgaagagta caatcgcatt 3720

tttactaata tactgttata ggaggacgct atggctccct tcttcgtttt cgcacaaaca 3780

gcaaacgtga tggttaatac ctgccgtcgt atgggcatct caccgacgga accacgcctt 3840

atccgtgaac cggagaactt aaagggcgtg agtttcccta aagagggtgg taagtacatc 3900

atcttcgtct gtggtgagac tgttccactc tggctccacc aatttgctgt gctgaacagc 3960

gtattgctgc tgggcatgga caagcacgtg atcgagtact accgtaagca ggacttccgc 4020

gtgaatgact acaacgctta cgaacaatag accgtatagt ttactgcaac atccatttta 4080

ctaaaattaa aggagctgaa aatggactca cgttacacct ttatcgacct agtcaaagac 4140

gtattcgcag cacttaagcg tgctaaagcc aacggtgtta ctttcaaagg tcaatctccg 4200

tcagctgtag cggctgacat ggctaagtac gacgctgact tagagaatgc cccggtagaa 4260

gacttggaga aggctgtggg gcttgtgcag agcattgacg gcacgtttaa gtcaggtatc 4320

gacttggaga aggctgtggg gcttgtgcag agcattgacg gcacgtttaa gtcaggtatc 4380

gaagagaagc ttgactcaat cctaacaccc tctgcggtga tcatggattg tgacttcgac 4440

gacatcctag aagataccga agtggcttac tgcgtcgatg caaacctaga cctgctgacg 4500

gttggtgcgt acaatgccaa gggcggttgc tgcgagtgct gtcgccctta ctaccgtgaa 4560

tccgtctacc tactacgcgc tatctgtatg cgtactggtg acgacattac taaaactgtt 4620

ctggaccgca aagcggctaa gaaccaagtt ggagacaagc aataatgcaa gtaccagtca 4680

acatcacagg tccagtggga tcacttgccc tagaatacat cggcggtaac gctgataagg 4740

tgattgagtt cgttaagaac tgcaccggga acacgccgac gtttaaagag ccggatgtgt 4800

tggagtaccc agttatcggt ggtacgcaga tgatcaccgt tggcagctgg ttggtatggg 4860

ataccactaa actacactgt atggatgaag tttactttag caagaacttc acgatcatct 4920

gggaacatcc acagcagcct gtcccggtgg acgagccaga aggtcgcgct atcgtctcat 4980

acgcggtcaa ccactcgact atcgcggaag acttggactt ctaccaccac aacgagtttg 5040

gcacgcgtaa ggtacgctgt atcaagtcga actacggtgc tgaaggtcat ccaatctacg 5100

gcttactaac gctagtgtgg ttagatggtg acgtggctat gtcgatgcct gttggtgagc 5160

tgggagacac agatgagttc tcaatggatt acctcggcgc ggtagaagct gctaagcaag 5220

ggcatcgtgt gtgtcgtaag ggttggaacg gcgctgacat gtgcctagtg tacgtgccag 5280

cggcagacat cccaacgaag ggaacaggtc ttactccgtt cttcggtgat accatgccta 5340

tgcgtgctca ttggttacta aaaaccgcca caggcgatgt agccagttgg tccccatcga 5400

cgtcggatag tctagcgacg gactgggaaa tctactcacc agaataattt tactaaaatg 5460

tattgactat aagaaacagg gggaatatac ttctccctgt tgtcgttttt actaacccaa 5520

aaaggagttc aacatggata acgttatgcc tcaccctatg ccaatcacct acagccgacc 5580

tgagcatcaa cgtcacgcta ttcagtttac taaaaataat gttggagcgt tgatccaagc 5640

cctgatccac ctcgacatcg tagagacctt gggtcttggt gaagaagctt ccgacgcggg 5700

ctttggtcgc cgtgagcgcg ttgttaagcc gtgtctttca gcggagctta gagattcgtt 5760

cttcgagcag ttcccacacc gtgcgaacac gtacgaggaa aatgagttct ctatctttga 5820

gggggagttt ctggtgcttc taggcgctgg tgatttctta gtgatgtcag agcaggagtt 5880

caactcaaca tacaagtggg gtcaggacga gcctgcgcag ctagcaccgt gtgcgactcc 5940

atcgcaagat gacttggcgg ctattgccgg ggaccgcgag ttctctgagc ctcgtgtagc 6000

tgcgctggag ccggaggtgg ctatcgctgt ggaagaggcg ctggaagaca ttgccgagcc 6060

aattgcagag ccagaagagt acggcatcga agagcgagag gagtctcctc gtgcaaagcg 6120

tatggctaag cgtaatcgtc gtgactagca ttgctagtac gtttttacta atctaataat 6180

caggagaaga gttatggact ctactttact aacagtaaca ccgaacaagc ccttccgtgc 6240

cgtgcagtac aacggtagta actgggcgca ggtacgtcag tttgctgcta agtttgctaa 6300

cgtagaaatc gcgatgaact tgaaagccga cagcccacta cttgcgcacc ttggcacagg 6360

caatatactc atccctaaag ggaactacgt agttgcttgg ggcaagtggg acctgcgcac 6420

gtattcgccg gaagagttta ctaaagtgtt tgctggcatc ccgtcaggac gtgaccaact 6480

acttgaggtt gagatggaca atgaccgtat cgtgatgtct attggtgtgg aagccatgcg 6540

catgagcatc gagtcgggtc aggcagactt cagcgtaggc ggcgcagtcc acgttgcaga 6600

cgtagacagc tttgggcacg agctgatcag ctacttgaaa gctgaagctg aagacggtgg 6660

tactccagtt catcgcatga tcgatagcgt agcgttggaa gcactagagc atggtgctca 6720

cggtatcatc gagttggacg aagaagactt agagcgagac atctaatggg cgtagtaact 6780

gtacagcgga ccaatccgat catcgcgctc caattcaccg gagataacca cgaagaggtg 6840

atcgagttca tgaagcaacg cggcgggacc accgtaacga tgcagtatga caatttacta 6900

aaaattgaat ccgactgctt cctatcatgg actatccgtc agggtgattg gattatccac 6960

gagaaagggt ccgtgtgtga ggtcatcttc ggagacttct tcccggatgc cttccgcgtt 7020

atcgagaacg gaccgctgtt tggtaagaag gtggtcatct ctggtacttt cggcaaggta 7080

actcgccacg ctgtaaaaga cctcctatct gctcgcggag ctaaggtgtt cggcagtgtg 7140

aacaagttca ccgacatatt ggtgtgtggc aaggactcag gctctaagct taccaaggct 7200

aaagagctgg gaatcagaat catctacgag tcggagattc tggaactact ctagcacttt 7260

tactaaaagc ctcctaacgg gggctttttt gtgtctgaag cattgacatg tgaaacagtg 7320

ggagtatact tcttagtaaa accaacgagg attaacaaat gcgtattatc tgtaaaatgt 7380

tcggtcacga cactccgtgg aaaactcccg cgagtgtaag ccctatgtgg actcctaagc 7440

aagtctctca agacatccta tccggcgaag tccgcgtaat cgacccaatc tataccgggt 7500

acatgttcca aggctacgag ggggtgtgtt cgcgctgtgg gcagactgtc ccggtatatg 7560

ctgagaacgt gccgatcatt gagcctagcc tagttcccac cgacgacgaa gagctactag 7620

accttaatcg tgaacaccgc cagctgtacc tattctggtg tgatgttact aaaaagcacg 7680

ctgatgcacg cctgtacctt actggtaggc agctaaagta cttatggcgc tgtatctaca 7740

aaattactaa agacaacaag aacctgagcc cgacggagtc cgatacttgt aactctatcg 7800

tgttctcgta ctggaaagcc aagaagagag gtaacagatg aaaaagttaa ttatagtact 7860

agcggtcaca tcactatccg ggtgcgcgtc cccggaacgt ctacacaatt tgaagtgtga 7920

ccagtgggtg tacgaacaag ttatggagca tggggaggac tacttcgatg ccgtgaaaga 7980

gtgtggctat aaaaagatcc ctatgtcgga cccactagac ggggcgtatc gtgtctacag 8040

cgttgggggt ggttactaat ggaagccatt tactacttca gcgactggcg tcatttttgg 8100

atgtggcttg actggaactt gtatgctaac ggcaatctat tcctgatact ctgctggtct 8160

tggatcttgg cgtcgagcct aatgggactc cgtgtcaagc tctggcaggc gctctgtaac 8220

atcctcgctg tatgccttac gtggatgttc gtttgttatg tatcctacgg aaccggactg 8280

gggtgaaaaa attactaaaa agtgtttgac actcatctga aacaacgtta ctatcctccc 8340

tgttgaatac atttactaat ctactaactc agcagggagt tttactaaaa tggctactat 8400

cactaatctt caaaaagtta tccttcacgc agttgctact atcaaagact gcacaggcta 8460

caacgtagct caccacttca tgcccgctaa tgacctccac tggaccgcct ctcaccagca 8520

ggtataccgc gaactacgcc gtatggagtc tgcggaccta gtggacagcc gcgaagtccc 8580

acaagacggc aagcctaaca agttcgtgta cactctgacc gagaagggta agaaagcgta 8640

tcaagaagct atcgcccacg agccatgcga ctacgctggt cttaacaccc aagctaccat 8700

ccacgcgctg ttcccaactg tcgagtacta cgcggcgttt ttgcagaagc gtaacgaaga 8760

gtgtgacgcc ctgtacttcc gcaagtgtga agacggtgtg tctaagctgg aagagattct 8820

tattgaccgt cgaatcttga tcctacatgc cgagtgtaca ttcgcgcaga agatgatcgt 8880

aatgctgtct gagcagcagg aagcgattga gggcgaatct gaagaagtgg tcaagtagtt 8940

tactaaactt aaaggagcag gcgtcatgcg tttcgagact atcaatattt accacaaata 9000

cgggaagtgg atcggcggtg gtcagctaag tagccgcagt ttggcggagc tgctttccgc 9060

tgcggtaaaa cctcaccaac gcgtggtctc aatggtttac catgagaacc acggcatctc 9120

acccaactac gacatagtcc tagaaaacca cgaggtgcag gtgctggaca acgtgcatga 9180

gaaccacacg tactacgcta acaagtgcgc taaggactgg caggaggatt ccctgttgaa 9240

cccagaagcc gaggctacgc aggctaagcc agcatcactg tctcgtagaa agtaactact 9300

aagccctcgc actgccgggg gcttttcgtt atactccacc gcaaaataag gaggtaataa 9360

tatggcttta tctgacgttt ttgacgccgt gtacggaggt ggttttagta cgccgggcaa 9420

ctttgtggct ggcgacgtgc tcgaccagat cgttgcctac accgaaccat cacgctcgga 9480

ggctggcagc acgctgtcat cgctgtacac gaaataccca aacttcaaga cgacctgtag 9540

gacggagctg ccaaagcaca tgcgcgttac tagcggtgag atcagcacta tcttctccca 9600

gttggacagt aattccgcag cgacactctc cacccttgct aatagtatcg cactgaagtg 9660

gattgagcac gtaaacgaca ataggcagct ctaatccgta taattactaa aaacggagat 9720

taacaatgcg taaagaagta tcctgcttta gtaagagtgg aaagctcact aaagacatga 9780

aagttatttt agacatagcc accactatcg agttggcaat cgtaaacaat cctgacaaag 9840

accgaatcca agttattagg gaaacggctc gacgtcttaa gcaatcaccc cagtaccgaa 9900

agctgtgggc acagtcgatc ctaaacaagt acctgaacgc cgacgaaacc gtatccaagg 9960

cacagcagga cactatcgac agcctgttct cgctccgggc ggctgggtac aaaacttact 10020

aaggagattg atgtgggagc acttcctaac aacaaccgca aggagtcgaa cgactcacca 10080

gacctttacg ttactaacca cccactggct attccggggc tgttctcccg ctggcgtcca 10140

gagagcggtt ctatcatcct tgagccctgc tgcggacttt gccacatttc ggataagctg 10200

gaggagttcg gtcacgtagt acacagcttc gacatgttta actgggggcg tggtaacgct 10260

cgtgaagagt atgacgctca gtcgtaccat tacggtgagt atgagtgcat tattactaat 10320

ccaccctaca accgagctat gccaatcgta gagaacctac tggagcagaa tctggctaag 10380

ggtgggtcta tggctatgct ggtacgtctg gagtttcaga cggggaaggc acgagcggcg 10440

gctcttgagc agaccccttt gaagtacatc ctaccgttcg cattccgtat cgagtgtgac 10500

aagggtgtag tcgtggagga gctggcggac ggctcttttg agacagagtt ggagaaatcc 10560

ccgaactcat caaactacgc ttggtttgtc tgggaggatg gttacgaggg ctaccctact 10620

acgatgtaca tccaccaaga catggctgag caaacactgg cagaacttac taatagagag 10680

gtttagtaaa atggaaaata ttccggctaa gcagatagct gacgacatcg aagagctgtt 10740

ggagaacgct gctgtgtaca ttcagcgtca ggttaatccc atgctcttga agatgcgtac 10800

agagcagtgc atcgaccttt acggatttgt gatggatagg gactccacgg ctactttctt 10860

ccactgcaat gtacctgcgg aggagatacg cggtatttac gccagcacgg gcgaggtact 10920

gatccgtaaa gaagtaccgc taacgcatgg tcgtttgtat ccgtcactgg ctctggctaa 10980

gaaccgtgct agtcaccgag tggctaaccg tattgagaag ctgcgagcgt acagtcgatc 11040

actgtcaaca gtcaccacga acgacgtcag caccatccaa gttaagtagt gtactctacg 11100

atgcaggggc ggcacgtctg cccttgcatt gtgctaaaga gttgaacgct gtgtacaacc 11160

acaagagtga atttttagtt agacgtatat ttgtatgaac cattttacta agagtaaggg 11220

agatgataag acgagagaga agaacggctg atcagcctgc cattatgtgg gaatgggaag 11280

acctaaagga caaccctaat aaagtcgtat ccgcactact tacaagctcg tacctgtatt 11340

acctgcgcgg cgacttacgt cccataatgc gagacgaaga ctttgaccta tgttgcaaat 11400

tactaagacg tagataccgt gaagtaacgc acatgcacaa gtccttgatc aagatgtccg 11460

acctacgcgc cggaaccttg tttagactcc gggaccacga ttacccaacc atcacgaagg 11520

tggtggctgt agaattgtct ctggggacga ttaacgacat gaggctacca acgcctcaac 11580

ccaccgttaa aaacgccacc acgagccgga gaactcgaaa taaagctgct cctaggcggg 11640

agcgtagtcg caagaagggg tttttataag atgactatca ccgggacgct aattatccta 11700

ctcctcatga tggaggcgct atcgtgggga ttctgggtac tatggcgtaa gttagtaaaa 11760

tcaggacgca ttcctaccat caagtttaat aagcgccgac actggtgtga ccttaagtac 11820

gtctacacgc gtgctgagtg gcgggggcat aggactccgt tcgtatgggt tactgagcgg 11880

gagtatccga taccagagtt tactaagaac cacccggacg agctggcgac agcggcggtt 11940

cgggactttg ttgcccagct cataaacgat agtgagttcg tactagatgc cgccgataaa 12000

gaggagatgt tctcgcagta cagtatctgg cttactaaac atgcaatgcc ggacaacggc 12060

tacgcgatca tagaagactg gcgagaccag ctaaacccag cgaaggatgg aattgacgat 12120

gaataacgat aaagtgaagg aattactaat tgagcagtac ggggcgctat tagaggagcc 12180

aacagatttc tttatacagc ctagagatga tgagttcttc ttcgagctaa acaacactaa 12240

ggcgtattca atccgtaacc cgtgggatgc gagccagccg aaggtcccta cggtggacac 12300

ctgcgttacc gctttcggtg agctgtgcgc agctgctggc attttcctaa acgaaactgt 12360

gcagggtacg gaatttgtgg gagacggtac tggtaagttc gacttcctaa aaatcccagt 12420

gcgcggggtg atcgtggcta tcacggttat cgaagctcca tcctcgcttg gggctgtgca 12480

agttatcggt cagactgcgg atcgttgctt tgctctggtt acggtctacc gcgaagactc 12540

acagataaat agtcagggca atgagttcat cgctcagact atgaatctga agactggcaa 12600

agtacacaca tgcctgttcc gtctagctgt cgctgaagac caagacacgt tcccagtgta 12660

cgttccgaag atggtgtcag tgaatggcta ctacaacgcg caggaagccg ttgacaacca 12720

agctgaagac gttaagaata tggtcccagt ggctgacttt actgagtagg tcactaggcg 12780

aaccggactt ccttcgggaa accaaagccc tcacatctag cggtgtgggg gctttttttg 12840

tatcctacgg ctgacaattt acttaacgag gactgcgtat ggcagacata aaggagggcg 12900

tgcgttcgaa actagaccta tatgaagtgc agctagggcg ctgcttttac tgctgtaaac 12960

cgatgtctcc gggctcttac caaccgagcg tccgtgccaa cggctttact agggaccact 13020

ttataccgaa gtgtgagggt gttcccggat ttcacaatat tgttttaagt tgtaagtact 13080

gcaacgagaa gaaggcagac aaggagcctt caaataaggc tctggctaaa tacgagttat 13140

tactaagagt aagggctgtg ttccctgacc ataatcacaa agactcggta cggctttctc 13200

gttggaagat taagtacttt taagtttagt aaaggagact atatgagttt ctggcgtaaa 13260

gaagagccag tgatggacga gaacggcaaa ctgatcaaag gcggcatgtt tgcccaccaa 13320

cgtgagtttt gggaatcaga gagtttcatt accgcgttag taaccgggta cggtggtggg 13380

aagaccttta ccgcaggcaa gatcagtatc tcaatggcac tggagaatcc cggcataccg 13440

tttatgtgtg tgtctccgtc gtacaaggta gctcgtaaga cgatggtaat tactatcaag 13500

gaactgctgc aaggtaagca gtctttgcta gagggtttta gttggaagta caacaaggcg 13560

gattgggagt tcttgatccg atacaaaggt cgtgagggta ttatctggat cggctcaggc 13620

gatgacccgg acgcccttaa aggtcctaac ttgtgtgggg ctctgattga cgaaccgttc 13680

atccaatcac gcgaggtatt cgagcagatg cttgctcgtg tccgtcaccc taacgcccgt 13740

actaaacgga tcatgctgac aggcaccccg gaggacttga actgggggta cgacatcact 13800

gagggtgagg agaaggagaa cttcgacgta cacttggtgc aggcatcttc gaaagaaaac 13860

aaagcacttg gtaaggagta cacggaacgc cttgagcgtg gtcttagtgc tcaggctgcg 13920

gagtcgtacg ttagtggtaa gttcgtaccg ctggctaagg gtcgtgttta ctacggcttc 13980

actcgtgagc gtaacgttat ggagttcccg gagattccta aaggagctaa gattggattt 14040

ggtctcgact ttaacgttaa cccgatgtcg gcggctatgt tctggtcttt gaatggacac 14100

atccacttct ttgacgagat cttactaccg aactcggaca cctacgacat gtgtgagaca 14160

atattggctg agtatggaca gtaccgccct atctgttacc cggatgctac cgggcgtaag 14220

cgtcagacta acgccgctgg cggtatgacc gacttccgta taattaggga tgagtacaag 14280

atcaagattg acgttggttc taccaacccg cctaagcgtg accgatacaa catagtaaat 14340

ggcaagctga atcctaagaa gggcttgcct actatgacta tctcaccgaa gtgcgtgaag 14400

atgatccgtt acttagagtc gtactcacac gagaaaatga agcaacaaga agagatgtcc 14460

cacatcttgg atgctatggg ctaccctgta gcgaggatgt tcccgctaca catgcgtgcg 14520

ggcgttacta aacttgcagg tcactaagga gattttacta atggctgaag aaaacaagaa 14580

ggctgagaaa gttacattca cagctaaaga gattgagcac aagaataaga tttacaccca 14640

gaagattaaa cgctgggaga agaaccgcct agtgtgtgct ggtacagacg ctgtgaagga 14700

ggctggcgtt aagctgttgc ctcagttgga aggtcagtca accgacgagt acgatgctat 14760

ggtagttcgt gctaacttct tccccggagc tctacgtgcc ctgcacggta ctaatggtat 14820

gatccacgcc aagactccaa ccatcacgtt cccggaaggc aagatgtctt atttagataa 14880

catcggtcta tcgggctcat ctatcgcgga agtggcagag gaaattaccg aggagcagat 14940

gctccaaggc tgggtgggca tctttgcgtt ctacaactcc gttccgggta caagcgagct 15000

aaagccgttc ctgtcagtaa tccgcgccga gaacatctgg gactggcgct tcggtgtggt 15060

aaatggcgtt aagcaactaa cgtacgtcaa gttcgttgaa tacgtcgagt cccctgactc 15120

caatatgttc ctatccgttg agattccgca agtaactcag ctgtggttag acaagaatgg 15180

taagcttaac taccgcattg tgcagaagca accgggcgag cagaagggtg aagcttcatg 15240

ggtccaaaaa cagggcggtg cagttttagt aagaggtaag gaagtcgagg gagttccttt 15300

ccgtttgatc ggtgcccgta agatgtcggg tcgtccagag gaagctccta ttgagcctat 15360

cgtagacgtc aacatctcac actatatcac ctccgccgac ttggagcagg gtcgccactt 15420

taccgcactg ccaacaccgt acatctgttc cgcatcgtta gacccagaca aggacttccg 15480

catcggtggt tacaactgct ggatcatccc ggagaacgaa gccaaggtag gtatgcttga 15540

gtacaccggg caaggtctgc tttcactgga gaaagccgat actgagaaga aatgggaaat 15600

ggcagtactg ggtgctcgca tgctccagaa cgacaagaaa gccaatgagt ctaaagacac 15660

ggtccgacta cgtcagacgg gtgagaacag tatcgttact aacgttgcac gccagtgctc 15720

cgtagcacta acctacctcc ttggtaaatt cattgccccg tggaccctcg tctctagcgg 15780

tacgaaagta ggcttcgtgc taaccacaga cttcttgacc atcgagatct caaccgagat 15840

gctgaactca atggctgcgc tggtggctac cgacaagatg tctatggaaa cattcttcta 15900

caacttgcag cgtggtggta tctacgaagc tggcactacc cttgagaaag agatgcagcg 15960

tatcgagaag cagtttgaaa agcagatgaa gcaacttggt cgtaagagtg acgtggtgga 16020

cttagacgat gaagaagacg agatcccgga cgatgagcag gctaagaagg acgagaagaa 16080

gactgcgact aatgaccacg tagacaatca agctaagagt aaggattagt aaatggcgaa 16140

ctttagtgac aatctggcgg tagtttacgc agaccaccag attgacctat accgctatga 16200

agccaaccag cggcaagaca ttgccgcttt cctactggca atggcggacg agataagaga 16260

ggcgttacta aaagctgact tcggctcaca gccgtttgtt actaaacgta aactcaatgc 16320

gttactaaaa gaaatcgaag caatcatcaa gcagtactac aaagacgctc gtgactacca 16380

gctaggcgag ctgaagcagt taagcgtaat cgaatacgct tgggctatgt ctgcgatgaa 16440

caacaccatt ggtgccagcc tgttcgagac cttccttact aatcacttcc tagagagtgt 16500

cgttagtaat gtcctgattg aaggtgctcc gtctaaagag tggtgggctc gtcaggctac 16560

agacacaatg gagaaatttg tagaccagat acgtatgggt gtagcactcg gtgagactaa 16620

cgatcagtta atcgaacggg tgaacggcaa gttcctccat aagttcggca agcgtaagat 16680

gtctgatggt aaggttaaac gctacggctt gtacgagggt ggaatactca agacatcgcg 16740

ctctaacgct gagactttag taagaagttc cgtgcaggct attgcgaatg atgctaagat 16800

gcgaatgtac gcagctaacg acgacctact gcatggttat caacagctat cggtactgga 16860

cttaaaaaca tctgacatct gtatcgccag aagtggtctt gcgtggacgg tagagggtaa 16920

gccgattggg aatcacaaga agcgtttccg aatcccccct ctacactgga attgccgatc 16980

actgctgtta ccgattctga aatcgtggca agatatgccg ggtaaagtgc gtactagcct 17040

tccgggaagt atgcaggcaa gtatggatgg tctagttagt gctgaccaaa cgtacgaaga 17100

tatgctcaag cgtcgtacag atgctgagat caagaagaag cttggaccgg gtagattcga 17160

gttgtggaaa cagggtaaac taacactacg cgatctcacc gaccaagatg atcgaccatt 17220

aacattgaaa gagtatcgag aaagtgctta aaatacacct cgattttact aacagtaaca 17280

gaaaggaaat acaaccatgc ttaaattccg tgtgcagaaa ctagacgacg tacaagaaga 17340

gttccgccac ctatacatcc aacaagctga cggctctttc ctactcggtg tagaaggtgc 17400

tgtggacaaa gctaaggtag acgagttccg taactcaaat accaacctgc aatctgctaa 17460

agagcagttg gagcagcaac tgcaacaaat ggctagtaag ttcgacggtc tagatccaga 17520

gcaagctaaa gaagcgatgg agatgatgaa caagatccgc gaccagaagc taatcgaaga 17580

aggcaagatc gaagaactta tcgaagcgcg tactaaagac atgcttacta accaccaaac 17640

tcgtgagcaa gagcttacga aatctatcaa agagtgggaa gagaagtttg gtggtcttca 17700

gtcaaactac cgcaagctta agatcggctc agacattctc ggtcagctag ataaaatcgg 17760

taagatccac ggttcttctc gtgacatcat taccgaccta gctgctcagg tatggcagtt 17820

gaacgagaaa gatgagttag tggcaatgaa gggcgaccag cgtgtgtaca gcccagcaga 17880

tgctactaaa ccactatcag ctgaagagtg gtgtatgcag ctggctaatg accgcccata 17940

cctgtttgag tcaactacct caatcagctc tggtcaaggc ggtcagggtg gtcaaggcgt 18000

taagggtgtt atcagcggtg atgacatgga cgcatttgag aacaaccttg aagcaatcgc 18060

atctggtgaa gttcaagtaa acgtctaact ctgaatttta attttagtaa acataaagcc 18120

accttcgggt ggctttttta gttttttatt aataaggggg ttgacgaact tagttgtttg 18180

ggttttataa tgtaaccaac aaaagcaaac ttgtttagcg gcggggtcgc ttctcaaggg 18240

tttcggggaa accatgcgaa ataacttatc aaacgacaac acgaggactt agcactatgg 18300

ctaatacttt agaagctgtc gccccgaagc tgttagctca gggcttaatg gcacttcgtg 18360

gcacgaacgt aatgccgact ttagtaaacc gcgattatga tcgtgatctt gcagccaagg 18420

gtctgacagt ggatattcca atcccttcag cggttcctac tcaggacgtt gcaccgggcg 18480

caacacctcc taacactggc gacgtagcgc caacagtagc gaaagttaca ctagacaagt 18540

ggaaagaagc acctttctac ctaacggata aagacgttaa gcaatcaatg aacggtatca 18600

tcccgcttca agcgtctgaa gctgttaagt ctcttgtaaa cgacgttaac gcggacatcc 18660

taggcaaata cacttctgta tacggcatgg taggtactcc gggcgtaact ccattcggtt 18720

caaacaccaa agaagcaact gacgctcgta ctaagctgaa catccagctt gcaccgggtc 18780

aagatcgtcg tttcgttatg gacccatctg cggaaggtaa tgcacttaac ctacgcgcat 18840

tcaacgacac taacttcgct gtgactgctc agcaagtacg tgacggtaaa atggctcgta 18900

aactaggctt cgattgggcg atggatcaac aagtacctgt tcacgctgct ggtctttcgg 18960

ctgcggctgt taacggtgct ggtcaaacag gcaaccaatt agccttcgac ggtggtgtag 19020

acgctgctga cggtggtact ggtgttaaag ctggtgacat cttcactatt gcgggtgacg 19080

ctcagactta cgcagttgta gcaactactg gcgctaaagc tggcactatg accgtgactc 19140

cggcgatcaa gaaagctccg gcggacgacg ctgtgatcac gttcaaagct actcacactg 19200

ttaacttggc attccaccgt gacgcgtttg cattcgcatc tcgtcctctg gctgaccaaa 19260

ctaacggctt gggcaacatc atccgtactg caactgaccc ggtaactggt ctagcactac 19320

gtcttgaaat cagccgtgag cacaaacgta ctcgtttctc gtacgaccta ctatggggtt 19380

catctctagt acgtccagaa ctggcagtgc gtgtagcggg ctaatcccgt ataggtcatg 19440

ggaggtaggg ttatcccgcc tccctatttt tttaagttta gtaaactgag agtacgaatt 19500

atgagtcaaa ttgacccgca agagatgctt aagcaaatct tagctgcatc tgataaccca 19560

gcagaactac tacgtggtgc gatccaagaa gctacaagcg gtgagaacgg cggcaacgct 19620

cttcaacagc ttgaattact tctagcatct gaccctaacc taaaacacca cctacgctct 19680

ggtatgcctg catccgtgca agaacaccta cgttacgtac ctacaatgat tgttacgttt 19740

actaatggtg ctttcggcgg caaaccaatg gtgatcaaca agtcggactt cgacgctgac 19800

cttcacgaag aagttgacgt tgctaaagct gttaaagcgg ctgctgcttc tgctgctaag 19860

caagaagagc ctgtcgtcga tgaaggcgct ggcgaagacg ctacaggcga aacggaagat 19920

cagaccgaag acgctcctaa gcagggtcgt agccgtaacc gccgtagcta atcagtaata 19980

cctattacta acgttactgt tgattggaag gaggaggctt agcttcctct tttttgtttc 20040

tggagattac taaaatggca atcacattaa ccgtacagac tggaatctac ccggtacgca 20100

atgccaacgc gttcgcgtct gtagagcagg ctgacaatta ccatgcgcag cgtggtaata 20160

cagagtgggc aggcttcgac gccgacaaga agaaagcttc actgatcaag ggtgcagact 20220

tcatcgctca agagttcaat ttccgtggtc gtccgatgta cagcgaagaa gacccgacta 20280

acccacagtt cctaccgttc ccccgccatg acttcgtaga caaggctggt cgttctatta 20340

ctggcacgcc ggaaggcgtt gttcgtgcta acatggagct ggcattgttt gccggacgtg 20400

gggaccttta ccctaaccag accacattac taaaaccggg tggtgctgtg actaaggtta 20460

gtaagaagac gggtccattg actactacgt acgagtacgc aaaccctgta gccgttctgc 20520

actcaacacc acactacaag aaggtgagca gctggcttaa ggactacgta tacactaccg 20580

ggcgtattca ccgatagggg attactaaaa tgtctaacgc ataccaagag cagatcgacg 20640

acgctctgga aagtatcaag gaagcaggca agcagttcga cttcgcgctt actacagtgg 20700

gcaacaaccc ggataagccg tggctaggtg gtacaaccac aacaaccgat gtcaagctgt 20760

gggcttgtgt ctttccggtt agcagtgctc cttctgcact acgtgagccg atgctcaaac 20820

agggcaccat gatcgaaact caaatgcgct acgtgctagc agctggtgaa ggaagaacta 20880

cacacccaaa cacaggtgac gtgctaaagt catttgaggg gaacgactgg gcaatcatgg 20940

catgtgcgcc tcttaccgtt aacggggagg ctgcgatcat ttacgagatg gtagttaaac 21000

gatgaacgac atagaagcac gagaacgcat taaccaagaa cttactaacg gctgggcgac 21060

ccattctgcc acggttgata ttgcctacga gggcgatggt tacaaaccac aaccgggcaa 21120

ggcttatatc gaagttattt tcagtgtaga atccacttca agccagtcgc tgggtgatga 21180

gggtaatcgc tcctttatcc gcgatggtgt tgtacacgtc aacgtgtaca ctccggctaa 21240

caacggcaac gccttcgcag ctcacatcta cgctatggct gtacgtgata ttttcgaagg 21300

taagcatttc ggggaccttt ggttctggga gtgtaaagca agtcctgccg ggaacgatgg 21360

gacatacaac gtagcgtacg caaattgtgc gttccgcttt cagcaaatca agtaggagaa 21420

ttactaatga gtgatactaa ccgcgtacgt cttgccattg tggaagagac tacaccgggg 21480

actgtcccta acaaccctaa gttggtacgc cagcgcgtta ctaacatgcc gtctctagcg 21540

gtaacgccgg agactatcga atcggaagaa ctggacccgt ccatgcagac aacagaccta 21600

atcaaagtag gtcaggctgt tggtggtgag ttcggtatgg agttcagcta cgacgctcag 21660

aagcaatcaa tgatcggcat catgcgtaac agctggaact cgttcacgca gtacaagggt 21720

gatgaagttg gcgctatctc agctggcaaa gttgctgtta ctaacgtagg cgctgcgcta 21780

cctgttggtg cgctggttct gttcaagaac ttcaacaact ctgctaacaa cggtgttaag 21840

cctgttacgg ctgcgaccac cacagctatt acagctacgg gcttagtagc cgacgcgtct 21900

aagacgggta aggaagccat tcacgtagtt ggtctgcaag gtggctctgg tcatctgact 21960

gtcaccgcaa acacgcttga agctacgtca gggtcggaca tcgactttac taactttaag 22020

ctacgtccgg gctccttgat caagctgggc ggtaaggacg cggctaaccg cttcgctacg 22080

gctaacgtga acgctatggt gcgcgttact aaaattgaag ccaagaagct tacgctagct 22140

gacctaccta ctggatgggc tgctgacaac ggtgctggta aaactattca agtctggttg 22200

ccagaggacg acattgtgaa cggcgtggct accaagtcat tcacgttcct gcaatcgttc 22260

ttggaccaca acccggtaac acaccagatc ttcaatggta tgcgtatcgg tactatgaac 22320

atggagatgc gctctaagca gatcgttacg ttcagcatca acgctcaggg tactaagggt 22380

gctatcgacg agacgggtgt ggcgggtgct actatcctgc cagcgtctac agatgtggta 22440

atcaacacct cgtctaacgt tgcggagcta cgcgaagctg gtcgtaagat tgaaggtccg 22500

aactacgtga caggcgttac actggcgctg aacaacaacc cacgtaacga cgacgcaatc 22560

ggctacgagt ctccggttaa catcggtggt ggtacgttcc agctgacagg cacgctaaac 22620

acgtactttg gcaacaagtc gctagttgag aaggtgatca acaactcgac aacgtcactg 22680

ctcctaaact tcaaagacgg tgaaggtcag cacgtggtgt tcgatatgcc acgcgttaag 22740

tactctagcg gctctccggc ggtatcaggc aagaacgcgg ctaataccgc agaccttggc 22800

tacacggctc tgaaacacct agactacggc tacacgctag gcattcagaa gttccgttac 22860

atggagtaaa tcgaagcact tgcgtcgagg cacaacctag aataagatca aagcgtccaa 22920

tacgggcgct tttttaatat tagtaaacaa ggaaatccca tgaagaagac taaccctttc 22980

gctctattcc gcacctctac taacgacgaa gtgaacggcg tagtggtaaa ctacggctca 23040

ttccgcgtta cagtggcgta cgctggcggc gctaacacag actacaaccg tctgcttatg 23100

aaacttggta agccgtactt gaagctaatc aagtcaggca acctgccaga agaagttacg 23160

aaagagatcg atgagaagct ttacacgcag accatcatca agaactttga agtagacgta 23220

gctgaagaag gtgcagaagc tccagtgtgg aaacgcggca ttccaactga agaaggtgaa 23280

gtcctagact acaacgaaga gaacttagta agcctactac gctcccttcc acacctattc 23340

gaagacatta agtcattcgc tcaggatatg tcgaactaca aacctgatct ggaagtagct 23400

aagggaaact aatagagttt ctaaagcatg agctggaata cggggacgat gatttcacgt 23460

ttatcattga agccgcaatc gaagccggag acccacttcc ggcttttgct tcagaagcgc 23520

cgaaactcct agacccatta gtaccaacct ataacatgtt ctgggatata taccgataca 23580

gacgtgttat cgccagaaat gattttgtcc cttgggaagt ccttgaacgg ttcgccgcta 23640

agtacgggat tgaagagctg ggagagtttc aagagttctg tgagttgttc cgcgagatgg 23700

agaaagttta cttagaccac ctagtagaac agtacgaaca ggaacagaag caggaccaga 23760

atcaacaagg gggacagcat ggcgaatttc tcggacatac ctaaagatct aaagcacttc 23820

gctttcaccc tccaagctaa taccgaaaca aggcagcgtg aagttgcctt gaccattacc 23880

agagagctta tgcagcgaaa ccctaagaag acgggtcgtt ctgctggcaa ctggcaggtc 23940

ggtatgaatc gccccaaact aattgcacag gctcctccgg tagcggagag ggcttcggat 24000

gaagaactta gtaaaagttc taaattgttc gtgcagaaac aacttagtaa agcgattaac 24060

gatattacta agggaactct acgcggggat gacccggtaa tctatattag taataccatc 24120

aactatgtag tttacctaaa caccacccga ccatcaccac aggctgctcc gggctggatt 24180

gaagcatcga tccgatttgg cgcagatagc actaagggag tgaaacttac gtaatggctg 24240

gtgaaagtta cattatcaag gtacggcaac acggtgcgcg ggaggtcaaa cgatcactcg 24300

atgacatcgg tgctagtgct aaacagacca cgagcacact ggaggggatg cgtaagggct 24360

tgaacctcct actgggtctc tacggtctaa cagcactaca gcagtatgcc gacgaatacc 24420

aaaacttgga tgcgaagctt aagatcgcaa cgcatagtac agaggagctg gctcgtgcgg 24480

aggaccagct tttccgcatt gcgaacagaa cgtattcctc attctcaagt accgttgacc 24540

tatacgcacg ttttgagcgt tctacacgga gtcttaagat ctcgcaggat aacctcctac 24600

agatcacaga gacggtgaac aaagccattg ccttatccgg tgcaagtact caggctgcta 24660

acgcggctat cttgcagtta ggtcagggta tggcggcagg cacactccgt ggtgacgaac 24720

ttaactcagt tctcgaacaa gcaccacgtc tagccgaggc aattgcttcc ggtatgggcg 24780

ttgctgttgg taacttgaag cgtcttggtg agcagggact tatcacaacg gaagaagtac 24840

tcaacgcact tgcgtctcag ggtgctgcta tcgacaagga gttcctaaac gtaaacctta 24900

ctattggtaa agcaatggag gttctgaata accagatcct acgtgccatt ggtcagctag 24960

atagtaaact aggcatctcg tctaccatcg ctaagtctat tgtaacgttg agccaaaacc 25020

tcgaccaagt tgcaggagcg gctcttggcg ctgctgctgg tcttgcagta atgtacgcgc 25080

cagctttact aaaaggtctg gtaactgtta cgcgattagt aaaagggcta actcttgcga 25140

tggtttctaa cccgttgggt gcgatggcgg tagctgctgc cacccttatt ggctatttga 25200

ccattatggg cgaccagatt aagcctctga gcgatagttt cgctactgtg gcggacttcg 25260

ctaccgctgg cttcgatatg gcgtcagaat cgctctcagg gctttacagc ttaattgagg 25320

atgacgtcaa ttctgcacta agcaccatgc gtaccacagc agacgagctg tttactaaga 25380

tgggaccgtt aatcgaccaa gcaatgtcta tcggttacga cgctatcaac aaagtgatcg 25440

gggcgttcgt tatcgctcgt aaggtcggcg ctcaactatt cgaattagta ctacagggtg 25500

cttccggtct atgggaaggt ctaacagcat tctacgactt ctctgtaggc ttggctaagt 25560

cgatgttcca gactgtcggc ggtattgcaa acacagtgtc gaagagcacg ggtattacgc 25620

tggagtcgct ggcacaagga ttcaagcaag tgcttaacat tgctatcggt gtgttcacgt 25680

taattccgcg cctagcattt actatggcga agaatgtagc gaagaacttc gaggcgctgt 25740

ttgcagatat tactaatatg ggctccgcta caatggaggc tctaaaagta gcgttctcag 25800

gtggtgactt tactgaagcg ttcaacaaga tccgggctca gtctaatggc gctattgacg 25860

gtatgggcga tgacatctct agcgttctta aggacacatt caaccaagac tatgttggcg 25920

atcttatcgc taagactgtc gagctgaaag agcaattcat ggacacgttc ggtgatgacc 25980

ttgcacaaga ctacctgagt gacgctcagc aatctctaag cgagttctac aaggagttca 26040

aggcacgtgc tgaagatgca gctactgccc gcctagcggc tgagcaggaa gttactaaag 26100

agtacgagaa gcagaacaag attgtaggtg gtggggatgc tttcagttct gaccagattg 26160

ataagtttac taagaagctt aatgcgatgc gtgagactct taatggactc gacccaactt 26220

gggaacgctc aactgagatc atcaacatct ggcgtgatac agcacttaac gcggtagaca 26280

agaactggga gcgttacgaa gagtacgcga acatggttga ggaagttacc cgcgagaaga 26340

tcaacaaagc ctactgggag cacatggacc aatccgaggc tgctttcgat ggtatgatct 26400

cagccgctca taaaatgcag aaggagtacg acaactctgc tgaactcatg aagaaaggca 26460

tgggtacagt gattgacggt cttactaacc agttcatgcg ctttgctatg actggtaagt 26520

ttagcttcaa agagctggct gctggcgttg ctgcttctat cgctgaaatg atgatgcgta 26580

tgctcgtcat gcgtgcggtt ctggctgcgt tgaacgtaat tccgggcttt gccgctgcta 26640

tggctggtgc cgacgttgct ggtcaggctg caaccgctgg cggggctgct gccaacttcg 26700

ccacatccac ggctgttcta cagcaaggta tgggtcaggc gcaggctgct aactcaaaga 26760

tgtacgctaa gggtggtgcc ttcagccgtg gtgttgagat gtttgctaac gggggtgcct 26820

ttactaacag tgtggtatct cgacctacaa acttcgcaat ggctggcggt cttggtatga 26880

tgggcgaagc gggaccagag gctattatgc cgctatcgcg tgggtctgac ggtaagttgg 26940

gtgttcaagc aagcggagcc gcttctccag aggtgaacgt gttctttgta caaagcgccg 27000

atgaagctgc ggagctaatg gctcaaaacc cgaaagcaat taacaaatta gtaagggcta 27060

ttgacgaggc taaaggataa atatggcata tcaaaaaggt actgctagca gtcatacaga 27120

cctgttagta aaactccaca cgttcgctac gtcgaacggg tggacatcac agaagaacga 27180

ggagtccggt ggcgagcacg agatcgtcct caagtccaca ggtacgggtg gaaacgattc 27240

gattgtactg gcgttccgct cgaacacact gcaagcgtct gatacatata acatacgatg 27300

ccgcgtgtct gctgcatacg ttacctctgc gtttgacaac ataacggggg cgtctccaga 27360

gtcagtactg tacgcgtgga atgccgatat ggagtactac tttattgtta gtaaagagcg 27420

gatcatgatt ctgtttacag tatctggcgt ctcgcagtac tactacggtg gtaacatgca 27480

ggtgtacacg tctcgtggac actgggtaag cccactctgc tgcttcgggg tagggaccag 27540

tgctacagct cgctggtccg agcagggtaa cgacttctcc ggatggcagt acgtccgtgg 27600

ttctaatccc aaccatatac tggatgacac aggtgcatgg actaaacccg attacttttt 27660

cccgaacatg gatacggcgc ttaccagtaa gatgcgtgcc tacgacaacg gcgacatccc 27720

actcctaccg atgttcgtga ggacagccgc tcgcggtatc gtaggagagc tgcatggtgc 27780

cttcgggtgt ggtgggttcg gtgcctctca cggacaggaa ttaacacact cctctggtag 27840

acgttttgta atcctacaga acgtgtaccg ggcgggtaca acagactaca tggcgatgga 27900

gctagcgtaa tgggatacga gaatgctact tacaactcaa gggtagagct tggtcagaag 27960

tttgccacgg ctattactaa tctggggggt actgtcaaac gtcagtacca gaacactgtc 28020

aacggagagc ctgcatacca agttatcgtg caagtggcgt ctgacgtgtt tatgcactat 28080

gactttcggt ggcaccgctg gtactcacgg agaggcacta cttaccaccg cgaggggcta 28140

tttactaaaa tgggaactgc ttacgttgac ggagctgaca cggtaagtgg gatagtcagc 28200

agcaactttg gtggtggtgg tggcgcacag gtatctacta actggagtca cctccaccag 28260

tacctaccca ttcccggaaa ggtacatgtt gctcgcaacg gcaataccgg ggaatttgta 28320

ctctgtatgg agaacccagc agactctaaa gcagtactgc atggggtagg gcgtatcata 28380

atgcaggaca caactttcgg ctccgtaccg cagtggtggt ttgatggtga gtaccactat 28440

cgagatgcca actcagccga tggggtgtac ttcatggaga atactgatgg gactggtcgt 28500

tcggctcggt ggtcagggca gctacagcag cgcgatagtg ctggtgctgt aactgggtac 28560

gaggcgatgg gggcggcaga taaagcttgg cagcgtccta tgaccgggta tcaaccacta 28620

gggaggcttg gtctctacag tgctattgtg ggagacctaa gttttgagcc gtcgcttacc 28680

agtatcttcc tgcctgcgct gtggtgcggt gtcatgggca cggactatac gatccgagcg 28740

cctaggggtg atcgtccaac gttcaagatt tgcaacatgc gttacttagg tgttgggcag 28800

gagattatct acaacaacca gtcgtaccga gtgttcccat gcttaggtaa gggtggtgag 28860

agcactccgg ggatcgcatt cttagtagga gcagcatcgt gagtaatgca gtacttttta 28920

gtaatgcgtt cggatacaca ctgaaggcag atccaccgcc taaagttaat acgcctgttt 28980

acatggctcc ctcctacctt cgggtaggag ttccgaatgc gcgaggtccg tggctaaaat 29040

ctaaggcatc ggtggcgggt gttccctatg gtggattctg ggactctttt aagaactccc 29100

tattcatcca gccaaacaag atagatttag ggcttgtgct tagtcccagt acgcattcca 29160

tccgtgtttg gagttcacat gagcagtcga tcactgctcc taacatagcg gctacaggca 29220

acggaggcat gtcgcttaca gggaccacct ctaatatcac actggacgcg ttcggcgggt 29280

acaaggacta tacgctggac gtgtcgctgt ctgtggctgg cgtaattgac gcgaagtatt 29340

catggaactt ccccggactc ccggagtctg tgagattact aaacgtaatc gggcagagga 29400

tcgtagtgtt tggcatccca ccacaaaaaa gaatggtgga gaagcttagt tggaagaccg 29460

atgttatacg taccctagac ggtgctgagc agcgcatacg tattcgtgag aacccaatgt 29520

ctactatccg ctacaagtca gtcacaggct atcttgatac ggcaggcgcg gaggctttga 29580

tctatcgact aggtcacgac gacgtggcga tcccactatg gcacgagggc gtacgttatc 29640

cgggagttct agctgcgggc gtgatccgga ttgacgttag cacggcggct tcatcgtttg 29700

ctgttgacga cttagtaata ctgtggaaag acttcttcac ctatgagact gccactatag 29760

ctacagtagc tgccgaccac ataacgctta agaagccgct tgtcggtact tgggaccgac 29820

cactggtagc ccctttagca tatggtctta tggacaacgt tacttttagt aaatataaga 29880

tcaacgtgtc tgagaataac gtggagtaca cccgcaacgc tactgtgagc atagctgggt 29940

ctgcaacgta cccactctac gatggaatcc ctgtgtacgg gaagctgctg tacatgagtg 30000

gtaagtcatc acccgctgct tatacaccat ctgttgactc gctggattat ggcattaagc 30060

ctagatccca gaagaagcgg tatgacttcc cagacgtaac gcgggacatc ttagtaaaaa 30120

tgtacagtcc gctagaggta atgaacttta agaagttcct gacctacttg ggcgggaagc 30180

agaagccatt ctggtacgtc agcgattggt gggatttcaa cccagtgggt aacacgccta 30240

ccactgagac cgtgatacgc atacaggaca tgggattcct agagtactac gcaggtgcgg 30300

ttaaccgtaa gtggctagcc attaagccga agggtggtgc ttgggtgtac cgtcaggtat 30360

cctcagttgc gaaaggcgcg gacattccgg gcgagcctaa gaacgaagct atcacaatag 30420

acacagcctt accctttact tttagtaaag acacggttga gaagattagc ttgatggtcc 30480

cagttcgact ccagcaagat gaggttactt tcaattggga tggtatggag catataacag 30540

ccagccttcg ggttgtggag gttactaaat aatgagctac acagataaag aggatagctt 30600

gtacgacggt agaccagtaa cggtctacca cttcaactat ggcaacgagg actactggta 30660

cacatcttcc gacgaggaca tactgtctcc cggcggtacg gctaccagca tccatattaa 30720

gcacgggact atgaaagaga ctacagagga taagagatcc actgttaaga ttgagataga 30780

tgctaatgtg cctctttcta aagctcttcg cataaacccg gcggactcgg tttgtcgtgt 30840

gaatatccta cgcgggcatc gaaatgacac tgatagacag tgggtttact actggcgtgg 30900

tacggtatcc gtcgttggga gagaggaagc ctctcagtta agtttagagt gtacgcacat 30960

gctagctact ctggcgtcag gtggtctacg gtctcggttt agctacacgt gcccgcacgc 31020

cctgtacgga ccgagttgta gggctacacg ctcagcggac aaactgagga cagcaactgt 31080

tactgcgatc tccggcgcta aggtatcgct gtctgggtgg agtactacga cttggtggaa 31140

tggtgggcag atgaccttcg tgaataacaa gtaccgtcgg tatataatgt cctccgatgc 31200

tacagggata ctgctggacg cagtacctat cggtcttgcc gtgggtatgg aagtgtcgtt 31260

agtagctggg tgtgaccgga ctaaggcaac gtgccagtcc aagttcaata acctagccaa 31320

ctatggcggc tacttagcag tgcccactaa aaacccgttc caagacggaa ttgcttaata 31380

ttagtaaaag gagtaattat ggctgttcag ttactctggt acgcagtaat acttattgta 31440

tcagtgatcg tgtctgtagc cttagcgccg aagcctccga agcctgactc ccctcagact 31500

aaggatatta acgcgcctac agcttcagag aatgagttta tcccggtagc cttcggcacc 31560

acgtggctgc aaaagccaaa cgtatgctgg tacggtaata cgggcaccga tgagattcgt 31620

aagagtggag gtaagaagta atgtcagata gggtggaact actccccggc gaagaggact 31680

ttagacttta catatcggac ttcgatcagg agtccgttaa gcagttcaat tgccatagcg 31740

ggatgcgtgc caagggcaaa gcctacggct tagattggtg ggattttctt actaatggaa 31800

ttatgttctc agagctggca gctcttgagg atggtcagat taaccgatgc ttagtggaga 31860

ttttaaatgg gcggaggcgg aagtaagagc caagtcgtcg ggtacaagta ctggggtgct 31920

atgcagctgg tactttgcca ccgagggctt gagtcgattg atagaatacg cgtaggcgac 31980

aaggtggtat ccagcgaggt gatcacccag tccaactgcc taaagtacat cgacaagtgg 32040

aacacgttcg gaggggataa gcgtgagggt ggtcctgtgg gattcgcccg gttcttcttc 32100

ggcagtgacg accagccagt gagctcgttg actgaaagta tgtatcagag cggtactgct 32160

ttccgtggtg tggtcagcgt ctccctaccg aagatgtact tctcagcaaa caacccgtac 32220

atcaagcctt ggtcgtttcg ggtgaagtct taccctgaca ctccggggtt agctcgtacg 32280

cacgtcaaga tcgatgctgg taacggtatc gtgaatatga acccagcgca cattatatac 32340

gcctgtttag taagtcgtag tgatcgctgg ggtctaggtc tcgactacac gaaggacatc 32400

gacgtgcctt catttgcttc ggctgcttcg gcgctctatg cagagaagtt cggtatcggt 32460

atagcgtggg agaacaactc cggcgttgat gacttcatca aagagattct acgccacata 32520

gacggggcta tatacgttga ccaagtgtcc ggcaagctta agctaaagtt gttccgtggt 32580

gactacactg ttagtgattt accagttctt gataagactg tagtaaaaga ccttgagaag 32640

tttgagtacc cccagtgggg taacatcacc actcaggtaa ctgtgagtta cgtggatgct 32700

gtagagggta aggagaagcc tgttaccgtg cataacatgg ctgctcgtga tgtgcagggt 32760

agggacgttc cagtggagat agacttcccc ggaattaaca cacgctggct agcttcccgc 32820

gttgctt 32827

Claims (8)

1. Vibrio alginolyticus phage vB _ ValS _ R12Z with the preservation number: GDMCC No. 61738-B1.

2. A phage composition comprising the Vibrio alginolyticus phage vB _ ValS _ R12Z of claim 1.

3. Use of the Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z according to claim 1 or the bacteriophage composition according to claim 2 for killing and/or preventing a microorganism of the genus VibrioVibrio alginolyticusATCC 17749^T、Vibrio alginolyticus JL2674、Vibrio alginolyticus BVA2、Vibrioowensii3186 one or more of the above.

4. Use of the Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z according to claim 1 or the bacteriophage composition according to claim 2 in the preparation of a medicament against a microorganism of the genus VibrioVibrio alginolyticusATCC 17749^T、Vibrio alginolyticus JL2674、Vibrio alginolyticus BVA2、Vibrioowensii3186 one or more of the above.

5. Use of the Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z according to claim 1 or the bacteriophage composition according to claim 2 for preparing an aquatic feed or feed additive against a Vibrio microorganismVibrio alginolyticus ATCC 17749^T、Vibrio alginolyticus JL2674、Vibrio alginolyticusBVA2、Vibrioowensii3186 one or more of the above.

6. A pharmaceutical agent against microorganisms of the genus Vibrio, comprising the Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z of claim 1 or the bacteriophage composition of claim 2.

7. An aquatic feed or feed additive against Vibrio microorganisms, comprising the Vibrio alginolyticus bacteriophage vB _ ValS _ R12Z of claim 1 or the bacteriophage composition of claim 2.

8. A method for preventing and treating the harmful infection of Vibrio alginolyticus in vitro, characterized in that the lysis of Vibrio alginolyticus phage vB _ ValS _ R12Z of claim 1 or phage composition of claim 2 is used to kill Vibrio alginolyticus in vitro, wherein the Vibrio is Vibrio alginolyticusVibrio alginolyticus ATCC 17749^T、Vibrio alginolyticus JL2674、Vibrio alginolyticus BVA2、Vibrioowensii3186 one or more of the above.

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