CN109082414A - A kind of staphylococcus aureus bacteriophage and application - Google Patents

Abstract

The invention discloses a kind of staphylococcus aureus bacteriophage and applications, bacteriophage of the present invention specifically can effectively eliminate staphylococcus aureus, there is the inside and outside antibacterial action of good body to drug-resistant S. aureus, preparation for the infection of clinical development prophylactic treatment drug-resistant S. aureus provides experiment basis, has great clinical application potentiality.In dermapostasis model, when MOI=10, Phage therapy group nude mice skin abscess average area is 47.32mm², average bacteriological discretion be 3.553 × 10⁷CFU/g；MRSA infected group nude mice skin abscess average area is 150.4mm², average bacteriological carrying capacity be 2.284 × 10⁸CFU/g.Dermapostasis area, the bacterium colony of Phage therapy group mouse, which count, is both less than MRSA infected group, and difference has statistical significance.

Description

A kind of staphylococcus aureus bacteriophage and application

(1) technical field

The present invention relates to a kind of staphylococcus aureus bacteriophage and applications.

(2) background technique

Staphylococcus aureus (Staphylococcus aureus, SAU) is clinically common pathogenic stronger Gram-positive cocci.After the advent of penicillin, infection caused by staphylococcus aureus is under control, but with penicillin Be widely used, constantly generate new drug-fast bacteria.Nineteen fifty-nine methicillin is applied to clinical treatment, has once efficiently controlled resistance to The infection of penicillin staphylococcus aureus, however Britain in 1961 is just separated to first plant of methicillin-resistant staphylococcus grape Coccus (Methicillin-resistant Staphylococcus aureus, MRSA), it has become in institute and community at present One of important pathogen of infection.It in recent years, is ST239 in the dominant clone type of the common Nosocomial S. aureus L-forms in China. And the S. aureus L-forms dominant clone type of Community-acquired is ST398 and ST59.

MRSA can cause skin, soft tissue infection and pulmonary infection.Its caused osteomyelitis is also that clinic is difficult to control The disease for the treatment of.In addition, bacteremia caused by MRSA and infectious endocarditis may result in heart failure and death.

In recent years, infection caused by MRSA increases year by year, also rises year by year to the drug resistance of antibacterials, multidrug resistant The ratio of bacterium obviously increases.All the time, vancomycin is all the choice drug of clinical treatment MRSA infection, is once referred to as and faces The anti-infectious last line of defense of bed.But being widely used with vancomycin, the sensibility of vancomycin just drop year by year It is low, clinically gradually generate vancomycin intermediary staphylococcus aureus (Vancomycin-intermediate Staphylococcus aureus, VISA) and vancomycin resistance staphylococcus aureus (Vancomycin-resistant Staphylococcus aureus, VRSA).It is extremely urgent to find new treatment means.

Bacteriophage (Bacteriophage) be it is a kind of can the microorganisms such as bacterial infection, fungi, actinomyces or conveyor screw disease The general name of poison by infection, proliferation, maturation and discharges filial generation etc. a series of process infects pathogen, reaches cracking, kills The effect of host strain.Early in 1915 and 1917, Twort and D, Herelle just respectively independently had found depositing for bacteriophage ?.Following period of time later, many scientists are directed to bacteriophage and have carried out every research and be applied to clinical infectious The treatment of disease also obtains certain curative effect.However, discovery and application from antibiotic such as penicillin the 1940s Since, antibiotic in a very long time all with its efficiently kill pathogen, it is highly-safe the features such as obtain whole world model Being widely popularized in enclosing, this causes to Phage therapy but also most countries terminate the research to phage preparation Research lies on the table.But due to the abuse of antibiotic, there are more and more antibody-resistant bacterium, drug resistance is also further Extensively, or even occur general drug resistant " superbacteria ", the research and development speed of new antibiotic drug resistance occurs far away from bacterium and dashes forward The speed of change, then, the research for Phage therapy attract people's attention again.

So far, the clinical case that bacteriophage is applied to human body therapy has thousands of examples；U.S.'s food in 2006 and medicine Product management board ratifies for the first time, bacteriophage is used as food additives, for killing the Li bacillus in meat products.However phagocytosis The application of body is not promoted but always, and in addition to the discovery for the antibiotic addressed above and application, Another reason is bacteriophage It is excessively high to host strain specific requirements.Phagocytosis physical efficiency confrontation pathogen include staphylococcus, streptococcus, Klebsiella Pneumoniae, Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus etc., the disease that can be treated include suppurative wound infection, skin Inflammation, pneumonia, pyothorax, enterogastritis, pyemia, osteomyelitis etc..Phage therapy have exponential growth, high specificity, easily in real time more Newly, Small side effects, the advantages that noresidue and period are short, at low cost, thus there is wide development prospect.

(3) summary of the invention

It is an object of the present invention to provide a kind of staphylococcus aureus bacteriophage SH-St 15644, which can be used in Staphylococcus aureus bacteriostatic agent and prevention or treatment are prepared by the drug of infection of staphylococcus aureus disease.

The technical solution adopted by the present invention is that:

The present invention provides a kind of staphylococcus aureus bacteriophage (Staphylococcus aureus phage ST 59- 9, call ST 59-9 in the following text), it is deposited in China typical culture collection center, preservation date is on June 28th, 2017, deposit number For CCTCC NO:M 2017394, preservation address: Wuhan, China, Wuhan University, postcode 430072.

The present invention also provides a kind of staphylococcus aureus bacteriophages in preparation staphylococcus aureus bacteriostatic agent Application.

The invention further relates to a kind of staphylococcus aureus bacteriophages in preparation prevention or treatment Staphylococcus aureus Application in bacterium infectious disease drug, the staphylococcus aureus are methicillin-resistant staphylococcus aureus.

Beneficial effects of the present invention: the present invention separates and purifies to obtain novel staphylococcus aureus bacteriophage SH-St 15644, specifically can effectively eliminate staphylococcus aureus, to drug-resistant S. aureus have it is good in vivo, Outer antibacterial action, the preparation for the infection of clinical development prophylactic treatment drug-resistant S. aureus provide experiment basis, have Great clinical application potentiality.In vitro in test, (count of bacteria 10 when MOI=10 and 1⁷CFU, bacteriophage, which counts, divides It Wei 10⁸PFU and 10⁷PFU), after bacteriophage and host strain co-culture 5 hours, compared with control group (MOI=0), OD_600nm Absorbance reading is decreased obviously (here with OD_600nmAbsorbance represent bacterial concentration), the average OD of MOI=10_600nm= 0.0125, MOI=1 average OD_600nm=0.094, the average OD600=1.155 of control group.

In dermapostasis model, when MOI=10, Phage therapy group nude mice skin abscess average area is 47.32mm², average bacteriological discretion be 3.553 × 10⁷CFU/g；MRSA infected group nude mice skin abscess average area is 150.4mm², average bacteriological carrying capacity be 2.284 × 10⁸CFU/g.Dermapostasis area, the bacterium colony meter of Phage therapy group mouse Number is both less than MRSA infected group, and difference has statistical significance.

(4) Detailed description of the invention

Fig. 1 is plaque morphology (A) of the bacteriophage SH-St 15644 on the double-deck agar plate and in Hitachi's transmitted electron Electron microscope (B) under microscope H-9500.

15644 biological characteristics of Fig. 2 bacteriophage SH-St, A are influence of the different temperatures to 15644 potency of SH-St, and B is Influence of the pH for 15644 cracking ability of SH-St, C are 15644 adsorption curve figure of bacteriophage SH-St, D SH-St 15644 one step growth curve figures.

Fig. 3 is the external cracking ability of 6 pnagus medius SH-St of embodiment 15644.

Fig. 4 is 15644 interior therapeutic ability of bacteriophage SH-St.

(5) specific embodiment

The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.It should be understood that These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

The present invention acquisition attached Ruijin Hospital of Shanghai Communications University and Shanghai Huashan Hospital's sewage, with double-layer agar technique point From, screening and purifying obtain a kind of novel staphylococcus aureus bacteriophage SH-St 15644, extract its DNA and surveyed Sequence carries out genetic analysis.Later, using the potency of double-deck agar plate method measurement bacteriophage, phagocytosis is analyzed using drop method 15644 host range of body SH-St, and pass through thermostabilization experiment, ph stability experiment, adsorption test and one step growth curve Measure its biological characteristics.In addition, also to carry out the observation under electron microscope to bacteriophage, its morphosis is understood.Most Afterwards, by constructing staphylococcus aureus dermapostasis model, the assessment of 15644 therapeutic effect of bacteriophage SH-St is carried out.This Staphylococcus aureus host strain used in inventive embodiments is methicillin-resistant staphylococcus aureus reference culture (ATCC43300)。

LB liquid medium: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L add distilled water to supply 1L, pH value after high pressure sterilization naturally, use.

Upper layer culture medium: 0.7% agar powder of LB liquid medium+mass concentration.

Bottom culture medium: 1.5% agar powder of LB liquid medium+mass concentration.

SM Buffer is NaCl 5.8g/L, MgSO₄·7H₂O 2g/L, gelatin 0.1g/L, pH7.5TrisHCl 50mL/L, solvent are water.

Methicillin-resistant staphylococcus aureus bacterium solution: by methicillin-resistant staphylococcus aureus (Methicillin- Resistant Staphylococcus aureus, MRSA) bacterial strain (ATCC43300) is seeded in LB liquid medium, and 37 DEG C It cultivates to OD₆₀₀=0.6 (10⁸CFU/mL), methicillin-resistant staphylococcus aureus bacterium solution is obtained.

The separation and screening of 1 bacteriophage SH-St 15644 of embodiment

The attached Ruijin Hospital of Shanghai Communications University and Shanghai Huashan Hospital's sewage sample is taken to be added a small amount of chloroform, 4 DEG C, 3500rpm is centrifuged 10min, takes supernatant, after suitably being diluted with SM Buffer, takes 10 μ l and 400 μ l methicillin-resistants golden yellow Color staphylococcus bacterium solution (OD₆₀₀=0.6) it mixes, upper layer culture medium is added and mixes, pouring into bottom culture medium, that the double-deck LB is made is flat Plate.It is inverted after solidification, and the overnight incubation in 37 DEG C of incubators.If there is plaque on plate, syringe needle picking allusion quotation is used Type, bright single plaque, add in 1mL SM Buffer and pulverize, and are placed in 4h or more on 4 DEG C of shaking tables, and 0.1mL chlorine is added It imitates in pipe, centrifuging and taking supernatant, 4 DEG C of preservations.(phagocytosis shape of spot, size phase after repeating purifying at least 3 times with double-layer agar technique Together) to get arrive 15644 culture solution of bacteriophage SH-St.

The purifying of 2 bacteriophage SH-St 15644 of embodiment

15644 culture solution (10 of bacteriophage SH-St that embodiment 1 is obtained⁶) and methicillin-resistant staphylococcus Portugal PFU/mL Grape coccus bacterium solution (OD₆₀₀=0.6,10⁸CFU/mL it) mixes, is cultivated at 37 DEG C, next day adds 2% chloroform, 4 DEG C of standing 1h, then 6500r/min, 15min, 4 DEG C of centrifugations.The DNase1 enzyme and RNase1 enzyme that respective final concentration is 1 μ g/mL, room is added in supernatant Temperature places 30min.After addition NaCl to final concentration of 0.5M, ice bath 1h, 4 DEG C, 11000g is centrifuged 10min.Supernatant is taken, is added Solid PEG8000 to final concentration of 10% (w/v) is statically placed in 4 DEG C overnight after mixing.Take preceding mixed liquor on the 1st, 4 DEG C, 8500r/ Min after being centrifuged 20min, abandons supernatant, and precipitating is resuspended with SM Buffer.Equal amounts of chloroform is added gently to mix 30 seconds, 4000g centrifugation 10min takes supernatant.Cesium chloride step gradients method purified phage.The bag filter for being 3000 with molecular cut off is with SM Buffer is after dialyzate is dialysed, to cross 0.22 μm of filter membrane, take filtrate, obtain the bacteriophage SH-St 15644,4 of purifying It DEG C saves and is extracted for transmission electron microscope observation and DNA.

The measurement of 3 bacteriophage SH-St of embodiment, 15644 potency

The potency of bacteriophage refers to the quantity of contained bacteriophage living in 1mL culture solution.Bacteriophage prepared by embodiment 2 SH-St 15644 is diluted to 10 with SM Buffer^-7、10^-8、10^-9After three gradients, 10 μ l and 400 μ l methicillin-resistant gold are taken Staphylococcus aureus bacterium solution (OD₆₀₀=0.6,10⁸CFU/mL) mixing is added in the culture medium of upper layer, then pours into bottom culture medium Double-layer plate is made, is placed in 37 DEG C of incubators to its solidification and stays overnight.Second day, count plaque number (calculation formula: titre (pfu/mL)=plaque number × extension rate × 100).

It is about 0.5-1mm that SH-St 15644, which infects the phagocytosis spot diameter that methicillin-resistant staphylococcus aureus is formed,.Spot It is rounded transparent.15644 potency 2 × 10 of bacteriophage SH-St prepared referring to A in Fig. 1, embodiment 2¹¹pfu/mL。

4 bacteriophage SH-St of embodiment, 15644 electron microscope observation

Bacteriophage SH-St 15644 1mL (potency 2 × 10 prepared by Example 2¹¹pfu/mL).4 DEG C, 40000g centrifugation 40min abandons supernatant.2% glutaraldehyde water solution of volumetric concentration, 200 μ l is added, sufficiently piping and druming stands the fixed bacteriophage of 30min. 44000g is centrifuged 40min, and supernatant is carefully sucked out, and bacteriophage, 4 DEG C of preservations, standby power transmission mirror is resuspended with 100 μ l PBS.Take 5 μ l weight Outstanding bacteriophage drop after its natural sedimentation 4min, sucks surplus liquid, adds 2% phosphotungstic acid of a drop on the copper mesh containing carbon film (PTA) 4min is dyed, extra dye liquor is sucked, to drying at room temperature.It observed, clapped with Hitachi transmission electron microscope H-9500 According to, and record the form size of phage particle.

Observation result is as shown in figure 1 shown in B, it is known that bacteriophage SH-St 15644 belongs to siphovirus, and head is in tall and thin Ellipse, head are about 75nm, wide about 40nm.Tail length about 250nm, noncontractile.Tail width is about 20nm.

5 bacteriophage SH-St of embodiment, 15644 host's spectrum analysis

Using drop method.37 DEG C, 200r/min, the methicillin-resistant staphylococcus aureus bacterial strain that table 1 is clinically separated It is seeded to Columbia Blood Agar culture medium (purchased from upper marine letter Ya Tesi diagnostic reagent Co., Ltd), 37 DEG C are incubated overnight. Picking single bacterium colony, is inoculated in 3mL LB liquid medium, and 37 DEG C, 200r/min shaking table culture 16 hours.Next day, with volume Bacterium solution is inoculated in LB liquid medium by ratio than 1:100, and 37 DEG C, 200r/min shaking table culture to logarithmic growth phase (OD₆₀₀ =0.6) it, takes 400 μ L of bacterium that upper layer culture medium is added, pours into bottom culture medium and double-layer plate is made.In double-layer plate table after solidification The bacteriophage (2 × 10 of 2 μ L embodiments 2 preparation is added dropwise in face¹¹Pfu/mL), after liquid on plate dries out, 37 DEG C of inversions were cultivated Night.The fragmentation pattern that bacteriophage is judged by plaque production, is shown in Table 1, and has 12 plants can be split in 37 plants of MRSA Solution.

The host range of table 1SH-St 15644

+:susceptible,-:resistant

6 bacteriophage SH-St of embodiment, 15644 biological characteristics

(1) thermal stability is tested

Phagocytosis body fluid (potency 2 × 10 prepared by embodiment 2¹¹Pfu/mL it after) diluting 10 times with SM buffer, sets respectively 1h is acted in 4 DEG C, 37 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C of water-baths.It is (same that bilayer LB plate is spread after action time immediately Embodiment 1), determine phage titre (test of 3 method of embodiment).

15644 thermal stability of SH-St, as shown in A in Fig. 2, abscissa indicates different temperature, and ordinate indicates opposite In 4 DEG C of potency (1.14 × 10⁹Pfu/mL percentage).It still keeps higher activity, 50 DEG C of activity after 40 DEG C of effect 1h It substantially reduces, 60 DEG C of whens lose activity.

(2) ph stability is tested

Phagocytosis body fluid (potency 2 × 10 prepared by embodiment 2¹¹Pfu/mL) 10 times are diluted with SM Buffer.Take pH be 2, 3, each 990 μ l of 4,5,6,7,7.45,8,9,10,11,12,13 SM Buffer adds 10 μ l bacteriophage dilutions.? After placing 1h in 37 DEG C, 10 μ l is taken to mix with 400 μ l methicillin-resistant staphylococcus glucose coccus bacterium solutions (OD600=0.6), After 37 DEG C of incubation 5min, with double-layer agar technique bed board (with embodiment 3).Next day determines pH stability according to plaque number. Each pH experiment is repeated 3 times.

It is tested and is found by ph stability, as shown in B in Fig. 2, abscissa indicates that bacteriophage is in different pH value, indulges Coordinate representation is relative to pH7.45 potency (1.4 × 10⁸Pfu/mL percentage).SH-St 15644 can stablize in pH6-9 Environment in, vigor highest when pH7.45, it is long-term to illustrate that the bacteriophage is suitble to for pH≤4 and then vigor completely loses when >=11 It is stored in neutral environment.

(3) adsorption experiment

Methicillin-resistant staphylococcus glucose coccus is cultivated to logarithmic growth phase.Bacteriophage is mixed with MOI=0.01 ratio Liquid (potency 2 × 10¹¹Pfu/mL) and methicillin-resistant staphylococcus glucose coccus bacterium solution (OD600=0.6, concentration are 10⁸CFU/mL).0 after mixing, when 1,2,3,4,5,10,15,20,25,30min, 100 μ l is respectively taken to add to 900 μ l SM In buffer buffer, mix.Immediately with 4 DEG C, 13000g is centrifuged 1min, sucts 100 μ l of clear liquid and adds to 900 μ l SM In buffer buffer, mix stand-by.Double-layer agar technique determines potency (with embodiment 3).

By SH-St 15644 to itself host's adsorption experiment the study found that abscissa expression is bitten as shown in C in Fig. 2 Thallus and the common incubation time of bacterium, ordinate indicate the bacteriophage percentage not adsorbed by host strain.As SH-St 15644 It mixes and is incubated for host, after 20min on most of phage adsorption to host.

(4) one step growth experiment

Take methicillin-resistant staphylococcus glucose coccus bacterium solution (OD₆₀₀=0.6) embodiment 2 of MOI=0.01 is added in 1mL Phagocytosis body fluid (the potency 2 × 10 of preparation¹¹Pfu/mL it) is managed in 1.5mL EP, mixes 37 DEG C of water-bath 1min.13000g centrifugation 1min abandons supernatant.It is resuspended and is precipitated with 3mL preheated liquid LB.After being mixed by inversion, 100r/min, each time point takes 100 μ l weight Suspension dilutes different multiples and (is diluted to 10 within 0-24 minutes^-3Gradient is diluted to 10 in 27-36 minutes^-4Gradient), it is flat with bilayer Plate method surveys potency (with embodiment 3).

One step growth curve shows that bacteriophage SH-St 15644 is grown in methicillin-resistant staphylococcus glucose coccus The time of generation bacteriophage is 21min, and incubation period is about 12min, and burst times are about 9min, and burst size is about 13PFU.

(5) measurement of external cracking ability

Take methicillin-resistant staphylococcus aureus bacterium solution (OD₆₀₀=0.6), using MOI=10,1,0.1,0 (as control) Phagocytosis body fluid (potency 2 × 10 prepared by ratio mix embodiment 2¹¹pfu/mL).Later, it is put into shaking table shaken cultivation 5 hours (100rpm, 37 DEG C).OD value was measured at 600nm every 30 minutes, recorded its variation, as a result as shown in Figure 3.

The DNA of 7 bacteriophage SH-St 15644 of embodiment is extracted and sequencing

Using phenol-chloroform-isoamyl alcohol method extracting 3 bacteriophage SH-St 15644 of embodiment gene.It is then sent to the Shanghai's style The sequencing of Sen Nuo company.Full length gene 45111bp (shown in SEQ ID NO.1), G/C content 33.35%.Share 64 open readings Frame.

8 bacteriophage SH-St 15644 of embodiment treats infection of staphylococcus aureus mouse recruitment evaluation

(1) experimental animal

Experimental animal is SPF grades of Female nude mices, and 4-6 weeks.It is purchased from through Medical College, Shanghai Communication Univ.'s Experimental Animal Center Shanghai Slac Experimental Animal Co., Ltd..Whole mouse feeding environments are room temperature, and free water is for food.

(2) building of staphylococcus aureus dermapostasis model

Picking monoclonal methicillin-resistant staphylococcus aureus strain (ATCC43300) on Columbia Blood Agar culture medium It is placed in 3ml LB culture medium, 37 DEG C, 200rpm overnight incubation (about 16 hours).1:100 is dilute by volume for overnight bacteria suspension Fresh LB liquid medium is released, 37 DEG C, 200rpm is cultivated to after mid log phase (about 4h), is detected using microplate reader OD₆₀₀Absorbance washed once with sterile PBS, adjust bacterium amount (OD=0.5) with sterile PBS later.

After ethobrom (3-5mg/25g nude mice) anesthesia, contain 1 × 10 in 100 μ l of its dorsal sc injection⁷CFU is resistance to Methicillin staphylococcus aureus suspension.

(3) dermapostasis nude mice is treated using SH-St 15644

(MOI=10) is treated in same position subcutaneous injection 100 μ L embodiment, 3 bacteriophage SH-St 15644 to after bacterium 1h, And control group (identical as treatment group to bacterium amount, but inject the sterile SM Buffer of 100 μ l when treatment) is set.Make for 24 hours after treatment With the length (L) and wide (W), calculating abscess area area (A)=π (L × W)/2 of gage measurement nude mice skin abscess.It later will be naked Mouse euthanasia, solution take dermapostasis, weigh.It is pulverized in sterile PBS, 12000g is centrifuged 5 minutes, siphons away supernatant, is used Precipitating is resuspended in PBS again and bed board carries out bacterium colony counting, and CFU/g is finally calculated.By comparing two groups of abscess areas and CFU/ Whether g is variant, to judge whether Phage therapy is effective.Experimental result: Phage therapy group nude mice skin abscess centre plane Product is 47.32mm², average bacteriological discretion be 3.553 × 10⁷CFU/g；MRSA infected group nude mice skin abscess average area is 150.4mm², average bacteriological carrying capacity be 2.284 × 10⁸CFU/g.Dermapostasis area, the bacterium colony meter of Phage therapy group mouse Number is both less than MRSA infected group (Fig. 4), shows that bacteriophage SH-St 15644 can effectively inhibit skin infection caused by MRSA. This research is for statistical analysis using 6.0 statistical software of GraphPad Prism.It tests to compare between each result and be examined using t.P < 0.05 has statistical significance for difference.Statistical difference is in each chart of this experiment with " * " mark, P < 0.05 *；**P< 0.01；***P<0.001.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications should also regard For protection scope of the present invention.

Sequence table

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agaagattta aagaaagatg tagacaaaat cactaaaatt gatgatgtag atacatctga 3780

attggttgaa acacctaaag aaagtatgtc actagaagaa aaagaaaaaa gagaaaaaat 3840

taaacgcgaa tgcgaaattt taaaaatgac aatgaattat taggaggaaa tgaaatgccg 3900

acattatatg aattaaaaca atccttaggt atgattggac aacaattaaa aaataaaaat 3960

gatgaattga gtcagaaagc aacagatcca aatattgata tggaagacat caaacaacta 4020

gaaacagaaa aagcaggttt acaacaaaga tttaacattg ttgaaagaca agtgcaagac 4080

attgaagaga aagaaaaagc gaaagttaaa gataaaggag aagcttatca atctttaagt 4140

gataatgaga agatggttaa agctaaggca gagttttatc gtcacgcgat tttaccaaat 4200

gaatttgaaa aaccttcaat ggaggcacaa cgtttattac acgctttacc aacaggaaat 4260

gattcaggtg gagataagct cttaccaaaa acactttcta aagaaattgt ttcagaacca 4320

tttgctaaaa accaattacg tgaaaaagct cgtctaacta acattaaagg tttagagatt 4380

ccaagagttt catacacttt agacgatgat gatttcatta cagacgtaga aacagcaaaa 4440

gaattaaaag caaaaggtga tacagtcaag ttcactacta ataaattcaa agtatttgct 4500

gcaatttcag atactgtaat tcatggatca gatgtagatt tagtaaactg ggttgaaaac 4560

gcactacaat caggattagc agctaaagag cgtaaagatg ccttagcagt aagtcctaaa 4620

tctggattag aacacatgtc attttataac ggatctgtta aagaagttga gggagcagac 4680

atgtatgatg ctattattaa cgctttagca gatttacatg aagattatcg tgataacgca 4740

acaatttata tgcgatatgc agattatgtc aaaattatta gtgttctttc aaatggaaca 4800

acaaatttct ttgacacacc agcagaaaaa gtatttggca aaccagtagt atttacagat 4860

gcagcagtta aacctattgt gggagatttc aattattttg gaattaacta tgatggaaca 4920

acttatgaca ctgataaaga tgttaaaaaa ggcgaatatt tgtttgtatt aacagcatgg 4980

tatgatcagc aacgtacatt agacagtgca ttcagaattg caaaagcaaa agaaaataca 5040

ggtccattac ccagctaagc cccaaaaggt taatgtaaca gctaaggcta aatcagctgt 5100

aatatcagcc gaataggggt gatgaaatga gtttagaaga aattaaattg tggttgagaa 5160

ttgactataa tttcgaaaat gatttaattg aaggtctcat tcaatcggct aagtctgaat 5220

tactattaag tggggttcca gattatgaca aagatgactt ggaatacccg cttttttgta 5280

cagcgattaa atatatcatt gcaagagatt atgaaagtcg tggatactca aatgaccaat 5340

ctagaagcaa ggtgtttaat gaaaaaggat tgcaaaaaat gattttgaaa ttaaaaaagt 5400

ggtaggtgat ttttaaatgg aatttaatga atttaaagat cgcgcgtatt tttttcaata 5460

tataaacaaa ggaccatatc cagatgaaga ggaaaaaatg aaattgtata gttgcttttg 5520

taaaatttat aatccttcta tgaaagatag agaaatttta aaagcgactg aatcaaaatc 5580

aggattaacc ataattgtca ggtcttctaa aactgaatat ctaccacaaa caaatcactt 5640

agttaaaatt gacagtgcat tatattccga taaattattc aacattgtag aaataagaat 5700

tgatacacca gatattggct ataatacagt ggttttatca gaaaaatgag tgtagaaatt 5760

aaagggatac ctgaagtgtt gaataaatta gaatcggtat acggtaaaca agcaatgcag 5820

gctaagagtg ataaagcttt aaatgaagca tctgaatttt ttataaaggc tttaaagaaa 5880

gagttcgaga gctttaaaga tacgggtgcc agtatagaag aaatgactaa atctaagcct 5940

tatacaaaag ttggcagtca agaaagggct gttttaattg aatgggtagg ccctatgaat 6000

cgcaaaaaca ttattcactt gaatgaacat ggttatacaa gagatggaaa aaaatataca 6060

ccaagaggtt ttggagttat tgcaaaaaca ttagctgcta gcgaacgtaa gtatagagaa 6120

attataaaaa aggagttggc cagataaatg aatatattaa acaccataaa aggaatttta 6180

ttatctgatg cagagctcaa aacacatata aattctagaa tatactatta caaagtcact 6240

gaaaacgctg aaacttccaa accctttgtt gttattacac ctgtttatga tttgccttca 6300

gactttatgt ctgataaata tctcagtgaa gaatacttaa ttcaaataga tgtagaatct 6360

tcaaataatc agaaaacaat tgatataaca aaacgaataa gatacctgtt atatcaacaa 6420

aatttaattc aagcatctag tcagttagat gcttattttg aagaaactaa acgttatgtg 6480

atgtcgagac gatatcaagg catacccaaa aatatatatt ataaaaatca gcgcatcgaa 6540

taggtgtgct ttttaatttt taaggaggaa ataagcaatg gcagaaggac aaggttctta 6600

taaagtaggt tttaaaagat tatacgttgg agtttttaac ccagaagcaa caaaagtagt 6660

taaactcatg acatgggaag atgaaaaagg tggtacagtt gacctaaata tcacaggttt 6720

agcaccagat ttagtagata tgtttgcatc taacaaacgt gtatggatga aaaaacaagg 6780

tactaatgaa gttaagtctg acatgagtat tttcaatatt ccaagtgatg atttaaacac 6840

agttattgga cgtactaaag ataaaaatgg tacatcttgg gtaggagaga atacaagagc 6900

accgtatgta acagtaattg gcgaatcgga agatggttta acaggtcagc cggtatatgt 6960

agccttactt aaaggtactt ttagtttaga ttcaattgaa tttaaaacac gaggtgaaaa 7020

agcagaagcc ccagaaccta caaaattaac aggtgactgg atgaatagaa aagttgatgt 7080

tgatggaacg tcacaaggta ttgtatacgg ttatcatgaa ggtaaagaag gagaagcaga 7140

attcttcaaa aaagtattcg ttggatacac ggacagtgaa gatcattcag aggattctgc 7200

aggttcgtta cccagctaat ccccaaaatg ttgaagtagc agttaattca aaatctgcaa 7260

cagtttcagc agaatagggg ctttcaaaat aaatcaaagg agaataattt atgactaaaa 7320

ctttaaaggt ttataaagga gacgacgtcg tagcttctga acaaggtgaa ggcaaagtgt 7380

cagtaacttt atctaattta gaagcggata caacttatcc aaaaggtact taccaagtgg 7440

catgggaaga aaatggtaaa gaatctagta aagttgatgt acctcaattc aaaaccaatc 7500

caattctagt ctcaggcgta tcatttacac cagaaactaa atcaattatg gtaaataccg 7560

atgacaatgt tgagccaaac attgcaccaa gcacagcaac gaataaaata ttgaaatata 7620

caagtgaaca tccagaattt gttactgtag atgaaaatac aggagcaatt cacggtgtag 7680

ctgaaggtac ttcagtaatc actgctatgt ctactgatgg aagcgataag tcaggacaaa 7740

tttcagtgac agtaacaaac ggatagggat ttaaggcgca gtatatctgc gtctttttta 7800

tttgaataaa aggagctaat acaatgatta aatttgaaat taaagatcgt aaaacaggaa 7860

aaacagagag ctatacaaaa gaagatgtaa caatgggcga agcagaaaaa tgctatgagt 7920

atttagaatt agtaaatcaa gagaataaaa aagaagcacc taacgcaaca aaaatgagac 7980

aaaaagagcg acagttatta gtagatttat ttaaagatga aggattgact gaagaagatg 8040

ttctgaacaa gatgagtact aaaacttata caaaagcctt acaagatata tttcgagaaa 8100

tcaatggtga agatgaagaa gattcagaaa ctgaaccaga agagatggga aagacagaag 8160

aacaatctca ataaaagaca ttttatcgaa cattaagaaa atacaacgtt tctgtatgga 8220

gcagtatggg tggacattaa ctgaagtcag aaaacagccg tatgtaaaac ttttagaaat 8280

acttaatgaa gagaataaag aagagactga agaaaaacaa agtgaacaaa aagtcattac 8340

aggtacggat ttaagaaaac tttttggaag ctagaaagga ggttaatatg aatgaaaaag 8400

tagaaggcat gaccttggag ctgaaattag accatttagg tgtccaagaa ggcatgaaag 8460

gtttaaagcg acaattaggt gttgttaata gtgaaatgaa agctaatctg tcagcatttg 8520

ataagtctga aaaatcaatg gaaaaatatc aggcgagaat taaggggtta aatgataggc 8580

ttaaagttca aaaaaagatg tattctcaag tagaagatga gcttaaacaa gttaacgcta 8640

attaccaaaa agctaaatcc agtgtaaaag atgttgagaa agcatattta aagttagtag 8700

aagccaataa aaaagaaaaa ttagctcttg ataaatctaa agaagcctta aaatcatcga 8760

atacagaact taaaaaagct gaaaatcaat ataaacgtac aaatcaacgt aaacaagatg 8820

cgtatcaaaa acttaaacag ttgagagatg cagaacaaaa gcttaagaat agtaaccaag 8880

ctactactgc acaactaaaa agagcaagtg acgcagtaca gaagcagtcc gctaagcata 8940

aagcacttgt tgaacaatat aaacaagaag gcaatcaagt tcaaaaacta aaagtgcaaa 9000

atgacaatct ttcaaaatca aatgataaaa ttgaaagttc ttacgctaaa actaatacta 9060

aattaaagca aacagaaaaa gaatttaatg atttaaacaa tactattaag aatcatagcg 9120

ctaatgtcgc aaaagctgaa acagctgtta ataaagaaaa agctgcttta aataatttgg 9180

agcgttcaat agataaagct tcatccgaaa tgaagacttt taacaaagaa caaatgatag 9240

ctcaaagtca tttcggtaaa cttgcaagtc aagcggatgt catgtcaaag aaatttagtt 9300

ctattggaga caaaatgact tccctgggac gtacaatgac gatgggcgta tctacaccaa 9360

ttactttagg gttaggtgca gcattaaaaa caagtgcaga ctttgaaggc caaatgtctc 9420

gagttggagc gattgcgcaa gcaagcagta aagacttgaa aagcatgtct aatcaagcag 9480

ttgacttagg agctaaaacc agtaaaagtg ctaacgaagt tgctaaaggt atggaagaat 9540

tggcagcttt aggctttaat gccaaacaaa caatggaggc tatgccaggt gttatcagtg 9600

cagcagaagc aagtggtgca gaaatggcta caactgcaac tgtaatggct tcagcgatta 9660

actctttcgg tttaaaagca tctgatgcaa atcatgttgc tgatttactt gcgagatcag 9720

caaatgatag tgctgcagat attcagtaca tgggagatgc attgaagtat gctggtactc 9780

ctgcaaaagc attaggagtt tcaatagagg acacttccgc agcaattgaa gttttatcta 9840

actcaggttt agagggttct caagcaggta ctgccctaag agcttcattt atcaggctag 9900

ctaatccaag taaaaataca gctaaggaaa tgaaaaaatt aggtattcat ttgtctgatg 9960

ctaaaggtca atttgttggc atgggtgaat tgattagaca gttccaagat aatatgaaag 10020

gcatgacgag agaacaaaaa ctagctacag tggctacaat agttggtact gaagcagcaa 10080

gtggattttt agccttgatt gaagcgggac cagataaaat taatagctat agtaaatcct 10140

taaagaattc caatggcgaa agtaaaaaag cagcagattt gatgaaagat aatctcaaag 10200

gcgctctgga acaattaggt ggcgcttttg aatcattagc aatcgaagtc ggtaaagatt 10260

taacgcctat gattagagca ggagcggaag gtttaacaaa attagttgat ggatttacac 10320

atctccctgg ttgggttaga aaagcttcag taggattagc actttttggt gcagcaattg 10380

gacctgcagt tcttgctgga gggttattaa tacgtacagt tggaagtgct gctaaaggat 10440

atgcgtcatt aaatagacgt attgctgaaa atacaatcct ttcaaatact aattcaaaag 10500

caatgaaatc tttaggtctt caaacattat ttcttggttc tacaacagga aaaacgtcaa 10560

aaggctttaa agggttagcc ggagctatga tgtttaattt aaaacctata aatgttttga 10620

aaaattctgc aaagctagca attttaccgt tcaaactttt gaaaaacggt ttaggattag 10680

ctgcaaaatc tttatttgca gtaagtggag gcgcaagatt tgcgggtgta gccttaaggt 10740

ttttaacagg acctataggt gctacaataa ctgctattac aattgcgtat aaagttttta 10800

aaaccgcata tgatcgtgtg gaatggttca gaaacggtat taacggttta ggagaaacta 10860

taaagttttt tggtggtaaa attattggcg gcgctgttag aaagctagga gagtttaaaa 10920

actatcttgg aagtatcggc aaaagcttca aagaaaagtt ttcaaaagat atgaaagatg 10980

gttataaatc attaagcgac gatgaccttc tcaaagtagg agtcaacaag tttaaaggat 11040

ttatgcaaac catgggcaca gcttctaaaa aagcgtctga tactgtaaaa gtgttaggga 11100

aaggtgtttc aaaagaaaca gaaaaagctt tagaaaaata tgtgcattat tctgaagaaa 11160

atagcagaat catggaaaaa gtacgtttaa actcgggtca gatatcagaa gacaaagcaa 11220

aaaaactttt gaaaattgaa acggatttat ctaataacct tatagctgaa atagaaaaaa 11280

gaaataaaaa ggaactcgaa aaaactcaag aacttattga taagtatagt gcattcgatg 11340

aacaagaaaa gcaaaacatt ttaactcgaa ctaaagaaaa aaatgacttg cgaattaaaa 11400

aagagcaaga actcaatcag aaaatcaaag aattgaaaga aaaagctttg agtgatggtc 11460

agatttcaga aaatgaaaga aaagaaattg aaaagcttga aaatcaaaga cgtgatatca 11520

ctgttaaaga attgagtaag actgaaaaag agcaagagcg tattttagta agaatgcaaa 11580

gaaacagaaa tgcttattca atagacgaag cgagcaaagc aattaaagaa gcagaaaaag 11640

caagaaaagc aagaaaaaaa gaagtggaca agcaatatga agatgatgtc attgctataa 11700

aaaataacgt caacctttct aagtctgaaa aagataaatt gttagctatt gctgatcaaa 11760

gacataaaga tgaagtaaga aaagcaaaat ctaaaaaaga tgctgtagta gatgttgtta 11820

aaaagcaaaa taaagatatt gataaagaaa tggatttatc cagtggacgt gtatataaaa 11880

atactgaaaa gtggtggaat ggccttaaaa gttggtggtc taactttaga gaagaccaaa 11940

agaaaaaaag cgataaatac gctaaagaac aagaagaaac agctcgtaga aacagagaaa 12000

atataaagaa atggtttgga aatgcttggg acggcgtaaa aagtaaaact ggcgaagcct 12060

ttagtaaaat gggcagaaat gctaatcatt ttggcggcga aatgaaaaaa atgtggagcg 12120

gaatcaaagg gattccaagc aaattaagtt caggttggag ctcagccaaa agttctgtag 12180

gataccacac taaggctata gctaatagta ctggtaaatg gtttggaaaa gcttggcaat 12240

ctgttaaatc gacaacagga agtatttaca atcaaactaa gcaaaagtat tcagatgctt 12300

cagataaagc ttgggcgcat tcaaaatcta tttggagagg cacatcaaaa tggtttagca 12360

atgcatataa aagtgcaaag ggctggctaa cggatatggc taataaatct cgcgcgaaat 12420

gggataatat ttctagtaca gcttggtcga atgcaaaatc cgtttggaaa ggaacatcga 12480

aatggtttag taactcatac aaatctttaa aaggttggac tggggatatg tattcaagag 12540

cccacgatcg ttttgatgca atttcaagtt cggcatggtc taacgctaaa tcagtattta 12600

atggttttag aaaatggcta tccaaaacat atgattggat tagagatatt ggtaaagaca 12660

tgggaagagc tgcggctgat ttaggtaaaa atgttgctaa taaagctatt ggcggtttga 12720

atagcatgat tggcggtatt aataaaatat ctaaagccat tactgataaa aatctcatca 12780

agccaatacc tacattgtct actggtactt tagcaggaaa gggtgtagct accgataatt 12840

cgggagcatt aacgcaaccg acatttgctg tattaaatga tagaggttct ggaaacgccc 12900

caggtggtgg agttcaagaa gtaattcaca gggctgacgg aacattccat gcaccccaag 12960

gacgagatgt ggttgttcca ctaggagttg gggatagcgt aataaatgct aatgacactc 13020

tgaagttaca gcgtatgggt gttttaccaa agtttcatgg aggtacgaaa aagaaagatt 13080

ggctagacca acttaaaggt aatataggta aaaaagcagg agaatttgga gctacagcta 13140

aaaacacagc gcataatatc aaaaaaggtg cagaagaaat ggttgaagca gcaggcgata 13200

aaatcaaaga tggtgcatct tggttaggcg ataaaatcgg cgatgtgtgg gattacgtac 13260

aacatccagg gaaactagta aataaagtaa tgtcaggttt aaatattaat tttggaggcg 13320

gagctaacgc tacagtaaaa atagctaaag gcgcatactc attgctcaaa aagaaattag 13380

tagacaaagt aaaatcgtgg tttgaagatt tcggtggtgg aggcgatgga agctatctat 13440

ttgaatatcc aatctggcaa agatttggac gctacacagg tggacttaac tttaatggcg 13500

gtcgtcacta tggtatagac tttggtatgc ctactggaac aaacgtttat gccgttaaag 13560

gtggtatagc agataaggta tggactgatt acggtggcgg taattctata caaattaaga 13620

ctggtgctaa tgaatggaac tggtatatgc atttatctaa gcaattagca agacaaggcc 13680

aacgtattaa agctggtcaa ctgataggga aatcaggtgc tacaggtaat ttcgttagag 13740

gagcacactt acatttccaa ttgatgcaag ggtcacatcc agggaatgat acagctaaag 13800

atccagaaaa atggttgaag tcacttaaag gtagtggcgt tcgaagtggt tcaggtgtta 13860

ataaggctgc atctgcttgg gcaggcgata tacgtcgtgc agcaaaacga atgggtgtta 13920

atgttacttc gggtgatgta ggaaatatta ttagcttgat tcaacacgaa tcaggaggaa 13980

atgcaggtat aactcaatct agtgcgctta gagacatcaa cgttttacag ggcaatccag 14040

caaaaggatt gcttcaatat atcccacaaa catttagaca ttatgctgtt agaggtcaca 14100

acaatatata tagtggttac gatcagttat tagcgttctt taacaacaga tattggcgct 14160

cacagtttaa cccaagaggt ggttggtctc caagtggtcc aagaagatat gcgaatggtg 14220

gtttgattac aaagcatcaa cttgctgaag tgggtgaagg agataaacag gagatggtta 14280

tccctttaac tagacgtaaa cgagcaattc aattaactga acaggttatg cgcatcatcg 14340

gtatggatgg caagccaaat aacatcactg taaataatga tacttcaaca gttgaaaaat 14400

tgttgaaaca aattgttatg ttaagtgata aaggaaataa attaacagat gcattgattc 14460

aaactgtttc ttctcaggat aataacttag gttctaatga tgcaattaga ggtttagaaa 14520

aaatattgtc aaaacaaagt gggcatagag caaatgcaaa taattatatg ggaggtttga 14580

ctaattaatg caatcttttg taaaaatcat agatggttac aaggaagaag taataacaga 14640

ttttaatcag cttatatttt tagatgcaag ggctgaaagt ccaaacacca atgataacag 14700

tgtaactatt aacggagtag atggtatttt accgggcgca attagttttg cgcctttttc 14760

attagtatta aggtttggct atgatggtat agatgttata gatttaaatt tatttgagca 14820

ttggtttaga tctgtgttta atcgcagaca tccttattat gttattactt ctcaaatgcc 14880

tggtgttaaa tatgcagtga atacagctaa tgttacatct aatttaaaag atggttcttc 14940

aactgaaatt gaagtaagtt taaatgttta taaagggtat tctgaatcag ttaattggac 15000

cgatagcgag ttcttattcg actctaattg gatgtttgaa aatggaattc ctcttgattt 15060

cacacctaaa tatactcata catcaaatca atttactatt tggaacggtt ctactgatac 15120

gataaatcca cgattcaagc acgatttgaa aatattaatt aatttaaatg cgagtggagg 15180

atttgaactg gttaactata caacaggtga tatttttaag tacaacaaaa gtatagataa 15240

aaacactgat tttgttttag atggtgtgta tgcatatcga gatataaata gagtgggaat 15300

tgatacaaat agaggcatta taacattagc gccaggtaaa aatgaattta agattaaagg 15360

agacgtcagt gatattaaaa ctacatttaa gtttcctttt atttataggt aggtgattta 15420

atggattatc atgatcattt atcagtaatg gattttaatg aattgatttg tgaaaattta 15480

ctagatgtag attatggttc ttttaaagaa tattatgaac tgaatgaagc taggtacatc 15540

acctttacag tttatagaac tactcataat agttttgttt ttgatttatt gatttgtgaa 15600

aacttcataa tttatcatgg tgaaaaatat acaattaagc agacagcgcc aaaggttgaa 15660

ggtgataaag tttttattga agttacggca tatcacataa tgtatgaatt tcaaaatcac 15720

tcagtggaat caaataagct tgatgacgac agtagcgaaa ctggtaaaac gccagaatac 15780

tctttagatg agtacttaag atatggattt gcaaatcaaa aaacttcggt caaaatgacc 15840

tataaaataa ttggagattt taagcgaaaa gtaccgattg acgaattagg taacaaaaac 15900

ggcttagaat actgtaaaga agcggtagac ctgtttggct gtataattta cccaaatgat 15960

acagagattg gtttttattc tcctgaaaca ttttatcaaa gaagcgagaa agtgattcga 16020

tatcaatata atactgatac tgtatctgca actgtcagta cattggaatt aagaacagct 16080

ataaaagttt ttggaaaaaa gtatacagct gaggaaaaga aaaattataa tcctattaga 16140

acaactgaca ttaaatattc aaatggtttt ataaaagaag gtacttatcg taccgcaaca 16200

attgggtcta aagctactat taactttgat tgcaagtatg gtaatgaaac agttagattt 16260

acaataaaaa agggctctca aggtggaata tataagttga ttttagacgg caagcaaatt 16320

aagcaaattt cttgttttgc taagtcggtt cagtctgaaa caatagattt aataaaaaat 16380

attgataaag gcaagcacgt tttagaaatg atatttttag gagaagaccc caaaaataga 16440

attgatatat cttcaaataa aaaagctaag ccttgtatgt atgttggaac tgaaaaatca 16500

acagtcttaa atttaattgc tgacaattca ggtcgcaatc aatacaaagc aattgtcgac 16560

tacgtcgcag atagtgcaaa gcagtttggg attcgatatg ctaatacgca aacaaatgaa 16620

gatatcgaaa cacaggataa gttgttagaa tttgcaaaaa agcaaataaa tgatactcct 16680

aagactgaat tagatgttaa ttatataggt tatgaaaaaa tagagccaag agatagcgta 16740

ttctttgttc atgaattaat gggatataac actgaattaa aggttgttaa acttgatagg 16800

tcacatccat ttgtaaacgc aatagatgag gtgtctttca gcaatgaaat aaaagatatg 16860

gtacaaattc aacaagcgct taacagacga gttattgcac aagataatag atataactat 16920

caagcaaatc gtataaatca tttatacact agtactttga attctccttt cgagacaatg 16980

gatataggga gtgtattaat ataatggcaa cagaagaagt taaaatcaaa gcgctacttg 17040

aaaacgataa acagtacttt ccagctacac attggaaagc tataaatggg ataccttatg 17100

caggcagtag tgatattgat ggattgcctc aagacggtat catttcggta gatgataaaa 17160

ataaattaga taatttaaaa ataggcgaag caggaattat tcaaaatagc attgtacaga 17220

aatccccaaa cggtaaattg tggaaaataa cagttgacga tagtgggaaa cttggtacag 17280

tgctatttta ttagaaagga aggtgcatta tggaaaattt gtatttaata aaggatttgg 17340

gagctttagc aggtcgagat tatagagcta aggaaataca aaacttacaa agaatagagc 17400

aatttgcgct tggcttgaca acagagttta agttgcatca gaaagctaaa acaattcaac 17460

acttcgctga gcaaatttat tataatggta gatcgcaagc agcagtaaac aaatctttac 17520

aaagtcaaat taacgcactt gttgtggcac cacgtaataa cagtgctaat gagattgttc 17580

aagctcgagt taatgtaaac ggcgaaacct ttgacacatt aaaagaacat ttagacgatt 17640

gggaaaccaa aactcaaatt aataaagagg aaactataag agaattaaat aagaccaaac 17700

aagaaattct tgatatcgag tatcgttttg aacctgataa gcaagaattt ttatttgtga 17760

cagaacttgc acctcttaca aatgcagtaa tgcaatcctt ctggtttgat aatagaacag 17820

gcatagtata catgacacaa gctagaaata atggctatat gctaagtcgt ttaagaccta 17880

atggtcaatt tatagacagc tcattgattg taggtggggg tcatggtaca cataacggtt 17940

atagatatat tgatgatgag ttatggattt atagttttat cttaaatggt aataatgaga 18000

atacattagt tcgtttcaag tatacgccta atgtggaaat tagctatggc aagtatggta 18060

tgcaagatgt atttacagga cacccagaaa aaccctacat cacccctgtc ataaatgaaa 18120

aagaaaataa aattctatac agaattgaga gacctagaag tcagtgggaa cttgaaaact 18180

caatgaatta tatagagata agaagtttag acgatgttga taaaaatatt gataaagttt 18240

tgcataaaat cagtatccct atgagactaa caaacgaaac ccaaccaatg cagggtgtga 18300

cttttgatga aaaatacttg tattggtata caggagacag taatccaaat aatagaaact 18360

atttaacggc tttcgattta gaaacaggag aagaagcgta tcaggttaat gctgactatg 18420

gtggaacact agattcattt cctggcgaat ttgcggaagc agaaggtttg caaatatact 18480

atgacaaaga tagtggtaaa aaagctttga tgctaggtgt tactgtcggt ggtgatggaa 18540

atagaacaca tcgtattttc atgattgggc aaagaggtat tttagaaata cttcactcaa 18600

gaggcgttcc ttttatcatg agtgacacag gtggtagagt taaaccttta ccaatgaggc 18660

ctgataaact taagaatctt gggatgttaa cagagccagg tctttactat ttatacactg 18720

atcatacagt tcaaatcgat gatttcccat taccaagaga atggcgtgat gcaggttggt 18780

tcttggaagt taagccacca caaactggcg gtgatgtaat tcagatattg acgcgtaata 18840

gttatgcaag gaatatgatg acttttgaaa gggttctttc tggaagaact ggagacattt 18900

cggactggaa ttatgtgcct aaaaatagtg gtaaatggga gagagtacct tcattcatca 18960

caaaaatgtc agatattaac atagtaggta tgtcgtttta tttaactacg gatgatacaa 19020

aacgttttac agattttcca actgaacgta aaggggtagc aggttggaac ttatatgtag 19080

aagcttctaa cacaggtggt tttgttcata ggctagttcg taatagtgtt acagcatctt 19140

gtgaaatatt attgaagaac tatgatagta aaacaagttc agggccatgg actttacacg 19200

aagggagaat tataagttaa tgagtaattt agagaaatct gtagctataa atttagaaaa 19260

cacagcgcat tatgaaaata tttcaaatct agatataact tttagaacag gagagagtga 19320

ttcttctgtt cttcttttta atatcattaa aaataatcaa ccgttattat tgagtgaaga 19380

aaatatcaaa gcacgaatag cgattcgagg taaaggagtc atggtagttg ctccactaga 19440

aatattagat ccatttaaag gtattttaaa atttcaatta cctaatgatg taattaaacg 19500

agatggaagt tatcaagctc aagtttcggt tgcagaatta ggtaattcag acgtggtagt 19560

tgtcgagaga actatcacat ttaacgttga aaaaagtttg tttagcatga ttccatctga 19620

aacaaaatta cactatattg ttgaatttca ggaattagaa aaaactatta tggatcgtgc 19680

gaaagcaatg gacgaggcta taaaaaatgg tgaagattat gcgagtctga ttgaaaaagc 19740

taaagaaaaa ggtctatcag atattcaaat agcaaaatct tcaagtatag atgaattaaa 19800

gcaacttgct aatagccata taaccgattt ggaaaataaa gctcagtctt attcaagaac 19860

atttgatgag cgaaagcgat atatggatga gaaacatgag gctttcaagc agtcagtgaa 19920

tagcggcggt ttagtcacaa gtggttcaac ttcaaattgg caaaaatcta agattactaa 19980

agacgacggt aaaataacac agattactgg atttgatttt aataatccag aacaaagagt 20040

aggagattca acccaattta tttacgtctc acaagccata aattatccaa gaggcgtaag 20100

cactaatggt attgtcgaat atttagtagt aacttccgac tacaaacgta tgacatatcg 20160

tccaaatggt acaaataaag tatttgttaa gagaaaagaa gcaggttctt ggtctgattg 20220

gtcagaatta gcccttaatg attacaatac gccttttgaa actgttcaaa acgcgcaatc 20280

aaaagctaat acggctgaaa gcaacgccaa actatacaca gatgacaagt ttaataaaag 20340

atattcagtt atttttgatg ggacggcaaa tggcgttggc tcaacattat atcttaatga 20400

aagtttagat caatttattt tgttaatttt ttatggaact tttccagggg gagattttac 20460

tgagtttggc aacccctttg gtggcggaaa aatttcattg aacccatcaa atttaccgga 20520

taatgatggt aatggtggag gcgtttatga gtttggatta actaaatcta gtcgtacatc 20580

tttaacgata tcgaacgatg tttattttga tttaggaagt caaagaggtt ctggtgcaaa 20640

tgcaaataga ggaacaatca acaaaattat aggagtgaga aaataatgca aatattagtt 20700

aacaagcgta atgagataat ttcatacgct atcattggtg gctttgaaga aggtattgat 20760

attgaaaatt taccagaaaa tttctctcaa gtttttagac ctaaagcctt taaatattca 20820

aatggggaaa tagtttttaa cgaagattat tcagaagaaa aagatgactt gcatcaacag 20880

attgacagtg aagaacaaaa cacagtcgct tctgatgaca tcttacgaaa aatggttgct 20940

agtatgcaga aacaagttgt tcaaagtaca aagttatcga tgcaagttaa taagcaaaat 21000

gcactaatgg caaaacaact tgtgacactt aataaaaaat tagaagaggt taaaggagag 21060

actgaaaatg cttaaattaa tttcaccaac attcgaagat attaaaacat ggtatcaatt 21120

gaaagaatat agtaaagaag atatagcgtg gtatgtagat atggaagtta tagataaaga 21180

ggaatatgca attattacag gagaaaagta tccagaaaat ctagagtcat aggttataat 21240

cttatggctt tttaatttga ataaagtggg tggtgtaatg tttggattta ccaaacgaca 21300

cgaacaagat tggcgtttaa cgcgattaga agaaaatgat aagactatgt ttgaaaaatt 21360

cgacagaata gaagacagtc tgagaacgca agaaaaaatt tatgacaagt tagatagaaa 21420

tttcgaagaa ctaagacgtg acaaagaaga agatgaaaaa aataagaaag aaaacgacaa 21480

aaatatacgc gatatgaaaa tgtggattct cggtttgata gggactatct tcagtacgat 21540

tgtcatagct ttactaagaa ctatttttgg catttaaagg aggtgattac catgcttaag 21600

ggaattttag gatatagctt ttggtcgtgt ttctggtttg gtaagtgtaa gtaatagtta 21660

agagtcagtg cttcggcact ggctttttat tttggataaa aggagcaaac aaatggaagc 21720

aaaagtaatg gcaagatacg ttgtattgat tttagcgtta gtcaatcagt ttttagcgaa 21780

taaaggtata agcccgatac cagtagatga agaaagtatt tcatcgatca tcttaacagt 21840

tattgcttta tacactacgt ataaagacaa tccaacatca caagaaggac gttgggcgaa 21900

tcaaaaatta aagaaatata aagcagaaag taaatataga aaagcaacag gacaagcacc 21960

tatcaaagaa gtgatgacac ctacaaatat gaacgacaca aatgatttag ggtaggtggt 22020

tgatatatgt taatgacaaa aaatcaagca gaaaaatggt tcgacaattc actagggaaa 22080

caatttaatc cagatggttg gtatggattt cagtgttatg attatgccaa tatgttcttt 22140

atgttagcga caggcgaaag gctgcaaggt ttatatgctt ataacatccc gtttgataat 22200

aaagcgaaga ttgaaaaata tggtcaaata attaaaaact atgacagctt tttgccgcaa 22260

aagttggata ttgtcgtctt cccatcgaag tatggtggtg gagctggaca cgttgaaatt 22320

gttgagagcg caaatttaaa tactttcaca tcatttggtc aaaactggaa cggtaaaggt 22380

tggactaatg gcgttgcgca acctggttgg ggtcctgaaa ctgtgacaag acatgttcat 22440

tattatgaca atccaatgta ttttattagg ttaaacttcc ctaacaactt aagcgttggc 22500

aataaagcta aaggtattat taagcaagcg actacaaaaa aagaggcagt aattaaacct 22560

aaaaaaatta tgcttgtagc cggtcatggt tataacgatc ctggagcagt aggaaacgga 22620

acaaacgaac gcgattttat acgtaaatat ataacgccta atatcgctaa gtatttaaga 22680

catgcaggac atgaagttgc attatatggt ggctcaagtc aatcacaaga catgtatcaa 22740

gatactgcat acggtgttaa tgtaggaaat aataaagatt atggattata ttgggttaaa 22800

tcacaggggt atgacattgt tctagagatt catttagacg cagcaggaga aaatgcaagt 22860

ggtgggcatg ttattatctc aagtcaattc aatgcggata ctattgataa aagtatacaa 22920

gatgttatta aaaataactt aggacaaata agaggtgtaa cacctcgtaa tgatttactg 22980

aacgttaatg tatcagcaga aataaatatc aattatcgtt tatctgaatt aggttttatt 23040

actaataaaa aagatatgga ttggattaag aagaattatg acttgtattc taaattaata 23100

gctggtgcga ttcatggtaa gcctataggt ggtttggtag ctggtaatgt taaaacatca 23160

gctaaaaacc aaaaaaatcc accagtgcca gcaggttata cacttgataa gaataatgtg 23220

ccttataaaa aagagactgg taattacaca gttgccaatg ttaaaggtaa taacgtaagg 23280

gacggctatt caactaattc aagaattaca ggtgtattac ctaataacgc aacaatcaaa 23340

tatgacggcg catattgcat caatgggtat agatggatta cttatattgc taatagtgga 23400

caacgtcgct atattgcgac aggagaggta gacaaggcag gtaatagaat aagtagtttt 23460

ggtaagttta gcacgattta gtatttactt agaataaaaa ttttgctaca ttaattatag 23520

ggaatcttac agttattaaa taactatttg gatggatgtt aatattccta tacacttttt 23580

aacattactc tcaagattta aatgtgcgta actggcaggt acttcggtac ttgcctattt 23640

ttttatgtta tagctagcct acgggctagt tttttgttat gatgtgttac acatgcatca 23700

actatttaca tctatccttg ttcacccaag catgtcactg ggtgtttttt cttacgatag 23760

agagcatagt tttcatacta ctccccgtag tatatatgac tttagcattc ccgtataaca 23820

gtttacgggg tgctttttat gttatactta cttttatata gtaggagtga actatatagc 23880

cgggcagagg ccatgtatct gactgttggt cccacaggag acatcttcct tgtcatcact 23940

cgatacatat atcttaacaa catagaaatg ttacattcgc tacaaccgta tcttaatcga 24000

tacggttata tttattcccc tacaaccaac aaaaccacag atcctattaa tttagggttg 24060

tggttatttt tgtgtttttt cggggcaaaa aaagggcaga ttatttgaaa aagggcaaac 24120

gcttgtggaa aatctaaaag gttaaaaata ataaagaact tggtataaca agggttttat 24180

acatttgcgt acaacgacga aatgtcaatt taccatcaca ttatgatgat atgtttattt 24240

caagaaaagc tttaacgcca gtgttctcaa gcgttttata aagcttgtaa aaaatataag 24300

ggcaaaaaaa gggcggattt aagctaactt ggaatgtttt cgagtttttg agttagttct 24360

ctatccattt tttcagttac atgagtatat atgcgaatgg ttgttttttc atctacatgt 24420

cctacccttt tcataattgc ttttaaagaa acattcattt ctactaataa agttatgtgt 24480

gtatgtctaa atgtgtgcgt ggtaactttc ttattcatat ttaaagcttt tgtagttttc 24540

ttaagcacac cggcgatttg attattacat aaaggattcc ctttttttgt tgtgaatatg 24600

aaccctctgt caacatagct cgaattccat cttttcaaca ttttgttttc cagtattatc 24660

tttttaaaaa tttctacggt tctagaattg atgctaatac ttctttttga acttatagtc 24720

tttgtagtgt ctttgtatcc gaatccttct tcgtatttaa tgcggtgaat tgtacctgtt 24780

atatttatag ttttgtttaa taaatctata tctttttcct gcagtgcttg tagttctcct 24840

atgcgcatac cagttaaagc ctgtacttct aagatgctgg caattaaaat gcgatttcgc 24900

ttgtgtaact tattatcatt tagtatatga tcacgtatct gtaggacttg gttcatttct 24960

aaatagttgt acattttaga ttcatctttt tcgatatcct ctattgtttt tcttctttta 25020

ggaattttga cattagttaa caaatattca tttggataat tgtaaaattt aactgcatat 25080

ttaatagctc ctttcatatc tccgagttga cgggttactt gattttgaga atagatatct 25140

gataatttat taataaatat ctgcatatat tttgtatcaa gtttgtttaa aagcaagttc 25200

tcagagcagt atcgtttaat gtttttaatt cttattttta tattattaag agtagtcaac 25260

tttgaacctg atgtttttat atgatattca agccattcat ctaatagcgc gtgaaaagtc 25320

aaagttttta attcgcttga cgacttgttg ttcagttttt cttttatttt ttcttctaaa 25380

cgaaacattg cttctttttg agattgtttt gtattcttgt tcaacacaac acttacgcgc 25440

ttccatttat ctgtgtatgg atctttgtac ttctcgtaat atctgtattt agtttcgtta 25500

tttttgtttt taaatttttc aatccacatg tttatacctc ctgtaggaac gtacgttctg 25560

taaatttgta aaaaataata agggtaggtg ggctacccaa aatttagtac taggtactaa 25620

atatgttata ataaaataaa aagtaggtga taagatgact caatttctag gggcgcttct 25680

tcttacagga gttttaggtt acataccata taaatatcta acaatgatag gtttagttag 25740

tgaaaaaaac aaggttatca atactcctgt attattgatt ttttctattg aaacatgttt 25800

gatatggttt tatagtttta taatttttaa taatgttgat ttaaaaaatt tgaatttaat 25860

tcagttgctt acaggtctaa aagcaaatat tttgtttcta tttatttttg ttttaacagt 25920

gtttgtattt aatcctttaa ttgttaaatt tattatctgg ttaattaata taaccagaaa 25980

gtttatgaaa ttggattgta taagcttatt agacaaaaga gacaagttgt ttaataacaa 26040

cggtaaacca gtatttatag ttataaaaga ctttgaaaac agaatcattg aagagggtga 26100

acttaaaacc tataattcag ctggtagcga tttcgattta ctagaggttg agcgacaaga 26160

tttcaaagta tctgatttac cgtcaaacga tgaattgtat attaaacata cacttgtaga 26220

ccttaaacaa caaattaaat tggatttata tttaatgaat gaatattaat cttttttctt 26280

agctttttct gataaagtgc tttttaagtt ttcgctggca cccggctttt caaaactttt 26340

gtttattggg ttactacggg tagcttcttg ttttttgttt ttatccgcca taaaattctc 26400

accaccattc aacgtctaca ctagtaggcg tttttgaatt tttttttaat cctttttctc 26460

gtcacgctat ataggtactt ttaatcgtaa ttaaagccaa gtttatttaa aatctctaaa 26520

taatctgacg actttgcccg taatgtttac gtcatttatt tttgacattg ggtagcttct 26580

atctttaatg gtaacgtaat tagataatcg ctttagggta aatgtgtcat ctgagttaaa 26640

tataatttca caaaaatatt catcagtcgc agttccgttt tcaggacagt aaacagtaat 26700

tatatctctg tcaaagataa agtccattac atcatcatcc tgatctaaaa tgtagcaaac 26760

agtcatatct tcattttcaa ctcttcttgc gctatttata gtagttattg gttgggcaac 26820

taatttgcct tttgcctctg tgtatctttt tggaaattcc acataattca ttaaatcgat 26880

tgttttattt gtttccatat tagctcttct ttcttatatt gttttattag ttaatattgg 26940

ctcctggtac tgcgctagca ccagctcttg cttcctcttc ttttcctttt ttgtaatctt 27000

caaaagcttt agcctgttca tctttagtcc agccaggcga aacgacatac tcatcattta 27060

aattagtatt ttgtgaatcg tttttattta tatttgcagg agtattatta gatggatctg 27120

aattattatt aagttgattg ttttgtgatt ggttttgatt ggttgcattg ttttgtacgc 27180

tattaatagg tggattttta tcttcaactt ttttcgtttt ttctttttgc ttttttggtt 27240

tgctatcttt atgtttttgt gtatgagact ttttattttc ttcttttttt ggtttttctt 27300

tttcaccaca agctgttaaa gctaatgtac ttactagtag cagtccgatt acttttttca 27360

tatgtatctc tcctttgttt atatttcctt atatttaaaa actctcaacg gctcaaatgt 27420

gatcgaatac tcgccatagt gagttccaat accatatatc tttttatatt gttctattgc 27480

ttctaatatg tattcttcgc ttaattgtag atactcagac aattcataca agttacgtac 27540

gccataatta taagcttcta caatttcgcg taatggaaca gctgagataa agccgtgtcg 27600

tcttgcgtaa ttttcgaact tgcgattgtt gaatttcgag taatcggcta tatcaccgta 27660

tgtaagttta ttatgtgcta attcttcaaa gagaatccct gctttttctc tatctgataa 27720

accacgcttt atcaaaatta aatctcctaa ccataccccg tctaaattat ctggaagcac 27780

atcagcctct cttatttcaa tataatcatg ttgtattaaa gtttcttcat ataatcccat 27840

ctgatacatc ctttacttac gtttacttct tatataatct gcataatcta aaactctttg 27900

ccactcgtca tcagttaatt ctccttctaa atgagctgca cgatgttgta cttcgttttc 27960

tgtttgtcta ttttttaata gtaaatattc tggggtaact ttcaatgcat tggcaatctc 28020

agctatatcc tccataggta tttttctgct accgttttca tatcgggata aggtagattt 28080

attgacacct atcttagttg caaaatcagt taaattcaca ttattctctt ttcgtagttg 28140

tttgattaat ttacctattt ccgctgaagt tctcatttca aatttacctc cgttttattt 28200

ataacagtat aataacactt ttccatatag gaaacaacta gcattttaaa agaataaaaa 28260

atatttttcg agatttttgt tgacaattag gaaacttagg tttagtattg agttaacttc 28320

aaaaaacgga ggtgagcaaa tgtatgagtt caacgtcaaa agaatgaaag ctgaacgcat 28380

tgctaaaggc atttcgattt ctgatatggc aaaaaaatta ggaatgacac caggaactta 28440

ttcaaaaaaa gaaaacgggc atattagaat taatgttgac gatttagcaa aagtaattga 28500

agtactagaa ttgccacaag ataagtgcgg tatttttttt acttatagag tttccaaaat 28560

gtcaacagaa caaaaacaaa catcttaaaa ggaggacact atggaacaaa taacgttaac 28620

caaagaagag ttgaaagaaa ttatagcgaa agaagttaga aatgctataa aaggcgagaa 28680

accaatcagc tcaggtgcaa ttttcagtaa agtaagaatc aataatgacg atttagaaga 28740

aatcaataaa aaactcaatt tcgcaaaaga tttgtcacta ggaagattga ggaagcttaa 28800

tcatccgatt ccactaaaaa agtatcagca tggcttcgaa tcaattcatc aaaaagctta 28860

tgtacaagat gttcatgatc atattagaaa attaacatta tcgatttttg gagtgacgct 28920

taattcagat ttgagtgaaa gtgaatataa cctagcagca aaagtttatc gagaaatcaa 28980

aaactattat ttatatatct atgaaaagag agtttcagaa ttaactatcg atgatttcga 29040

ataaaggagg ttcaacaaat gttacaaaaa tttagaatcg ctaaagaaaa aagtaaatta 29100

aaactcaatt tactaaaaca tgcaaacagt aatttagaaa caagaaacaa ccctgaactg 29160

ttgcgagcag ttgcagagtt gcttaaagag attaatcgat aaattctatg aattcgattt 29220

tagctgaagc gatagctact attttgtctc caacaaaagt atatgagcca ttagtgaaca 29280

aggaactttt aattttttct tttgatattt caacagttcc gcgatgacct gactttatca 29340

ctttttctaa attatcgatt tcaacaaatt tatcattaga aagatataaa caagctttca 29400

tacttatcac ctccttaggt tgataacaac attatacacg aaaggtggaa caacaaatga 29460

acattcaaga agcaactaag atagctacaa aaaatcttgt ctctatgaca cggaaagatt 29520

ggaaagaaag tcatcgaact aagatattac caacaaatga tagtttttta caatgcatca 29580

tttcaaatag cgatgggaca aaccttatca gatattggca accttcagcc gatgacctca 29640

tggcaaatga ttgggaagtt ataaacccaa ctagagacca ggaattattg aagcaatttt 29700

agaaatgcta tcaatgatac tttttaaatt gtttttaaac tcattttcaa agtaaacaac 29760

agtcttgtct gaaattgtta catgataaat agtgttacta gcatacacgc cgtttaggaa 29820

cccagagttt ttaagtttat ttaaatcgta ttttacatct tcgaaatgta gtttttgaaa 29880

atactttgta tgtatatctt tagcacttcc aaaattattg caggttaatt taaccgaacc 29940

taactttaca cattctaaat aatctttgta gagtacggac aagatatatt gttggtcttt 30000

agtaagtgta tcaaattcat cagatatcaa gggcatgtta tcacctcctt aggttgataa 30060

caacattata cacgaaagga gcataaacaa atgaacacaa gatcagaagg attgcgtata 30120

ggcgtcccac aagtttctag caaagctgat gcttcttcat cctatttaac ggaaaaggaa 30180

cgtaacttag gagcggaaat attagagctt attaaaaaaa gtgattacag ctacttagaa 30240

ataaacaaag ttttctatgc attagataga gaacttcaat acagggcgaa taataacaaa 30300

ctttaacatt tatctaaagg agtgatagag atgccaaaaa tcataatacc accaacacca 30360

gaaaacacat atcgaggcga agaaaaattt gtgaaaaagt tatacgcaac acctacacaa 30420

atccatcaat tgtttggagt atgtagaagt acagtataca actggttgaa atattaccgt 30480

gaagataatt taggtgtaga aaatttatac attgattatt cagcaacggg aacattgatt 30540

aatatttcta aattggaaga gtatttaatc agaatgcata aaaaatggta ttaggaggat 30600

attaaatgag cgacacatat aaaagctact taatagcagt actgtgcttc acagtcttag 30660

cgattgtact catgccgttt ctatacttca ctacagcatg gtcaattgcg ggattcgcaa 30720

gtatcgcaac attcacattt tataaggaat acttttatga agaataaaga aactgctact 30780

tgttggagca agtaacagtg caagatgagc aattgtctta aataattata taaggagtta 30840

ttaatatgac cttacaacaa aaaatactat cacattttgc aacatatgac aatttcaatt 30900

ctgatgatgt tgttgaaaat tttgggatat ctaaaacaca tgcaaaatcc actctttcaa 30960

aacttaagaa aaaaggaaag attgaaatgg aaagttgggg tatctggcgt gttattgaat 31020

cgcaattgca tttaagtgta gtcgaacgta aaaaagaaat tttagaagaa caatttgaat 31080

tgttagcaag attaaatgaa caaagtgatg accctagaga aatagaagaa cgtatcaagt 31140

taatgattcg tctagctaac caattttaag gaggagttaa tcaatggcag tattagaaga 31200

tatttttgaa gaattaaaac tattaaataa gaatttacgt gtgttaaaca ctgaactgtc 31260

aactgtagat tcatcaattg tacaagagaa agttaaagaa gcaccaatgc caaaagaaga 31320

aacagctcaa ctggaatcaa ttgaagaagt taaggaaact tctgctgatt tgactaaaga 31380

ttatgtttta tcagtaggaa aagagttcct taaaaaagca gacacttctg ataagaaaga 31440

atttagaaat aaacttaacg aacttggtgc ggataagcta tctactatca aagaagagca 31500

ttatgaaaaa attgttgatt tcatgaaggc gagaattaat gcatgaaact agatcactca 31560

aatagagctc atgcaaagct tagtgcaagt ggagcaaaac aatggctaaa ctgtccaccg 31620

agtattaagg caagtgaagg tattgcagat aaaagttcag tttttgctga agaaggtaca 31680

ttcgctcatg agttaagtga gttatatttc agtcttaaat atgaaggcct aacacagttt 31740

gagtttaata aagcttttca aaattataag cgaaatcaat attacagtga agagttgcgc 31800

gaatatgttg aagagtacgt agctaatgta gaagaaaagt ataacgaggc tttgagtaga 31860

gatgacgatg taatagcttt atttgaaaca aaattggatt taggtaaata cgtccctgaa 31920

tcttttggta ctggtgatgt cattatattt tcaggtggtg tacttgaaat tattgacctt 31980

aaatacggta aaggcattga agtttcagct atagataatc ctcaacttag attatatggc 32040

ttgggcgcat atgaactgct tagtttaatg tatgacattc atacaattcg catgactatc 32100

atacaacctc gaatagataa cttttctact gaagagttac caatatcaag attacttcaa 32160

tggggaaccg attttgttaa accattagcc agacttgctt ataacggtga aggtgagttt 32220

aaagcaggta gtcattgtag attctgtaag ataaagcatt catgtagaac acgtgcagaa 32280

tacatgcaaa atgtgcctca aaagccacca catttgttaa gtgatgaaga gattgcagaa 32340

cttttatata aactgcctga tatcaaaaaa tgggctgatg aagtagaaca atatgcacta 32400

gatcaagcaa aagaaaatga taaaaactat cctgggtgga agcttgtaga aggtcgttcg 32460

cgaagagtga taactgatac aaaagcaacg cttgaaaagt tagttgaaga gggttataaa 32520

cctgaagata ttacagaaac caagttactt agcattacga atttagaaaa attaatcggc 32580

aaaaaagcat tttctaaaat tgcagaaggc tttatagaaa agccgcaagg taaattaaca 32640

cttgctactg agtcagataa aaggccagct ataaagtcat ctgctgaaga tgattttgac 32700

aaactataaa aatttaaaag gacggtatat aaacatgaaa gcaaaagtat taaataaaac 32760

taaagtgatt acaggaaaag taagagcatc atatgcacat atttttgaac ctcacagtat 32820

gcaagaaggg caagaagcaa agtattcaat cagtttaatc attcctaaat cagatacaag 32880

tacgataaaa gccattgaac aagctataga agctgctaaa gaagaaggga aagttagtaa 32940

atttggaggc aaagttcctg caaatctgaa acttccatta cgtgatggag atactgaaag 33000

agaagatgat gtcaattatc aagacgctta ttttattaac gcatcaagca aacaagcacc 33060

tggtattatt gaccaaaaca aaattagatt aacggattct ggaactgttg taagtggtga 33120

ttatattaga gcttcaatta atctatttcc tttcaacaca aatggtaata agggtatcgc 33180

agttggattg aacaacattc aacttgtaga aaaaggcgaa cctcttggcg gtgcgagtgc 33240

agcagaagat gatttcgatg aattagacac tgatgatgag gatttcttat aagtcaatag 33300

gtggggtttt agccccactt taattttaaa gaaattgagg tgtcaagaat ttgaaattta 33360

tgaatataga tattgaaaca tatagcagta atgatatttc gaaatgtggt gcctataaat 33420

acacagaagc tgaagatttc gaaattttaa ttatagctta ttcaatagat ggtggagcga 33480

ttagtgcgat tgacatgact aaagtagatg atgagccctt tcacgctgat tatgagacat 33540

ttaaaattgc tctttttgac cctgctgtaa aaaagtatgc attcaatgct aatttcgaaa 33600

gaacttgtct tgctaaacat tttaataaac agatgccacc tgaagaatgg atttgcacaa 33660

tggttaattc aatgcgtatt ggcttacctg cttcgcttga taaagttgga gaagttttaa 33720

gactacaaaa ccaaaaagat aaagcaggta aaaatttaat tcgttatttc tctatacctt 33780

gtaaaccaac aaaagttaat ggaggaagaa caagaaactt gcctgaacat gatcttgaaa 33840

aatggcaaca atttatagat tactgtattc gagatgtaga agtagaaatg acgattgctc 33900

ataaaattaa agactttcca gtaactgcaa ttgaacaagc atattgggtt tttgaccaac 33960

atataaacga cagaggtatt aagctttcta aatcattgat gttaggagct aatgtgctcg 34020

ataagcagag taaagaagaa ttgcttaaac aagctaaaca tataacaggt ttagaaaatc 34080

ctaatagtcc tacacagtta ttggcttggt taaaggatga acaaggatta gatataccta 34140

atttacaaaa gaaaacggtt caggattact taaaagaagc cacaggaaaa gctaaaaaaa 34200

tgctagaaat tagattgcaa atgtctaaaa ccagtgtgaa aaaatacaac aaaatgcatg 34260

acatgatgtg cagtgatgaa cgggtaagag gtctgtttca attttacggt gccggtactg 34320

gaagatgggc aggtagaggt gtacaacttc agaatttaac aaagcattat atttcagata 34380

ctgaattaga aatagcaaga gatcttatta aagaacaacg ttttgacgat ttagatttat 34440

tactgaatgt tcatcctcaa gacttattaa gtcaattagt taggacaaca tttacttctg 34500

aagaaggtaa tgaactagca gtaagtgatt tttctgcaat agaggcaaga gtcatagcat 34560

ggtatgcaaa agaacgatgg cgtttagatg tgttcaacac acacggaaag atatatgaag 34620

catcggcttc tcaaatgttt aatgtcccgg tagaaagcat aactaaaggc gaccctctca 34680

gacaaaaggg aaaagtgtcc gaattagctt taggttatca aggtggcgct ggagctttaa 34740

aagcaatggg tgcattggaa atgggcattg aagaaaacga gttacaaggt ttagttgata 34800

gttggcgtaa cgcaaatcct aacatagtta atttttggaa ggcttgccaa gaggctgcaa 34860

ttaatactgt aaaatctcga aagacgcacc atacacatgg acttagattt tacatgaaaa 34920

aaggctttct aatgattgaa ctacctagtg gaagagcttt agcttatcca aaagcttcag 34980

ttggtgaaaa tagttggggt agtcaagttg ttgaatttat gggcttagat cttaaccgta 35040

aatggtcaaa gttaaaaaca tatggtggga agttagtcga gaatattgtt caagcaactg 35100

caagggattt acttgcgatt tctatagcta ggcttgaagc atcaggtttt aaaatagttg 35160

gtcatgtcca tgatgaagta attgtagaaa tacctagagg ttcaaatgga cttaaggaaa 35220

tcgagactat catgaataag cctgttgatt gggcaaaagg attgaatttg aatagtgacg 35280

gatttacttc tccgttttat atgaaggatt aggagtgtga ttgaatgcaa catcaagctt 35340

atatcaatgc ttctgttgac attagaattc ctacagaagt cgaaagtgtt aattacaatc 35400

agattgataa agaaaaagag aatttggcgg actatttatt taataatcca ggtgaactat 35460

taaaatataa cgttataaat atcaaggttt tagatttaga ggtggaatga tggctagaag 35520

aaaagttata agagtgcgta tcaaaggaaa actaatgaca ttgagagaag tttcagaaaa 35580

atatcatata tctccagaac ttcttagata cagatacaaa cataaaatgc gcggcgatga 35640

attattgtgt ggaagaaaag actcaaaatc taaagatgaa gttgaatata tgaagagtca 35700

aataaaagat gaagaaaaag agagagaaaa aatcagaaaa aaagcgattt tgaacctata 35760

ccaacgaaat gtgagagcgg aatatgaaga agaaagaaag agaagattga gaccatggct 35820

ttatgatgga acgccacaaa aacattcacg tgatccgtac tggttcgatg tcacttataa 35880

ccaaatgttc aagaaatgga gtgaagcaca atgagcataa tcagtaacag aaaagtagat 35940

atgaatgaaa tacaagacaa tgttaagcaa ccagcgcatt acacatatgg cgacattgaa 36000

attatagatt ttattgaaca agttacggca cagtacccac cacaattagc attcgcaata 36060

ggtaatgcaa ttaaatactt gtctagagca ccgttaaaga atggacatga ggatatggca 36120

aaagcgaagt tttacgttga tagagtgttt gacttgtggg agtgatgaca atgacagata 36180

gcggacgtaa agaatactta aaacattttt tcggctctaa gagatatctg tatcaggata 36240

acgaacgagt ggcacatatc catgtagtaa atggcactta ttactttcac ggtcatatcg 36300

tgccaggttg gcaaggtgtg aaaaagacat ttgatacagc ggaagagctt gaaacatata 36360

taaagcaaag tgatttggaa tatgaggaac agaagcaact aactttattt taaaagggcg 36420

gaaacaatga aaatcaaaat tgaaaaagaa atgaatttac ctgaacttat ccaatgggct 36480

tgggataacc ccaagttatc aggtaataaa agattctatt caaatgatgt tgagcgcaac 36540

tgttttgtga cttttcatgt tgatagcatc ttatgtaatg tgactggata tgtatcaatt 36600

aacgataaat ttactgttca agaggagata taacaatgaa aatcaaagtt aaaaaagaaa 36660

tgagattaga tgaattaatt aaatgggcgc gagaaaatcc ggatctatca caaggaaaaa 36720

tatttttttc aacaggattt agtgatggat tcgttcgttt tcatccaaat acaaataagt 36780

gttcgacgtc aagttttatt ccaattgata tccccttcat agttgatatt gaaaaagaag 36840

taacggaaga gactaagttt gattgtctag tagaactaaa cgatattgaa ggttttgaaa 36900

tatatgaaaa tgattcaatc agagagttga tagacggtac ttccagagcg ttttatatac 36960

taaacgaaga taaaactatg acattaattt ggaaagatgg ggagttgcta gtatgatgtt 37020

gaaatttaaa gcttgggata aagataaaaa agttatgagt attattgacg aaatcgattt 37080

taatagtggg tacattttga tttcaacagg ttataaaagt ttcaatgaag taaaactatt 37140

acaatacaca ggatttaaag atgtgcacgg tgtggagatt tatgaagggg atattgttca 37200

agattgttat tcgagagaag taagttttat cgagtttaaa gaaggagcct tttatataac 37260

ttttagcaat gtaactgaat tactaagtga aaatgacgat attattgaaa ttgttggaaa 37320

tatttttgaa aatgagatgc tattggaggt tatgagatga cgttcacctt atcagatgaa 37380

caatataaaa atctttgtac taactctaac aagttattag ataaacttca caaagcatta 37440

aaagattgtg aagagtacaa gaagcaacga tatgagctta ttggggttat agcgaagtta 37500

cgagattgta acaaagaact ggagaagaaa gcaagcgcat gggataggta ttgcaagagc 37560

gttgaaagag atttaataaa caaattcggt aacgatgatg aaagagttaa attcggaatg 37620

gaattaaaca ataaaatttt tatggaggaa gacacaaatg aataaccgtg aacaaataga 37680

acaatccgtt ataagtgcta gtgcgtataa cggcaatgac acagagggat tactaaaaga 37740

gattgaggac gtatataaga aagcgcaagc gtttgatgaa atacttgagg gaatgacaaa 37800

tgctattcaa cattcagtta aagaaggtat tgaacttgat gaagcagtag ggattatgac 37860

gggtcaagtt gtctataaat atgaggaggc acaggaaaat gactaacaca ttaacaattg 37920

atcagttaca agagttatta caaatacaaa aggagttcga cgatagaata ccaacgctga 37980

acttacgaga tagcaaaata gcatatgtag ttgaattctt tgaatggttt aatacattgg 38040

aaacgtttaa aaactggaag aagaaaccag gtaagccgtt agacgtacaa cttgatgaat 38100

tagctgacat gttggcgttt ggattgagta ttgcgaatca agtaggagtg tcatcagaag 38160

agataaaaga agcgattgaa tcaagtttta aagatacaga atttcacaaa atgtttaatt 38220

ttaaagataa agaatttgct caagacgcag ttgttagtac accacagata atatttaaag 38280

aattttatcc cgaccaattg gcaattgtta tagcgataga catagcttac aacttatatt 38340

caatcaacca actcattgac gcatacaaaa agaaaatgaa aaggaatcat gaaagacaag 38400

atggaacagc agacgcagga aaaggatacg tgtaaagaca tattagatcg ggtcaaggag 38460

gttttgggga agtgacacaa tacttagtca caacattcaa agattcaaca ggacgtaaac 38520

atacacacat aactaaagct aagagcaatc aaaggtttac agttgttgag gcagagagta 38580

aagaagaagc gaaagagagg tacgaggcgc aagttaaaag aggtgcagtt attaaagtgg 38640

gtcagttgta tgaaaatata agggagtgtg ggaaatgatt aaaaaactta aaaatatgga 38700

tgggttcgac atctttattg ttggaatact gtcattattc ggtataaccg cattgctact 38760

tgttgtcgca ttgcctatct atacagtggc tagttaccaa aacaaagaag tacatcaagg 38820

gacaattaca gataaatata acaaaagaca agataaagag gacaaattct atattgtatt 38880

agatgataaa caagtcatcg aaaactcaga cttattcttc aaaggaaagt ttgatagcgc 38940

agacatacaa gctaggttaa aagtaggtga taaagtaaaa gttaagacga ttggatatag 39000

aatacacttt ttaaatttat atccggtctt atacgaagta aagaaggtag ataaacaatg 39060

attaaacaaa tactaagact attattctta ctagcgatgt atgagctagg taagtatgta 39120

acggagcaag tatatattat gatgacggct aatgatgatg tagaggagcc gagtgacttt 39180

gaaaaaatca gagctgaagt ttcatgttaa tagctattat catttttgaa ttaattatat 39240

taatgtgttt agcaatagca ctggaggtgt tgtaaatatg tggattgtca tttcaattgt 39300

tttatctata tttttattga tcttgttaag tagcatttct cataagatga aaaccataga 39360

agcattggag tatatgaatg cttatctttt caagcagtta gtaaaaaata acggtgttga 39420

aggtttagaa gattatgaaa atgaagttga acgaattaga aaaagattca aaagctaaag 39480

agaggcgttg gcttctctgc tctatctaaa ataatgaaag gagccgaaca tgttagacaa 39540

agtcactcaa atagaaacaa ttaaatatga tcgtgatgtc tcatattctt atgctgctag 39600

tcgtttatcc acacattgga ctaatcataa tatggcttgg tctgacttta tgcagaagct 39660

agcacaaaca gttagaacta aagaagattt aactgagtac aataaaatgt ctaagtctga 39720

acaagcagat ataaaagacg ttggcggatt tgtcggtggt tatttaaaag aaggtaaacg 39780

acgtgctggt caagtcatga atcgttcaat gttaacactt gatatcgatt atgctgctca 39840

agatatgact gacatattat ctatgtttta tgattttgca tattgtttat attcaacaca 39900

taagcataga gagataagtc caagactgcg tttagtgatt cctttaaaac gaaatgtaaa 39960

tgcagatgag tatgaagcta ttgggcgtaa agtcgcagat atcgttggca tggattactt 40020

cgatgataca acttatcaac ctcataggtt aatgtattgg ccttcaacta gcaacgatgc 40080

ggaatttttc tttacgtatg aagatttacc tttgttagat ccagatacaa tattaaatga 40140

atatgttgat tggactgaca cattagaatg gccaacgtct tcaagggaag agagtaagac 40200

taaaagatta gcagataagc aaggcgaccc agaagaaaag ccgggaattg ttggtgcatt 40260

ttgtagagcc tatacgatag aagaagctat agaaactttt attcctgatt tatacgaaaa 40320

acattctact aaccgttata cctatcatga aggttcaact gcaggtggat tggtgttata 40380

cgaaaataac aagtttgcct attctcatca taatacggat cccgtaagcg gtatgcttgt 40440

gaacagtttt gatttagtac gcatacactt atatggtgct caagatgaag aaactaaaac 40500

agatactccg gttaatcgac tacctagtta taaagcaatg cagcaaatag cgcaaaatga 40560

tgaggttgtt aaaaagcaat taattaatga caaaatgtct gatgcaatgc aggatttcga 40620

tgaaatagaa aatagcgatg atgcatggtc tgagacgtta gaaattactt cgaaaggtac 40680

tttcaaagct agtatcccaa atatagaaat tatattgcgt aatgatccaa atttaaaagg 40740

aaaaatagcc tttaacgaat ttacgaaaca aattgaatgt ttagggaaag tgccatggaa 40800

tactaatttt aagacacgtc aatggcaaga cggtgatgat agcagtttaa gaagttatat 40860

cgaaaagatt tatgacatac accattcagg taaaacaaaa gatgccatta taagcgtagc 40920

aatgcaaaat gcttatcatc cagtaaggga ttatctaaat aaaatatcgt gggatggaca 40980

taaacgtctt gaaaagttat ttatcaaata cttaggtgtt gaagacactg aagtgaatag 41040

aacaactacc aaaaaagcat tgactgctgg aatcgctaga gtaatggagc ctggatgtaa 41100

atttgactat atgcttacac tttatggtcc tcaaggtgta ggtaaatctg ctttgctaaa 41160

aaaattaggt ggtgcatggt tttctgacag tttagtttct gttacaggta aagaagctta 41220

tgaggcctta caaggcgttt ggctaatgga aatggcagaa cttgcagcta caagaaaagc 41280

tgaagttgaa gctattaagc atttcatatc taaacaagtt gaccgatttc gtgttgctta 41340

tgggcattat attgaagatt ttccaaggca atgtattttc attggtacaa ctaataaagt 41400

tgatttctta agagatgaaa ctggtggaag acgtttttgg ccaatgactg taaatccaga 41460

gagagttgaa gtgaactggt ctaaactaac caaagatgag attgaccaaa tttgggcaga 41520

agctaaacac tattatgaac aaggagaaga tttattcctt aaccctgaac tagaagaaga 41580

aatgcgttca atacaaagca aacatactga ggaatctcca tatacaggca ttattgatga 41640

atatcttaac acaccaattc ctagcaattg ggatgactta actatctttg aacgaagacg 41700

attttatcaa ggtgatgttg atatgttacc aacaggaaat gtagattacg ttaaaagaaa 41760

taaagtctgt gcgcttgaag tgtttgttga atgttttggt aaagataagg gagatagtag 41820

aggatctatg gaaattagaa agatttcaaa catcttaaga caattagaca attggtctgt 41880

atatgatggt aataaaagtg ggaaaattcg atttggaaaa gattatggtg tacagatagc 41940

ttatgtaaga gatgaaagtt tagaggattt aatataagaa atattgaata aatatgcatt 42000

ttagagtgtt gtatcagatg ttgcatcatt ttttgagtga tgcaacacgg gagtgtaaaa 42060

agtaatcgta ggtgttgtat catttttggt gatgcaacat tgatgaaaca aatgatacaa 42120

cacctctttc ctttctagct gtagggttca accctgtttg tttccaatgt tgcatcaaat 42180

tcactataaa gtttaaaaag tagtgttagg gagtaaaggg gtataggggt aaccctctaa 42240

cagctatttt taaaagtttg gcaagaattg atacaacatc ggaacacaaa tataaatttt 42300

gtatacaagg tgaataaatg aaagaatcga cattagaaaa atatttagtg aaagagataa 42360

caaagctaaa cggtttatgt ttgaaatggg tcgcacctgg aacaagaggt gtgccagata 42420

gaattattat tatgccagaa ggaaaaacat attttgtaga aatgaagcaa gaaaaaggaa 42480

agttgcatcc tttacaaaaa tatgtgcata gacaatttga aaatagagat cataaagtat 42540

atgtgttatg gaataaagaa caagtaaata cttttatcag aatggtaggt ggaacatttg 42600

gcgattgact tcaaaccaca tagctatcaa aagtatgcaa tagataaagt tattgataat 42660

gagaaatacg gtttgttttt agatatgggg ctagggaaaa cagtatcaac acttacagca 42720

tttagtgaat tgcagttgtt agacactaaa aaaatgttag tcatagcacc taaacaagtt 42780

gctaaagata catgggttga tgaagttgat aagtggaacc atttaaatca tctgaaagtg 42840

tctttagtct taggaacacc taaagaaaga aatgatgcat taaacacaga ggctgatatc 42900

tatgtaacca ataaagaaaa tactaaatgg ttatgcgatc aatataaaaa agaatggcca 42960

tttgatatgg ttgtaattga tgaactgtct acatttaaaa gtcctaagag tcaaaggttt 43020

aaatctatta aaaagaaatt accactcatt aatagattta taggattaac aggaacacct 43080

agcccaaata gtttacagga tttatgggct caagtttatt tgatagacag aggcgaaaga 43140

cttgagtctt cattcagtcg ttatcgagaa aggtacttta aaccaacaca tcaagttagc 43200

gaacatgttt ttaactggga gctaagagac ggatctgaag aaaagatata tgaacgaata 43260

gaagatatat gtttaagcat gaaagcgaaa gattatctag atatgcctga cagagttgat 43320

actaaacaaa cagtagtctt atctgaaaaa gaaagaaaag tatatgcaga attagaaaaa 43380

aactatattt tagaatcgga agaagaagga acagttgtag ctcagaatgg ggcatcatta 43440

agtcaaaaac tacttcaact atctaacggt gcagtttata cagatgatga agatgtaaga 43500

cttatacatg ataagaagtt agataagtta gaggaaatta tagaggagtc tcaaggccaa 43560

ccaatattat tgttttataa cttcaaacat gataaagaaa gaatacttca aaggtttaag 43620

gaagcaacca cattagagga ttcaaactat aaagaacgtt ggaatagtgg agacattaag 43680

ctgcttatag cacatccagc aagtgcaggg catggattaa acttacaaca aggtgggcac 43740

attattgttt ggtttggact tacatggtca ttggaattat accaacaagc aaatgcaaga 43800

ttatatagac aaggacaaaa tcatacgact attattcatc acattatgac cgataacaca 43860

atagatcaaa gagtatataa agctttacaa aataaagaac taacgcaaga agaattgatg 43920

aaagctatta aagcaagaat agctaagcat aagtaatgga ggtataagat gggaaaggca 43980

tcatacgata ttaagccagg tacatttaaa tatattgagt cagagatata taacctacaa 44040

gagaacaaga aagagataaa tagattgaga atggagatac ttaacccaac gaaagagcta 44100

gacactaaca ttgtgtatgg accgttgcaa aaaggtgaac cagttagaac aactgaacta 44160

atggcaacaa ggttattgac taataagatg ttacgaaacc tagaagaaat ggtcgaagca 44220

gttgaaagtg aatacttaaa gttacctgaa gatcataaga aagtaataag gttaaagtat 44280

tggaatagag ataagaagct aaagatagag caaataggag atgcatgtca catgcatcgt 44340

aatacagtta ctacagtacg aaagaacttt gttaaagcgg tagcgtatca tgcgggtatc 44400

aaataacatt gtgcaaagat tgtgcaaaag gcctacaaat ctgtagtaat atgatagtat 44460

cggaaagatg tataaagtta tctaaaagtt atacgacata agtaaacgag gcacatcgct 44520

atgcggtgtg tcttttgtta tgcaatcgaa gaggtgtaag agttgatcaa gcgtaataac 44580

atttataagc atggtcgtaa gtcatatcaa tacgattggt tctatcattc aaaagcatgg 44640

aagaagttaa gagagatagc attagataga gataattatc tttgtcaaat gtgtttacgc 44700

gaagatattg taacagatgc aaacattgta catcacatta tttatgttga tgaagatttt 44760

aacaaagctt tagacttaga taatctaatg tcagtttgtt atagttgtca taacaaaatt 44820

catgcaaatg ataaaaacaa agataaatat aaaaataaaa agtttcgagt tgaaaaaatt 44880

taaataaaaa attataaaat aatttttaat cccccctacc tggtggggag ttccattata 44940

caccgggaac cggtgaaggc aggaacgaga gcagcgcaga taatttttca tgaaaggggg 45000

ttttcttatg aaattaacaa aaaaacagct gaaagaatat attgaagact acaaaaaatc 45060

tgacgatatg ttaattaatt tatatttaga aacatatgaa ttttattgtc g 45111

Claims (3)

1. a kind of staphylococcus aureus bacteriophage, it is deposited in China typical culture collection center, preservation date in June, 2017 28 days, deposit number CCTCC NO:M 2017394, preservation address: Wuhan, China, Wuhan University, postcode 430072.

2. a kind of the answering in preparation staphylococcus aureus bacteriostatic agent of staphylococcus aureus bacteriophage described in claim 1 With.

3. staphylococcus aureus bacteriophage described in a kind of claim 1 is in preparation prevention or treatment infection of staphylococcus aureus Application in disease medicament.