CN114231500B - Mutant staphylococcus aureus phage and application thereof - Google Patents

Abstract

The invention belongs to the technical field of animal infectious disease prevention and treatment, and particularly relates to a mutant staphylococcus aureus phage and application thereof. The staphylococcus aureus phage 4PHCISA25 isolated by the invention is preserved in China Center for Type Culture Collection (CCTCC) NO: m20211354. The invention provides the function of the bacteriophage in preventing and treating staphylococcus aureus and a biofilm thereof as a bactericide, and provides a bactericide product with application potential for clinically treating staphylococcus aureus infection diseases. The staphylococcus aureus phage of the invention has wide cracking range and strong cracking capability, and can forcefully kill methicillin-resistant staphylococcus aureus.

Description

Mutant staphylococcus aureus phage and application thereof

Technical Field

The invention belongs to the technical field of animal infectious disease prevention and treatment, and particularly relates to a mutant staphylococcus aureus phage and application thereof.

Background

Staphylococcus aureus (Staphylococcus aureus, s.aureus) is a g+ bacterium that can cause a variety of infectious diseases in humans and domestic animals, such as local abscesses, sepsis, and the like. Staphylococcus aureus can attach to and persist in host tissues (e.g., bone and heart valves), causing osteomyelitis and endocarditis. The bacterium is also one of the common pathogens causing bacterial food poisoning and nosocomial infections, and is related to sanitation and safety relatives in the food industry and the medicine field. In recent years, staphylococcus aureus is resistant to methicillin to form "superbacteria" (MRSA) due to the use of antibiotics, resulting in the onset of a large number of patients infected with "superbacteria" and 21.8% of infected patients die each year. MRSA strains are prone to the formation of Biofilm (BF), the biofilm matrix protecting the host from phagocytosis and other innate and adaptive immune and inflammatory systems. Staphylococcus aureus is 10-1000 fold less sensitive to a large number of antibacterial agents through its highly structured extracellular matrix biofilm-mediated resistance. A large number of antibiotics lack the ability to penetrate cell membranes and bacterial biofilms. These make it increasingly difficult to treat staphylococcus aureus infections. To date, there has been no approved drug specifically targeted to biological membranes in clinical trials. Therefore, the prevention and treatment of staphylococcus aureus infection becomes one of the urgent tasks in the international medical and agricultural safety fields at present.

With the overuse and misuse of antibiotics in the agricultural, pharmaceutical and food fields, the current risk of antibiotic resistance is caused. Among the many options being explored, the resumption of natural viral enemy-phage therapy as a bacterium is an attractive alternative or complementary therapy. Phage-comparison antibiotics have the following advantages: as an obligate bacterial virus specific to bacterial hosts, it is not toxic to non-target microorganisms and its killing activity is limited to a narrow range of pathogenic strains, avoiding collateral damage to the human and animal healthy commensal microbiota. Phage provide a dynamic dose that increases when the target bacterial host strain is present and decreases with the number of target bacteria. Phages are generally capable of killing bacteria independently of their multi-drug resistant phenotype, exhibiting effectiveness against antibiotic resistant bacteria. It is well known that phage treatment is highly safe, phage is composed mainly of proteins and nucleic acids, has been repeatedly tested for phage intravenous injection, and has no major side effects observed. Clinical trials reported so far have shown that they do not show significant toxicity. Based on the advantages, the phage has wider prospect in application.

In addition to virulent phages that ultimately lead to the lytic cycle of bacterial death, some phages can also undergo the so-called lysogenic cycle, a condition in which the viral genome integrates with the bacterial chromosome and replicates, and can actively contribute to horizontal gene transfer leading to the spread of antibiotic resistance and virulence genes, a type of phage known as temperate phages. Thus, temperate phages are not considered good candidates for phage therapy. More than 200 staphylococcus aureus phages have been reported in the National Center for Biotechnology Information (NCBI) database by 2018. Bioinformatic analysis of 127 of the longtail viridae phages, in which the presence of lysogenic determinants in the genome of the majority (125 phages, 98.4%) was temperate phages. Therefore, genetic engineering of lysogenic determinants of the warm-banded bacteriophage of staphylococcus aureus, to obtain virulent phage, is a useful strategy for further application of control of staphylococcus aureus. Studies have shown that pyrophosphates can be used for DNA mutation of extrachromosomal elements including plasmids and viruses. Phage deletion mutants can be easily isolated by exposing phage particles to multiple rounds of chelators (e.g., ethylenediamine tetraacetic acid, EDTA), sodium citrate, or sodium pyrophosphate. Sodium pyrophosphate destabilizes the phage capsid by binding to cations located on the surface of phage particles, which can lead to conformational defects and ultimately to loss of viability resulting in mutant phage. Thus, the urgent need for random mutation to transform into virulent phage is a technical problem that needs to be solved in the art.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and a mutant staphylococcus aureus phage (4 PHCISA 25) is obtained by screening, and the phage has no integration effect after mutation and can not mediate bacterial drug resistance and virulence gene level gene transfer. The phage 4PHCISA25 is expected to be used as a novel staphylococcus aureus control microbial inoculum, and shows that the phage 4PHCISA25 can effectively inhibit 29 methicillin-resistant staphylococcus aureus.

The second object of the invention is to provide an application of a mutant staphylococcus aureus bacteriophage in degrading a staphylococcus aureus biofilm.

The invention further aims to provide an application of the mutant staphylococcus aureus phage in skin abscess bacteriostat.

In order to achieve the technical purpose, the invention adopts the following technical measures:

the invention uses staphylococcus aureus as host bacteria to separate a mild bacteriophage with strong cracking capability and wide cracking range, and the mild bacteriophage is mutated into a virulent bacteriophage by sodium pyrophosphate. The biological characteristics of the phage are researched, and a theoretical basis is provided for the clinical application of the phage in veterinary medicine. The applicant named staphylococcus aureus phage 4PHCISA25,Staphylococcus aureus bacteriophage 4PHCISA25 delivered to China, wuhan university Chinese typical culture Collection, with a collection number of CCTCC NO: m20211354.

The phage 4PHCISA25 provided by the invention is a long-tailed phage of the order of the tailed phage, and the titer is kept stable under the conditions of 4-60 ℃ and pH of 5-9. The activity is stable when stored at 25 ℃ for 6 months. The activity was stable after 9 months of storage at 4 ℃. The strain has a certain resistance to ultraviolet light, and the survival rate is kept relatively stable. Intolerance to alcohol, namely, the inactivation can be carried out in the presence of alcohol for 10min.

The invention also provides application of the staphylococcus aureus phage 4PHCISA25 in preventing and treating staphylococcus aureus biofilm. And application in preparing a staphylococcus aureus infection disease bacteriostat, for example, the bacteriostat can be used for inhibiting skin abscess caused by methicillin-resistant staphylococcus aureus.

The beneficial effects of the invention are as follows:

(1) The prior art shows that the phage mutated by sodium pyrophosphate does not have an integration effect, and the phage 4PHCISA25 provided by the invention has high application safety.

(2) The phage of the invention has good thermal stability. The phage 4PHCISA25 provided by the invention is better than the thermal stability of staphylococcus aureus phage SH-St 15644 (stable at 4-40 ℃) in patent document 201810715693 0 and the thermal stability of staphylococcus aureus phage qdsa001 (stable at 4-50 ℃) in patent document CN 2015109752592.

(3) The phage has strong bacteriostasis capacity under the condition of different MOI. In the invention, the phage 4PHCISA25 has good antibacterial effect on staphylococcus aureus under the condition of MOI= (0.01-100).

(4) The phage of the invention has a broad host spectrum. The phage 4PHCISA25 provided by the invention has good cracking effect on 29 methicillin-resistant staphylococcus aureus.

(5) The bacteriophage of the present invention has high capacity of degrading biomembrane. After the bacteriophage 4PHCISA25 plays a role in a strong adhesion biological film formed by staphylococcus aureus for 4 hours, the biological film is obviously inhibited.

(6) The phage of the invention has good in vivo therapeutic effect. The bacteriophage 4PHCISA25 in the invention obviously enhances the healing speed of the skin of the mice, the abscess area and the healing time are obviously smaller than those of MRSA infected groups, and the difference has statistical significance.

In conclusion, the staphylococcus aureus phage 4PHCISA25 provided by the invention can effectively inhibit and lyse staphylococcus aureus, and the biological preparation (biological agent) prepared by using the phage provided by the invention can be used as a bactericide, can target a biological film, and can be widely applied to the fields of cultivation, medicine and food safety.

Drawings

Fig. 1: plaque morphology of staphylococcus aureus phage 4PHSA25 on double-layer agar plates.

Fig. 2: electron microscope image of staphylococcus aureus phage 4PHSA 25.

Fig. 3: in vitro bactericidal results for staphylococcus aureus phage 4PHCISA25 at different MOI.

Fig. 4: one-step growth curve of staphylococcus aureus phage 4PHCISA 25.

Fig. 5: the pH stability results of Staphylococcus aureus phage 4PHCISA25 are shown.

Fig. 6: graph of the results of the thermostability of staphylococcus aureus phage 4PHCISA 25.

Fig. 7: UV and alcohol stability results for Staphylococcus aureus phage 4PHCISA 25. Reference numerals illustrate: FIG. 7a is a graph of phage UV stability results; FIG. 7b is a graph showing the results of phage alcohol stability.

Fig. 8: staphylococcus aureus phage 4PHCISA25 inhibition Staphylococcus aureus biofilm results.

Fig. 9: in vivo treatment results for staphylococcus aureus phage 4PHCISA 25. Reference numerals illustrate: figure 9a of figure 9 is a plot of the area of subcutaneous abscess in MRSA-infected mice; figure 9b of figure 9 is the formation of skin abscesses in mice 24h after infection; the 9c plot in fig. 9 is the colony count of skin abscess tissue 24h after infection.

Detailed Description

Description of sequence Listing

SEQ ID NO. 1 is the nucleotide sequence of a Staphylococcus aureus phage.

The invention will be further explained in detail with reference to specific examples. The scope of the invention should not be limited to the examples.

EXAMPLE 1 isolation and screening of Staphylococcus aureus phage 4PHSA25

The detailed steps are as follows:

(1) Sample collection

The pig endometritis swab sample is collected in a pig farm in Guangxi Zhuang resident autonomous area.

(2) Isolation of staphylococcus aureus phages

The pig endometritis swab sample was soaked overnight with physiological saline at 4℃and centrifuged at 8000r for 10min and the supernatant was filtered through a 0.22 μm filter. This filtrate was a pre-treated sample for phage isolation and was stored overnight at 4 ℃. In 10mL of TSB liquid medium (tryptone 17g, sodium chloride 5g, soytone 3g, glucose 2.5g, dipotassium hydrogen phosphate 2.5g, pH 7.3.+ -. 0.2) in a volume ratio of 1:100 ul of host bacteria (Staphylococcus aureus SA 25) were added at a ratio of 100 and incubated at 37℃to proliferate to log phase. 10mL of the pretreated fecal sewage sample and 40mLTSB liquid culture medium are added into the logarithmic phase bacterial liquid, and the culture is carried out for 3 to 4 hours at 37 ℃ in a shaking way, so that phage are further proliferated. The proliferated phage solution was centrifuged at 8000r for 10min at 4℃and the supernatant was filtered with a 0.22 μm filter. Plaque was observed by a double-layer plate method. The method comprises the following specific steps: mu.L of host bacterial liquid, 1mL of filtered phage sample and 6mL of semisolid culture medium are taken and added into a10 mL sterilized centrifuge tube, the mixture is inverted and evenly mixed, the TSA solid plate prepared in advance is poured into the centrifuge tube, and the upper agar is solidified and then is inverted and cultured overnight at 37 ℃.

(3) Purification of phages

A single large and transparent plaque was picked up from the upper agar layer where the plaque was formed with a sterile white tip, the phage-carrying tip was blown and mixed well in 1mL SM buffer, and left to stand in a refrigerator at 4 ℃. After standing for several hours, the phage solution is mixed by shaking, diluted by 10 times, 300 mu L of SA25, 1mL of phage diluent and 6mL of semisolid culture medium are added into a10 mL sterilized centrifuge tube, the mixture is inverted and poured into a TSA solid plate prepared in advance, and after the agar at the upper layer is solidified, the mixture is inverted and cultured for 12 hours at 37 ℃. The procedure was repeated more than 5 times until the plaque size morphology was uniform.

EXAMPLE 2 Electron microscopic observation of phage 4PHSA25

Phage were visualized by phosphotungstic acid negative staining (Clokie and Kropinski 2009). Firstly concentrating phage, carrying out mass multiplication on phage 4PHSA25, adding 1mL of phage stock solution into 100mL of host bacterial liquid (staphylococcus aureus SA 25) in logarithmic phase, and shake culturing for 3-4h until bacterial liquid becomes clear. Centrifuging at 8000r at 4deg.C for 10min, and filtering the supernatant with 0.22 μm filter for sterilization. The filtered phages were further trimmed with a long needle with 5mL30% sucrose solution, concentrated phages by centrifugation at 30000r for 2h at 4℃and the supernatant discarded, and the pellet was resuspended with 200. Mu.L TEN solution. The concentrated resuspended phage was sent to a transmission electron microscope platform, stained with conventional phosphotungstic acid (PTA), dried, and observed for phage morphology using transmission electron microscope. Length measurements were made with image processing software ImageJ.

As a result, as shown in FIG. 2, the phage 4PHSA25 isolated by the present invention belongs to the family of long-tailed phages of the order of the end phages, the head is oval-like, the diameter of the head is 80 nm.+ -.5, the width is 40 nm.+ -.5, the length of the tail is 260 nm.+ -.5.

Example 3: extraction of phage genome and whole genome sequencing

Phage genomes were extracted using proteinase K/SDS methods. 1mL of concentrated phage stock solution after super separation is added with 20ul RNase, 2ul DNaseI and 10ul Buffer, and the mixture is evenly mixed and then is cracked for 1h at 37 ℃. 10% SDS was added to 0.5% by volume of phage concentrate 1/5, 50ul proteinase K was mixed upside down and then subjected to a 56℃water bath for 1h. Adding 1mL of protein extract of phenol-chloroform-isoamyl alcohol (prepared in situ and stored in dark place) with a volume ratio of 25:24:1, mixing the mixture, centrifuging the mixture for 10min at 13000r, sucking the supernatant into a new 2mLEP tube with a 1mL broken gun head and a 200uL broken gun head, and repeating the process for 3 to 4 times. The last time the collected supernatant was transferred to a10 mL EP tube, pre-chilled absolute ethanol was added in a volume ratio of 1:2, -20 degrees standing overnight. The flocculent precipitate is separated into 1.5mL centrifuge tubes by a broken gun head for the next day, and centrifuged for 5min at 13000 r/min. Washing the DNA precipitate with 1mL of 75% absolute ethanol, centrifuging for 5min at 13000r/min, and repeating for 3-4 times; after the DNA was dried to be transparent on a super clean bench, the DNA was resuspended in 50ul deionized water and the DNA was dissolved at 4 ℃. The DNA samples obtained were submitted to commercial sequencing companies for sequencing, stored at-20℃for later use.

As a result of the whole gene sequencing of the phage 4PHSA25, the phage genome is double-stranded DNA, and the total length of the sequence is 45314bp (see SEQ ID NO:1 of the sequence table). Annotation analysis of sequencing results: phage 4PHSA25 contains integrase, repressor protein and lysogenic module (Lysogeny module), and can be judged as a lysogenic phage 4PHSA 25.

EXAMPLE 4 mutation assay of phage 4PHSA25

Wild type phage 4PHSA25 (10) ⁸ PFU/mL) was mixed with 300mM sodium pyrophosphate (pH 7.4), incubated at 42℃for 40min, and then mixed with the Staphylococcus aureus host strain. 1mL of SM buffer (Na Cl 5.8g, mgSO4.7H2O 2.0g, gelatin 0.1g,1.0mol/L Tris-HCl (pH 7.5) 50.0 mL) was added to phage platesAnd incubated at room temperature with gentle shaking (60 rpm) for 1h. The collected liquid was centrifuged at 10,000rpm at 4℃for 10 minutes to allow the supernatant to contain the phage which remained. Phage in SM buffer were again treated with sodium pyrophosphate at the same concentration and incubated under the same conditions, and the whole mutation process was repeated 30 times. The putative phage deletion mutant forms a lytic plaque that is carefully observed and compared to the lytic plaque formed by the wild-type phage. Each putative "clear lysis plaque" was picked and suspended in 200. Mu.L SM buffer. PCR sequencing of whether a phage lysomodule (lysogene module) generates a mutation site, sequencing of successfully mutated phages, and screening of the integrated strain is performed as follows: taking 300uL of host bacteria (such as staphylococcus aureus SA 25) in the earlier logarithmic phase, adding 1mL of mutant phage (4 PHCISA 25), paving a double-layer plate, incubating at 37 ℃, picking up the monoclonal after the monoclonal grows out, carrying out PCR detection, and purifying by 3 rounds.

Leu 1 base substitution in phage 4PHCISA25 repressor gene, 1 base substitution of TTA codon to termination codon TAA. PCR (Forward primer F; gttcgtgtacgtttgagaatgg, reverse primer R; ctcctgtaagaagaagcgc reaction System: 2X Rapid Taq Master Mix. Mu.L, 1. Mu.L each for upstream and downstream primers, 2. Mu.L for template, ddH) for Gene amplification Using integrase Gene (1200 bp) ₂ O6. Mu.L was used for a total of 20. Mu.L system. The reaction conditions were 95℃for 5min,95℃for 15s,55℃for 15s,72℃for 30s, and 30 cycles, and 72℃for 10 min.) after 3 rounds of purification, the mutant phage was not screened for an integrative strain, indicating that the mutant phage obtained by screening no longer had an integrative effect, and therefore, the invention successfully screened for a virulent phage.

Example 5 determination of phage 4PHCISA25 host spectrum

30 staphylococcus aureus strains (for example, the conventionally applied strains such as SA1, SA2, SA3, SA4, SA5, SA6, SA7, SA8, SA9, SA10, SAA, SA12, SAB, SAC, SA, SA16, SA17, SA18, SA19, SA20, SA21, SA22, SA23, SA24, SA25, SA26, SA27, SA28, SA29, SA30, laboratory preservation, practice of the invention is not limited to the above strains) and 8 other bacteria (for the conventionally applied strains such as Salmonella typhimurium 1344, salmonella paratyphi 201007, E02, E.faecalis EF3964, E.pasteurella type A HB03, E.pasteurella type D HB05, B.bauhinensis D8D9, kp9, laboratory preservation, practice of the invention is not limited to the above strains) were selected for host analysis of phage 4PHCISA25 (see Table 1), and the following steps are performed:

the above strains (30 staphylococcus aureus strains and 8 other species strains as described above) were cultured separately to the logarithmic growth phase. mu.L of bacterial liquid cultured to logarithmic phase and 6mL of semi-solid culture medium are added into a10 mL sterilized centrifuge tube, and the mixture is inverted, poured into a TSA solid plate prepared in advance, and placed at room temperature for drying. Phage 4PHCISA25 was diluted 10-fold, 10uL phage dilutions were sequentially added dropwise to the upper agar surface, after the liquid surface was dried, the strain was incubated upside down overnight at 37℃for observation of its lytic ability, and the test was repeated three times to average. The ratio of phage titer of other strains to phage titer of the host bacteria was EOP (Efficiency of plating) with phage titer of the host bacteria being 1.

As a result, as shown in Table 1, the phage 4PHCISA25 of the present invention can cleave 29 strains out of 30 strains of Staphylococcus aureus, and exhibits broad spectrum. In addition, phage 4PHCISA25 was unable to infect other gram positive and gram negative bacteria, indicating that this phage has a higher host specificity for Staphylococcus aureus.

TABLE 1 determination of the phage 4PHCISA25 host profile of the invention

Table 1 notes: "+++". Representation of EOP is more than 0.1, forming transparent dense plaques; "++" means EOP 0.1-0.001, forming a lighter dense plaque; "+" indicates that EOP < 0.001, single plaques were formed; meaning that no plaque is formed and no cleavage occurs.

EXAMPLE 6 evaluation of phage 4PHCISA25 Schizosaccharomyces cerevisiae ability

(1) Staphylococcus aureus strain SA25 was picked and inoculated as a single colony in 5mL TSB medium (conventional medium), and shake cultured in a shaker at 220r/min at 37℃for 15h. The following day in 5mL TSB liquid culture medium according to the volume ratio of 1:100, and then culturing and shaking in a shaker at a temperature of 220r/min and a temperature of 37 ℃ for 3 hours. The culture solution is diluted by 10 times and 100ul of the culture solution is coated on a flat plate for counting for standby.

Bacterial count (CFU/mL) calculation formula = number of individual colonies x dilution gradient x 10

(2) Taking phage 4PHCISA25 stock solution (phage 4PHCISA25 stock solution preparation method comprises taking Staphylococcus aureus SA25 as host bacteria, taking 100ul Staphylococcus aureus SA25, 100ul 10) ⁶ PFU/mL 4PHCISA25 was mixed with 6mL semi-solid medium, poured into TSA solid plates prepared in advance (conventional) after mixing, and after the upper agar solidified, cultured upside down at 37℃for 12h to proliferate phages. Adding 5mL of SM buffer solution (conventional) into the upper agar, taking 5mL of proliferation solution into a centrifuge tube, centrifuging for 10min at 4 ℃ for 12000r, and filtering the supernatant by a 0.22 mu m filter to obtain phage stock solution. Phage were diluted 10-fold to obtain 10ul spot titers for use.

Phage titer (PFU/mL) =number of plaques x dilution x 100

(3) The log phase staphylococcus aureus bacterial solution was taken, 100uL of log phase host bacteria were mixed with 100uL of different MOI (0.01, 0.1, 1.0, 10, 100) phages in a 100 well plate and shaken at low speed at 37 ℃. Meanwhile, a control group is set, an equal volume of TSB culture medium or phosphate buffer (PBS, PH=7.5) is added as a negative control, and an equal volume of staphylococcus aureus bacterial liquid is added as a positive control. Determination of OD by full automatic growth curve instrument ₆₀₀ Values were measured every 1h up to 12h and the test repeated three times.

The results are shown in FIG. 3: phage 4PHCISA25 showed potent bactericidal properties when moi= (0.01-100) infected with staphylococcus aureus, and significantly delayed bacterial re-proliferation compared to the positive control without phage. After 8 hours, the absorbance of the test group increased, which indicates that the staphylococcus aureus bacterial load increased, the bacterial strain with resistance to phage 4PHCISA25 could not completely kill the bacteria, but the test group was significantly lower than the positive control group, and the inhibition ability to host bacteria was also stronger when the MOI value was high enough.

Example 7 determination of phage 4PHCISA25 one-step growth curve

A single colony of staphylococcus aureus SA25 was picked and inoculated into 5mL of TSB medium and shake-cultured in a shaker at 220r/min at 37℃for 15h. The following day in 20mL TSB liquid culture medium according to the volume ratio of 1: 100. Aureus was inoculated and incubated at 37℃for 3-4h to achieve the logarithmic phase, phage 4PHCISA25 prepared in example 6 was added at optimum MOI=0.01, mixed well and incubated at 37℃for 10min, centrifuged at 8000r/min for 10min, and the pellet was washed 3 times to thoroughly wash out unadsorbed phage. The pellet was resuspended in an equal volume of TSB medium, incubated on a shaker at 37℃with shaking, 200ul samples were taken every 10min from 0min, centrifuged at 12000r/min at 4℃for 10min, and the supernatant was aspirated into a fresh 1.5mLEP tube for determination of phage titer by 10-fold dilution spot method.

The results are shown in FIG. 4: the incubation period for phage 4PHCISA25 was about 10min; the cracking period is 10-100 min; the burst period is about 90 minutes; the amount of cleavage was about 470PFU/cell.

Example 8: determination of the pH stability of phage 4PHCISA25

Taking 1.5mL centrifuge tubes, adding 900uL PBS buffer solutions with different pH values (3-12) respectively, and adding 100uL (10) ¹⁰ PFU/mL) phage were added to the same for 1h in a 37℃water bath, and the titers of the phages from the different pH treatment groups were determined by spot method at 10-fold dilution. Each test was repeated three times.

The results are shown in FIG. 5: phage 4PHCISA25 has higher activity in the pH range of 5.0-9.0, while biological activity is significantly reduced at pH below 5.0 or above 11.0. At pH values of 3 and 12, the phage is completely inactivated and loses the ability to infect the host.

Example 9 determination of the thermal stability of phage 4PHCISA25

1mL (10) was added to a 1.5mL centrifuge tube ¹⁰ PFU/mL) phage, respectively regulating a constant-temperature water bath (4 ℃, 20 ℃, 40 ℃,50 ℃, 60 ℃,70 ℃ and 80 ℃) and respectively acting for 20min, 40min and 60min after the temperature of the water bath is stable, and measuring the phage titer at different times by a spot method by 10-fold dilution. Each test was repeated three times.

The results are shown in FIG. 6: the titer of the phage 4PHCISA25 basically keeps the original activity at 4-50 ℃, the titer is reduced from 50-60 ℃, the phage is incubated for 40min at 70 ℃ and 20min at 80 ℃, and the phage is basically killed. Phage 4PHCISA25 has poor tolerance to high temperature (above 70 ℃) and is suitable for survival in the environment below 60 ℃.

Example 10 determination of UV, 75% alcohol stability of phage 4PHCISA25

Ultraviolet stability measurement: 1mL (10) was added to each well in a 6-well plate ¹⁰ PFU/mL) phage solution is placed under (20 w,90 cm) ultraviolet lamp to act for 5min, 10min, 15min and 30min respectively, after the action, the phage solution is placed in dark to be stable for 30min, 3 repeats are set for each group, and the titer of phage in different irradiation groups is measured by spot method by dilution of 10 times.

Alcohol stability determination: a1.5 mL sterile EP tube was taken and 990ul of 75% alcohol was added to 10ul (10 ¹⁰ PFU/mL) phage stock solution addition treatment, while setting control to add 10ul phage stock solution to 990ul ddH ₂ O. Samples were taken every 10min for measurement up to 30min, 3 replicates were set for each group, and the phage titers were measured at 10-fold ratio dilutions using spot method.

The results are shown in FIG. 7: the survival rate of phage 4PHCISA25 under ultraviolet (20 w,90 cm) irradiation at different times is kept relatively stable, and phage titer is as high as 10 under ultraviolet irradiation for 30min ⁹ PFU/mL, shows that the phage has some resistance to UV light (FIG. 7 a). Phage 4PHCISA25 showed substantially no detectable presence of phage under 75% alcohol for 10min, indicating intolerance of this phage to alcohol (FIG. 7 b).

Example 11 inhibition of Staphylococcus aureus biofilm by phage 4PHCISA25

The test method is as follows:

(1) Staphylococcus aureus was cultured for 15h at a volume ratio of 1:1000 with shaking, and the next day the staphylococcus aureus was diluted with NaCl-Free medium (conventional) at a volume ratio of 1:100.

(2) 200uL of diluted bacteria solution is added to each well of a 96-well plate, a culture medium control group is simultaneously arranged, and the culture medium control group is subjected to standing incubation at 37 ℃ for 24 hours.

(3) After incubation, the culture was gently aspirated and each well was washed 1 time with 200ul PBS.

(4) 100. Mu.L (10) of each well was added to each of the 96-well plates in the test group ⁹ PFU/mL) phage dilutions; control groups were added 100. Mu.L of NaCl-Free medium to the biofilm-forming wells. The culture is carried out for 4 hours at 37 ℃ in a constant temperature incubator.

(5) After incubation, 96-well plates were washed 1 time with PBS and air dried. The mixture was fixed with 200uL of methanol for 30min. Methanol is discarded, PBS is washed for 1 time, and the mixture is naturally dried.

(6) Stain with 200 μl of 1% crystal violet for 15min. The crystal violet was discarded, washed 3 times with PBS until the solution was colorless and dried.

(7) Adding 150uL of 33% glacial acetic acid, shaking with a horizontal shaking table for 15min, and performing OD ₅₉₀ nm reading.

The results are shown in FIG. 8: when the concentration of phage 4PHCISA25 was 10 ⁸ At PFU/mL, after phage addition (accession numbers SA1, SA9, SAA, SAC, SA) OD ₅₉₀ nm was significantly reduced with 64.66% reduction in SA1, 45.65% reduction in SA9, 47.77% reduction in SAA, 69.47% reduction in SAC, 43.28% reduction in SA 21. From this, it was found that phage 4PHCISA25 showed a good inhibitory effect on the biofilm formed by Staphylococcus aureus.

EXAMPLE 12 evaluation of the Effect of phage 4PHCISA25 on treatment of Staphylococcus aureus infected mice

(1) Test animals

SPF-class female balb/c mice at 6 weeks were purchased from university of Yichang three gorges, hubei.

(2) Construction of staphylococcus aureus skin abscess model

Using cottonThe back hair of the mice was removed by dipping sodium sulfide with a swab, wiping the surface with alcohol cotton balls to remove sodium sulfide and disinfect the back skin. The unhairing area is about 2cm multiplied by 2cm and occupies about 15% of the body surface area. Preparation of Staphylococcus aureus liquid according to the protocol of example 6, and adjustment of the bacterial load to 1X 10 ⁸ CFU/mL. The mice were anesthetized with inhaled ether and 100. Mu.L (1X 10) was subcutaneously injected with a 1mL syringe ⁸ CFU/mL) staphylococcus aureus suspension. After 24h local infection forms subcutaneous abscesses.

(3) Treatment of skin abscess nude mice using 4PHCISA25

1h after injection of the bacterial liquid, 100. Mu.L of phage 4PHCISA25 treatment (MOI=10) was injected subcutaneously at the same site, and a positive control group (100. Mu.L of sterile SM buffer was injected) was set. Daily photographs were taken to record the treatment effect of the mice, the length (L) and width (W) of skin abscesses of the mice were measured using a measuring scale, the area of abscesses area (a) =pi (l×w)/2 was calculated, and an area of abscesses curve of back skin infection of the mice was drawn.

The phage subcutaneous injection alone does not cause skin injury to mice and can be safely applied. The skin abscess area and cure time of phage 4PHCISA25 treated mice were smaller than those of MRSA infected group (fig. 9 a), significantly enhancing the skin healing rate of mice. Phage 4PHCISA25 treated group significantly inhibited skin abscesses in mice for 24h (fig. 9 b). Average bacterial cut-out of skin abscess was 7.81×10 in phage-treated mice ⁷ CFU/g; the average bacterial load of skin abscess of mice in MRSA-infected group is 8.87×10 ⁸ CFU/g; the phage treated group resulted in a 1.1log reduction in bacterial numbers after 24 hours compared to the infected group, the differences being statistically significant (see figure 9 c). The phage 4PHCISA25 of the invention is shown to be a powerful biological bactericide for treating skin infections caused by MRSA.

Sequence listing

<110> university of agriculture in China

<120> a mutant staphylococcus aureus phage and use thereof

<141> 2021-12-15

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taaattatcc aagaggcgca agcactaatg gtactgtcga atatttagta gtaacttccg 480

actacaaacg tatgacatat cgtccaaatg gtacaaataa agtatttgtt aagagaaaag 540

aagcgggttc gtggtctgat tggtcagaat tagcgcttaa tgattacaac acaccttttg 600

aaactgttca aaacgcgcaa tcaaaggcta acacggctga aagcaatgcc aaactataca 660

cagatgacaa gtttaataaa agatattcag ttatttttga tgggacggca aatggcgttg 720

gctcaacatt atatcttaat gaaagtttag atcaatttat tttgttaatt ttttatggaa 780

cttttccagg tggagatttt actgagtttg gtaacccctt tggtggcgga aaaatttcat 840

tgaacccatc aaatttaccg gataatgacg gtgacggtgg aggcgtttat gagtttggat 900

taactaaatc tagtcgtaca tcattaacta tatcaaacga tgtttatttt gacttaggaa 960

gtcgaagagg ttctggtgca aatgcaaata gaggaacaat caacaaaatt ataggagtga 1020

gaaaataatg caaatattag ttaacaagcg taatgagata atttcatacg ctatcattgg 1080

tggctttgaa gaaggtattg atattgaaaa tttaccagaa aatttctctc aagtttttag 1140

acctaaagtc tttaaatatt caaatgggga aatagttttt aacgaagatt attcagaaga 1200

aaaagatgac ttgcatcaac agattgacag tgaagaacaa aacacagtcg cttctgatga 1260

catcttacga aaaatggttg ctagtataca gaaacaagtt gttcaaagta caaagttatc 1320

gatgcaagtt aataagcaaa atgcactaat ggcaaaacaa cttgtgacac ttaataaaaa 1380

attagaagag gttaaaggag agacggaaaa tgcttaaatt aatttcacca actttcgaag 1440

atattaaaac atggtatcaa ttgaaagaat acagtaaaga agatatagca tggtatgtag 1500

acatggaagt tatagacaaa gaagaatatg caattattac aggagaaaaa tatccagaaa 1560

atatagagtc ataggttaaa agcttatggc tttttaattt gaataaagtg ggtggtgtaa 1620

tgtttggatt taccaaacga cacgaacaag attggcgttt gacgcgtttg gaagaaaatg 1680

acaagactat gtttgaaaaa tttgacagaa tagaggatag tctgagaacg caagaaaaaa 1740

tttatgacaa gttagataga aatttcgaag aactaaggcg cgacaaagaa gaagatgaaa 1800

aaaataaaga gaaaaatgct aaaaatatta gagatatcaa gatgtggatt ttaggattaa 1860

tagggacgat cctaagtaca tttgttatcg cattattaaa aactattttt ggtatttaaa 1920

aggaggtgat cgccatgctt aaaggaattt taggatatag cttttgggcg tgtttctggt 1980

ttggcaaatg taaataaaga ttaaaggtca gtgcttcggc actggctttt tattttggaa 2040

aaaaggagca aacaaatgga tgcaaaagta ataacaagat acatcgtatt gatcttagca 2100

ttagtaaatc aattcttagc gaacaaaggt attagcccga ttccagtaga cgatgagact 2160

atatcatcaa taatacttac tgttgtcgct ttatatacta cgtataaaga caatccgaca 2220

tctcaagaag gtaaatgggc aaatcaaaag ttaaagaaat ataaagctga aaacaagtat 2280

agaaaagcaa caggacaagc acctattaaa gaagtaatga cacctacgaa tatgaacgac 2340

acaaatgatt tagggtaggt ggttgatata tgttaatgac aaaaaatcaa gcagaaaaat 2400

ggtttgacaa ttcattaggg aaacaattca acccagatgg ttggtatgga tttcagtgtt 2460

atgattacgc caatatgttc tttatgttag cgacaggcga aaggctgcaa ggtttatatg 2520

cttataatat tccatttgat aataaagcaa ggattgaaaa atatggggaa ataattaaaa 2580

actatgatag ctttttaccg caaaagttgg atattgtcgt tttcccgtca aagtatggtg 2640

gcggagctgg gcacgttgaa attgttgaga gcgcaaattt aaacactttt acatcgtttg 2700

gccaaaattg gaatggtaaa ggttggacaa atggcgttgc gcaacctggt tggggtcctg 2760

aaactgttac aagacatgtt cattactacg acgacccaat gtattttatt agattaaatt 2820

tccctgacaa agtaagtgtt gggaatagag ctaaaagcgt tattaagcaa gcaactgcca 2880

aaaagcaagc agtaattaaa cctaaaaaaa ttatgcttgt agccggtcat ggttataacg 2940

atcctggagc agtaggaaac ggaacaaacg aacgcgattt tatccgtaaa tatataacgc 3000

caaatatcgc taagtattta agacatgcag gtcatgaagt tgcattatat ggtggctcaa 3060

gtcaatcaca agacatgtat caagatactg catacggtgt taatgtagga aataataaag 3120

attatggatt atattgggtt aaatcacagg ggtatgacat tgttctagag attcatttag 3180

acgcagcagg agaaaatgca agtggtgggc atgttattat ctcaagtcaa ttcaatgcgg 3240

atactattga taaaagtata caagatgtta ttaaaaataa cttaggacaa ataagaggtg 3300

taacacctcg taatgattta ctgaacgtta atgtatcagc agaaataaat atcaattatc 3360

gtttatctga attaggtttt attactaata aaaaagatat ggattggatt aagaagaatt 3420

atgacttgta ttctaaatta atagctggtg cgattcatgg taagcctata ggtggtttgg 3480

tagctggtaa tgctaaaaca tcagataaaa accaaaaaaa tccaccagtg ccagtaggtt 3540

atacacttga taagaataat gtgccttata aaaaagagga tggtaattac acagttgcca 3600

atgttaaagg taataacgta agggacggct attcaactaa ttcaagaatt acaggtgtat 3660

tacctaataa cgcaacaatc aaatatgacg gcgcatattg catcaatggc tatagatgga 3720

ttacttatat tgctaatagt ggacaacgtc gttatatagc gacaggagag gtagacaagg 3780

caggtaatag aataagtagt tttggtaagt ttagcacgat ttagtattta cttagaataa 3840

aaattttgct acattaatta tagggaatct tacagttatt aaataactat ttggatggat 3900

gttaatattc ctatacactt tttaacatta ctctcaagat ttaaatgtgc gtaactggca 3960

ggtacttcgg tacttgccta ttttttttat gttatagcta gccttcgggc tagttttttg 4020

ttatgatgtg ttacacatgc atcaactatt tacatctatc cttgttcacc caagcatgtc 4080

actgggtgtt ttttccttgc gatagagagc atagttttca tactactccc cgtagtatat 4140

atgactttag cattcccgta taacagttta cggggtgctt tttatgttat aattacatgc 4200

gtatatagta ggagtgaact atatagcccg gcagaggcca tatatctgac tgttggtctc 4260

acaggagaca tcttccttgt catcactcat atacatatat cttgctaaca tagagttgtt 4320

atagtcgcta cgccacccat actagttact gggtggttgt ttttgttcgc cattatgttc 4380

tgtctacatc tttttgcgca agttgtgtat cataatactt aattgtgtta aaagaggtgg 4440

aaatatgaaa ggtgatatat acataccgat aatatcatct attttatctg gcgggatatc 4500

gtattatgct gcaacggtgg ttcataaatt taacaaaagg tataaaagga aagaaaaggc 4560

tgttgaaatg gcagacgagt tttcaaaatt aatttcgaaa aggagttttg atatagggga 4620

gataaacaag aaaattttag aaaatgttgg tttgcaagat aaaattaatg aattagagaa 4680

taagatgaat ttaagttttg ataatcacga actaagaaca gtctttactg ataaagagat 4740

tgcaaaatat cacacttata aaaaagtcac caataccgaa tttataaaaa ttttgcttcg 4800

tactttcaag aacgagaatt ataaagaaga gtgtttacgg gctataattt tagaatttga 4860

caacaagaaa acaattaaag aaaatatagg agataaacag tggacttttt cgacaggtga 4920

aaaagaaatt gttattaaaa gtaatgacgt attgcaaggt ataaatacac tcattaagaa 4980

gtataatgaa acccatatat attgcatgaa taaattagaa tatttttcta tgcattttac 5040

caataacata gcagatagta atacggttta tcagtcgttg catcaaatct atttaaaaac 5100

aatattgagt ctttacattg atatctcatc aacaaataaa atagggcatg aaaaatttta 5160

tgttaattta atagaatttt acaatgaatg gaataacaaa aagataaaat ttaaaaagaa 5220

aacagaaaag cgtataaata aaaacagaaa ctcattttta aaaactgaaa aattgaaata 5280

aactagaaaa tatagtatca ttatggtata taaaggagtt gatttttatg tggagtcctg 5340

tttgcgaatt aggagaaaca aaagaatcag ataaaaaaag aactaacaat aacgactaat 5400

ttttaaacta ctattatatt tacagataac agagtaaccg tatctttatt gatgcggtta 5460

tttttatacc cacacaacca aaaaaaccac accgcctatt aatttaggag tgtggttgtt 5520

tttgtttttt ttagggcaaa aaaagggcag attatttaaa taagggcaaa cacgagtgga 5580

aaatatttaa agttaaagtg ataaaaactt tgatattaca gcgtttttaa acgttcgtgt 5640

acgtttgaga atggccgttt taccattagg tgttaatgtg tcataatgat acagataacg 5700

ccttaattac agtgttttca aggtattcag aatgtattgt aaggtaaaaa agggcataaa 5760

aagggcagat ggattttagt atataagctt ttctaatttt cgatctaatt ctctatccat 5820

tttctccgtt acatgggtgt atacttttat ggtcgttttt tcatcggtat gcccaaccct 5880

tttcataatt gcttttaacg atatattcat ttccaccaat aaacttatat gcgtgtgtct 5940

taaagtatgg gtagtgacct ttttatttat attcaatgat tctgcagcta aagataatag 6000

tttgtttatt ttatcaattt gtaaaggatt tcctttacta gttgtgaata agaatcctct 6060

atcaatataa cttggtttcc attgtttcat ctttttattt tctaacatta tcttttttaa 6120

aacctttgct gttctggaat tgatggtaat gtttcttttt gaacctgtag tttttgtagt 6180

atccttatat ccaaacccag tattacattt aattctatga agagttccgt taatatcaat 6240

tgttttattt tttaggtcaa catctttaat ttgaagtgct aataactcgc ctatacgcat 6300

tcccgttaaa gcttgtactt ctagagcacc agcaattaaa atacgagttc tgtaatgcaa 6360

actgttataa ttcaatataa aatcgcgtat ttgaatgact tgttgtaatt caagatagtt 6420

gtacatttta gcttcgtctt tttcaatatc ttcaatcgtt tttcttttct ttggaagttt 6480

gacaccggtt agtagatgtt catttgggta atcgtagaat ttaactgcgt atttaatggc 6540

ttctttcatg tgtccaagtt ggtaccttaa ttgatttgta gaatagatgc ttgacaactt 6600

attaataaac atttgcatgt attttgtatc aagcttgttt aaaagtaagt ctttagaact 6660

gttctttttt atatttttga ccctcgtttt gatattatta agagtagtca cctttgaacc 6720

tgacgttttt atatgatatt caagccattc ctctaataga gtgtgaaaag tcaaagtttt 6780

taattcgctt gacgacttgt tgtttagttt ttcttttatt ttttcttcta aacggaacat 6840

agcttctttt tgtgattgtt ttgtatcttt gttcaagaca acacttacac gtttccattt 6900

atctgtgtat ggatctttgt acttctcgta gtatctgtat ttagtttcgt tatttttgtt 6960

tttaaatttt tcaatccaca tgtttatacc tcctgtaaga acgtacgttc tgtaaattta 7020

taaaaaataa taagggcagg cgggctaccc aaaatttagt actaggtact aaatatgtta 7080

taataaaata aaaagtaggt gataagatga ctcaatttct aggggcgctt cttcttacag 7140

gagttttagg ttacatacca tataaatatc taacaatgat aggtttagtt agtgaaaaaa 7200

acaaggttat caatactcct gtattattga ttttttctat tgaaacatgt ttgatatggt 7260

tttatagttt tataattttt aataatgttg atttaaaaaa tttgaattta attcagttgc 7320

ttacaggtct aaaagcaaat attttgtttc tatttatttt tgttttaaca gtgtttgtat 7380

ttaatccttt aattgttaaa tttattatct ggttaattaa tataaccaga aagtttatga 7440

aattggattg tataagctta ttagacaaaa gagacaagtt gtttaataac aacggtaaac 7500

cagtatttat agttataaaa gactttgaaa acagaatcat tgaagagggt gaacttaaaa 7560

cctataattc agctggtagc gatttcgatt tactagaggt tgagcgacaa gatttcaaag 7620

tatctgattt accgtcaaac gatgaattgt atattaaaca tacacttgta gaccttaaac 7680

aacaaattaa attggattta tatttaatga atgaatatta atcttttttc ttagcttttt 7740

ctgataaagt actttttaag ttttcgctgg cacccggctt ttcaaaactt ttgtttattg 7800

ggttactacg ggtagcttct tgtttttttg tttttatccg ccataaaatt ctcaccacca 7860

ttcaacgtct acactagtag gcgttttttg atttttatat taaagggcta taaaaagctg 7920

ttaatacttc aattctttaa tccacatata tttaaaagtg aggtagtagg taataaatat 7980

aagacttaaa gttaagattg cttttttcat gtcaatttct cctttgttta tatttatatt 8040

aaatcactaa atagacgtta ttaatcacaa tacaattaat tgattgtaag atacttagtc 8100

gtataattct atatacctat tagtaaattc ttctgctgtt atttctccat tttctttttg 8160

ttgttgaagt ttagaagctt ctttttgaat tgcatcatat ttttcacgag aatacccata 8220

tttttccatc tctttataat tagcttcgtt tatttgttct tgttgctgag gtgtgacaca 8280

accaccaact gtgcattgtg taccatcagg ttttgtgtaa cctataacgt cacctgcgcc 8340

ttgtgcttgg taccaagtat taccatctgc atctaccatg ccgttaacat tgtgaccatt 8400

ttttactctt tgtgatattt cgtctttagt taaaggtcta ttggtttgtt gatcgttgtt 8460

aacgtttgtg ttgttctcgt tgtttacttg attattgtta tcgttttgat tagcattttc 8520

ttttttcgct tctgcttttt ctttagtttc tttcttttta tctttgttat ctttctttgt 8580

ttcagttttt ttgctttcct ctttcttatc gccgtcgtgg ctaccacaag cgcctaaaac 8640

taacgcactc gctaatgtta aacctaataa tcttttcatt ttaatttctc ctttgtttat 8700

atttctttat atttaaaaac tctcaatggc tcaaatgtaa ttgagtattc gccgtagtga 8760

gtcccaatac catatatctt tttatattgt tctattgctt ctaatatgta ttcttcactc 8820

aattgcagat actcagacaa ctcatacaag ttacgtacac cataattgta agcttccaca 8880

atttcgcgta acgggactgc tgagataaag ccgtgtcgcc ttgcgtaatt ttcgaacttg 8940

cgattgttga atttcgagta atcggctata tcaccgtatg taagtttatt atgtgctaat 9000

tcttcaaaga gaattcctgc cttttctcta tctgataagc cacgctttat taaaattaaa 9060

tctcctaacc ataccccatc caaattatct ggaagcacat cagcctctct tatttcaata 9120

taatcatgtt gtattaaagt ttcttcatat aatcccatct gatacatcct ttacttacgt 9180

ttgcttctta tataatctgc ataatctaaa actctttgcc attcatcatc tgtcaattct 9240

ccttcaaggt gagctgcacg atgttgtact tcatcatcgt tttcttcaac ccaccccatt 9300

aaatatgcag gattaacatt taatgcagta gctatacttt ctatagtatc gttttttaaa 9360

tttttgatat ttccgctttc ataacgctgt acagtagctt cagttttacc aatttttctt 9420

cctagttcgg ccaaagtcat accttgtttt tctcttgatt gtttcattct ttttgaaaag 9480

cacatcgtaa tacagctcct tttacttgat agttctatta taaggaaaac tttcggcatt 9540

tgcaatattt ttctaaaaaa ctttcgtaaa gtgcttgacc tctttcgtaa catcatgata 9600

agattactta cgtaatacga aaggtggtga aaagaaatgc ctatagatgc taaacttttg 9660

aaatctaaaa tggctttgaa agaacataac atcaaaaccc tttctgaaga aattggtgtt 9720

aatagagata ctttatctaa tatgattcac gggagaacga aaccgtcgta cccggtaata 9780

aatggtattt attttgcgtt agaattgaca cctcaagaag gaagagatat tttttttaac 9840

gaagacttac gcaaaaagaa agttttaact taaggaggaa actgaaatgc aagcattaca 9900

aatagtagaa cagaacgaaa cacattatgt agacagtaga gaagttgcgg aaatgatagg 9960

aaagcgacac gacaatttag taagagacat taaaggttat atcaaggttt tagaggactc 10020

ctcaaaattg agtagtcata atttctttga agaaagcacc tatgttaatt cacaaaacaa 10080

agtacaacct tgttacctac taaccaaaaa aggttgcgac atagtagcaa acaagatgac 10140

aggtagtaaa ggcattttgt ttactgcaac ttatgttgat gcatttcata aaatggatga 10200

atacattaaa caacaagcac agcttaatgt accacaaaca ccaatgcaag cattagagat 10260

gatgttcaaa gcacaaaaag accaagaaca gtttaacaaa caaatgcaac aagaaatcac 10320

aggcattcgt cacattgtcg gtattgaaac gaaaaactgg cgtaacgaca caaacaaaat 10380

gttatctgca attgcgcaac atttaggtgg cggagcaatg caccagaaag ttaagtctga 10440

agcatataaa gctttagaag aaaaaggacg ctgtaattta aaaattcgta tgcagaaccg 10500

caaaggcaaa atgctagcga atggtgcaac gaaaacccag attaacaagt tgtcaaaatt 10560

agatgtgatt actgatgaac ctagattggt tgagatatac atttcagtga ttaagagtat 10620

ggcgattaaa tacggtgtag atattagcca atttgaaatt taaacaaaca tcttaaaagg 10680

aggaacaaca aatgttacaa aaatttagaa tcgctaaaga aaaaagtaaa ttaaaactca 10740

atttactaaa acatgcaaac agtaatttag aaacaagaaa caaccctgaa ctgttgcgag 10800

cagttgcaga gttgcttaaa gagattaatc gataaattct atgaattcga ttttagctga 10860

agcgatagct actattttgt ctccaacaaa agtatatgag ccattagtga acaaggaact 10920

tttaattttt tcttttgata tttcaacagt tccgcgatga cctgacttta tcactttttc 10980

taaattatcg atttcaacaa atttatcatt agaaagatat aaacaagctt tcatacttat 11040

tacctcctta ggttgataac aacattatac acggaaggag gagcaacaat acaagctcaa 11100

aacaaaaaaa gtcatctatt actactacga cgaagaaggt aatagacgac caatagatat 11160

tcaaattaat gacggatatg aactgatggt ccaatctcat ttcatcaacg acaccattga 11220

agaaatacca tacttaaata ataacttata tgcattggtt gatggttatg aatttaagtt 11280

aagttaaatt tttgagaaag atattgaaaa gctaattttc ccataaggtt aagagacata 11340

ctggatgttt tgttaacgac tcttttaact tcgttccaag ttttattgtc tctaatatta 11400

tcgagaaatt catggccaga ccaagtgatg tcattaataa tccaagaaac gaccctgcct 11460

tcgatgaatt tcagatcgca accaataaat ttagcttctt ctaattttaa aagtgaatac 11520

attactgttt caaaatcata tttatcaaaa ataatattat cgttgaaatt atgtcgagta 11580

agtggttcac ctattttctt attagattct atttctaaga gcaagagtct aacgcaatcg 11640

tggttaagtt tcatcctatc acctccttaa caggagtata gcagtaagga tcataaacat 11700

cttaaaagga ggaacaacaa atgaacattc aagaagcaac taagatagct acaaaaaatc 11760

ttgtctctat gacacggaaa gattggaagg aaagtcatcg aactaagata ttaccaacaa 11820

atgatagttt tttacaatgc atcatttcaa atagcgatgg gacaaacctt atcagatatt 11880

ggcaaccttc agccgatgac ctcatggcaa atgattggga agttataaac ccaactagag 11940

accaggaatt attgaagcaa ttttagaaat gctatcaatg atacttttta aattgttttt 12000

aaactcattt tcaaagtaaa caacagtctt gtctgaaatt gttacatgat aaatagtgtt 12060

actagcatac acgccgttta ggaacccaga gtttttaagt ttatttaaat cgtattttac 12120

atcttcgaaa tgtagttttt gaaaatactt tgtatgtata tctttagcac ttccaaaatt 12180

attgcaggtt aatttaaccg aacctaactt tacacattct aaataatctt tgtagagtac 12240

ggacaagata tattgttggt ctttagtaag tgtatcaaat tcatcagata tcaagggcat 12300

gttatcaacc taaggaggtg ataacaacat tatacaagaa aggagcataa acaaatgaac 12360

acaagatcag aaggattgcg tataggcgtc ccacaagttt ctagcaaagc tgatgcttct 12420

tcatcctatt taacggaaaa ggaacgtaac ttaggagcgg aaatattaga gcttattaaa 12480

aaaagtgatt acagctactt agaaataaac aaagttttct atgcattaga tagagaactt 12540

caatacaggg cgaataataa caaactttaa cattatacac ggaaggaaag atagaaatgc 12600

caaaaatcat agtaccacca acaccagaaa acacatatag aggcgaagaa aaatttgtga 12660

aaaagttata cgcaacacct acacaaatcc atcaattgtt tggagtatgt agaagtacag 12720

tatacaactg gttgaaatat taccgtgaag ataatttagg tgtagaaaat ttatacattg 12780

attattcacc aacaggcact ctgattaata tttctaaatt ggaagagtat ttgatcagaa 12840

agcataaaaa atggtattag gaggattatc aaatgagcga cacatataaa agctacctaa 12900

tagcagtgct atgcttcacg gtcttagcga ttgtactcat gccgtttcta tacttcacta 12960

cagcatggtc aattgcagga ttcgcaagta tcgcaacatt catattttat aaggaatact 13020

tttatgaaga ataaaaaaac tgctactcag agcaatgagt aacagtgtca aacatatcta 13080

ataaagaaat aaaaaatatg ttttcaatat aaaacgaaat acggaggatg tcaactatga 13140

ctaaaaaata taaagacatg acgcaggaag aaataaaaga cttattatct gaaaaaaccg 13200

cagaattata tgaattagcg aaagaaatta agggagaaag taaatttgat attttgcttt 13260

tctcatcaat aggagttatc gacggagatt atttagcagg ttcaagttct gtgattggtc 13320

atactttcga tcttgcttcc ttattggata gcactaagag ttataaagac attgtcaatg 13380

ttctccaaat gtgtaaatca caaaaatttc tcggtattga tgacagcaag gaggactaaa 13440

acaatgtatt acgaagtagg cgaaatcata cgcaaaaata ttcatgttaa cggattcgat 13500

tttaagctat tcattttaaa aggtcatatg ggcatatcaa tacaagttaa agatatgaac 13560

aacgtaccaa ttaaacatgc ttatgtcgta gatgagaatg acttagatat ggcatcagaa 13620

ttattcaacc aagcaataga tgaatggatt gaagagaaca cagacgaaca ggacagacta 13680

attaacttag tcatgaaatg gtagaggggg attaactaat ggctaatcta tatgagctat 13740

cagaagcatt taaaaagttg tctaatcaag atgaattaga tccaacatta ttaaaagaca 13800

cattagattc tatccaagca gaaatgaatg tcaaagtaga taacatcgtc aattggagac 13860

gtgaaacatt aggtgacata gatgtcatag ataaagagat taagcgactt caaaatttaa 13920

aaaaacaaaa acaaaattta actgatcgat taaaagatta tttaaaagag atgttagaaa 13980

cacaggaagt agatagttac cgcacagcta ctaatcatat ttacaagcgc aaaaacgggg 14040

ctagtaaaaa tattatcgat gaaaaactta ttccaaagga ttattggcta tcacaagccc 14100

cgaaacttaa ttctaagcaa ctaatcgatg atttgaaaga tgggaaagat attcctggcg 14160

ttgaattaaa ggtaacagaa agcctggtga ttaagtgatg aataaatcag aaacagttgt 14220

agaaataaac aaagctatgg ttgcgtttcg taaagaagta aaacaaccgc tcaaagataa 14280

aaataatcca tttttcaaat caaaatacgt acctcttgag aacgttgtag aagccattga 14340

cgaggcggca acacctcatg gactgtctta tactcaatgg gctttgaacg atgtagacgg 14400

gcgcgtagga gtcgctacaa tgcttatgca tgaaagcggt gaatatatcg agtatgatcc 14460

tgtatttatg aatgcagaaa agaatacgcc acaaggagca ggctcgttaa taagttatct 14520

taaacgttat tcgctatctg cgattttcgg tattactagt gaccaagacg atgacggaaa 14580

tgaagcaagt ggaaaaaata ataatccaaa acagcaaact agaacgcaat gggcaagtag 14640

cgaaactata gggattttaa ggaaagaggt tataagtttc gctaaattga taaagggcac 14700

ggataaagaa gctccacaaa atatagtaga acaaaaattc gacataaata actataaatt 14760

aacagaaaaa caagcagcag aagctattca aaaaatacga aacaacgcaa aaacaattac 14820

tggaggaaaa caataatgtt aaacagaaca gtattagtag gacgcttaac aaaagatcca 14880

gaatatagaa caacgccaaa tggtgtgagt gttaccactt tcactatcgc agttaacaga 14940

acatttacta acgctcaagg agaacgtgag gcagacttta ttaactgtgt aacttttaga 15000

aaacaagcag aaaatgtaaa taattattta tccaaagggt cgctggcagg tgtagacggg 15060

cgactacaaa cacgtagcta cgaaaacaaa gtcgggcaac gtgtatttgt gacagaagta 15120

gtagcggaca atgttcaatt cttagaaccg aagaataaca accaacaaca aaacaacaat 15180

tatcaacaac agaataatgc atataacgca ccacagaata gacaacaatc aaataacccg 15240

tttgctaatg ctaatagtcc tatagacatt agttcagacg acctcccgtt ttgatgaggt 15300

gtttatatga aagaaatttg gaaaggtgtt gttggatatg aaggtatata cgaagtcagt 15360

agttacggaa ggattagaac gcataaaaaa taaaaccact tataccgaga aacacggtat 15420

tagacattgg aaacaacgtt atttaaaaga taaaatgccc aacggtagag atgtaagagt 15480

aacgctttgg aaaaacggag agtgcaaata ttttctagtt catagattag tagctttcgc 15540

ttttatacca gtcgttaaag gaaaagagtg tattaaccat atagacggta atcctaaaaa 15600

taataacgtt gaaaatctcg aatggtgtac ttataaagaa aatactaatc atgctttcga 15660

aaatggactt aatactagca atatggctgt aaaactaaca aatcacttag ggatagaata 15720

cgaatttata agcatggcga gggcttcaaa atttttatgt aggtgtcacg gatacgtaag 15780

cgataggttg aaaactggtc atgaacaatt aaccgatatt aacggaaatt attttaaagc 15840

agagaagttg gtttaaatgg ctcaaatcaa aaactatatc actcaagatg acggcacaac 15900

aacggtcgtt atcgaggatg ccgagctagg agacaaagaa acgttattac tagataacgg 15960

ctacgaagtc gaatgtgatt tacgaatcga agacccattc aaaataacag acaagcaacg 16020

aagaaaaata tttgcgcttt gcaacgacat agagagccac acaggccaac cacgtgacta 16080

tatgaggtat ttattccaag aatatgtaac ggttctgtat ggctatgaca agagcatttc 16140

gttaagtgac tgtacacgga tgcaagcgaa tcaaattatc gaggtaacac ttgattggat 16200

atttcacaac gacataccgc ttagctacaa aacaagcgac ctactaaagc aagataaatc 16260

attcttgtac tggtcaacgg ttaaccgcaa ctgtgtaata tgcggaaagc ctcacgcaga 16320

cctagcgcat tatgaaacag tcggcagagg catgaacaga aacaagatga atcactatga 16380

caaacatgta ttagcgttat gtcgcgaaca tcacaacgag caacatgcga ttggcgttaa 16440

gtcgtttgat gataaatatc acttgcatga cgcgtggata aaagttgatg aaaggctcaa 16500

caaaatgctg aaaggagaga aaaatgaata agttactaat agatgactat ccgatacaag 16560

tactaccgaa attagctgaa ttaataggat taaacgaagc aatagtattg caacaaattc 16620

attattggtt aaacaactca aaacataaat acgatggtaa aacttggata ttcaattcat 16680

atccagaatg gcaaaagcaa tttccgtttt ggtctttgat tactataaaa agaacaatat 16740

acagtcttga aaaacaaaac ttgttgctca taggcaatta taacaaagct aaatttgata 16800

agacgaagtg gtacagcatt aattatcaaa ctattgaagg tatgatacga ccatcgtatc 16860

aaaatgatac gacgagtgta tcaaaaagag acgatggagt gtatcaaaat gatacgacca 16920

ataccataga ctacacagag actaacaaac atagagagac agacgacgtc tcaaagtcat 16980

ttaagtatat tagtatcaat ttagaaatta tacaaaaccc tttaaaagca gaacagttag 17040

aacacgaaat taaatcattt aagcaagatc agttcgaaat agtaaaagtc gctaccgatt 17100

actgtaaaga aaataacaaa agtctaaatt atctattaac tgtattaaag aactggaata 17160

aagagggtgt ttcagataaa gaaagtgctg aaaacaaatt gaaacctcgt aaaactaaaa 17220

aagaaactac cgatgatgtc atagcgcaaa tggaaaaaga attgagtgat gactaatgcc 17280

gatgagcaaa acacaagcat tagaaattat taaaaaagtt aggtacgtct acaacattga 17340

ttttgataaa ccgaagttgg aaatgtggat tgatgtatta agtcaaaacg gagattatca 17400

gccaactgta aaagcagtag atggatatat caacagtaac aacccgtatc cgcctaactt 17460

accagcaatc atgcgtaagg cacctaaaaa agtatctatc gagccggtag acaacgaaac 17520

cgctacacac caatggaaaa tgcagaatga ccccgaatat gtcagacaaa gaaaaatagc 17580

gctagataac ttcatgaata agttggcaga atttgggggc gataacgaat gaattacgga 17640

caatttgaga ttgaaagtac aataatcgct acgctactta aacaaccgga cgtattagaa 17700

aagataaggg ttaaagatta catgtttacg aacgaaaagt ttaaaacctt tttcaattat 17760

gtaatggacg ccggaaagat agatcatcaa gaaatctatt taaaagcaac taaagataaa 17820

gagtttttag atgcagatac tataactaaa ctttataact ccgatttcat tggatacggc 17880

ttctttgaac gttatcaaca agaattattg gaaagttatc aactcaacaa agcgaatgaa 17940

ttggtcactg agttcaaaca acaacctacg aaccaaaact ttaacaactt gattgatgaa 18000

ctcaaggatt taaaaacgat tactaacaaa aaagaagatg gaaccaagaa gtttgttgag 18060

gagtttgtcg aagagttata cagcgatagc cctaagaagc aaattaagac gggttacaag 18120

ctcatggatt acaaaatagg gggattagag ccatcacaat tgatcgtcat cgcagcgcgt 18180

ccctcggtgg gtaagacagg ttttgcatta aacatgatgc tgaacatagc acaaaatgga 18240

tacaaaacat ctttcttcag tctcgaaaca accggcacat cggtattgaa acgtatgtta 18300

tcaacaatta ctggcattga gttaacaaag ataaaagaaa tcaggaactt aacgccggat 18360

gacttaacaa agttaacgaa tgcgatggat aaaatcatga aattaggcat cgatatttct 18420

gatgaaagta atatcacacc gcaagatgtg cgagcacaag caatgaggca ttcagacagg 18480

caacaagtta tttttataga ttatcttcaa ctgatggata ctgatgcgaa agttgataga 18540

cgtgtagcag tagaaaagat atcacgtgac ttaaagataa tcgctaacga gacaggcgca 18600

atcatcgtac tactttcaca actgaatcgt ggtgtcgagt ctagacagga taaacgacca 18660

atgctatcgg acatgaaaga atcaggcgga atagaagcag atgcgagttt agcaatgcta 18720

ctttaccgtg atgattacta taaccgtgac gaagatgacg gtattacagg taaatctatt 18780

gttgaatgta acatagccaa aaacaaagac ggagaaactg gaataattga atttgagtat 18840

tacaagaaga ctcagaggtt tttcacatga atatcatgca attcaaaagc ttattgaaat 18900

cgatgtatga agagacaaag caaagcgacc cgattgtagc aaatgtctat atagaaactg 18960

gttgggcagt taacagattg ttagacaata acgagttatc gccttttgat gattatgaca 19020

aagttgaaga gaagatcata aatgaaatca actggaagaa aacgcacatt aaggagtgtt 19080

aaaaatgccg aaagaaaaat attacttata ccgagaagat ggcacagaag atattaaggt 19140

catcaagtat aaagacaacg taaatgaagt ttattctctc acaggagccc atttcagcga 19200

cgaaaagaaa attatgactg atagtgacct aaaacgattc aaaggcgctc acgggcttct 19260

atatgagcaa gaattaggtt tacaagcaac gatatttgat atttagaggt ggcacatgga 19320

aatagaaatt aaatttaacg aaacgttcga ggcacctatg ggctcgcctc gtccacgctt 19380

tcgtaataca ggtagatttg ttcaaactta catgcctacg tcttacacaa agcataaagc 19440

gtatatacaa gggcaaatgc ctaagttaaa tctagagcgc gcactaaaaa tagaattaga 19500

cttttacttt ccattgctta aatcgtggtc gaagaaaaag aaaattgaaa tggttggaca 19560

gtataaagtg actaagccgg atatcgataa cttaattaaa acggtattag atgcttgtaa 19620

tggtcatgta tggaaagatg ataaccaaat tacagaaata actagctcaa agcgttatgg 19680

actagaacca aaaataatca tgcgagttga ggaagtgatc taatgcaaca acaagcatat 19740

ataaacgcaa cgattgatat aagaatacct accgaagttg aatatcagca ttttgatgat 19800

gtggataaag aaaaagaaac gctggcagat tacttatatg acaatcctga cgaaatacta 19860

gagtatgaca atttaaaaat tagaaatgta aatgtagagg tggaataaat gggcagtgtt 19920

gtaatcatta ataataaacc atataaattt aacaattttg aaaaagaaat aatggcaaag 19980

cgtgggataa atgctggaat tgtttctaaa cgtgttagag gttgttggga gttttcagaa 20040

gctttagacg cgccttatgg catgcaccta aaagaatata gagaaatgaa acaaatggaa 20100

aagattaaac aagcgagact cgaacgtgaa ttggaaagag agcgaaagaa agaggctgag 20160

ctacgtaaga agaagccaca tttgtttaat gtgcctcaaa aacattcacg tgatccgtac 20220

tggttcgatg tcacttataa ccaaatgttc aagaaatgga gtgaagcata atgagtgtaa 20280

tcagtaacag aaaagtagat atgaacgaaa tgcaagataa tgttaagcag ccgtcgcatt 20340

acacatacgg agacattgaa attatagatt ttatcgaaca agtaacggca cagtatccac 20400

cacaattagc attcgcaata ggtaatgcaa ttaaatactt gtctagagca ccgttaaaga 20460

atggtcatga ggatttagca aaggcgaagt tttacgtcga tagagtattt gacttgtggg 20520

agtgatgacc atgacagata gcggacgtaa agaatactta aaacattttt tcggctctaa 20580

gagatatctg tatcaggata acgaacgagt ggcacatatc catgtagtaa atggcactta 20640

ttactttcac ggtcatatcg tgccaggttg gcaaggtgtg aaaaagacat ttgatacagc 20700

ggaagagctt gaaacatata taaagcaaag tgatttggaa tatgaggaac agaagcaact 20760

aactttattt taaaagggcg gaaacaatga aaatcaaaat tgaaaaagaa atgaatttac 20820

ctgaacttat ccaatgggct tgggataacc ccaagttatc aggtaataaa agattctatt 20880

caaatgatgt tgagcgcaac tgttttgtga cttttcatgt tgatagcatc ttatgtaatg 20940

tgactggata tgtatcaatt aacgataaat ttactgttca agaggagata taacaatgaa 21000

aatcaaagtt aaaaaagaaa tgagattaga tgaattaatt aaatgggcgc gagaaaatcc 21060

ggatctatca caaggaaaaa tatttttttc aacaggattt agtgatggat tcgttcgttt 21120

tcatccaaat acaaataagt gttcgacgtc aagttttatt ccaattgata tccccttcat 21180

agttgatatt gaaaaagaag taacggaaga gactaagttt gataggttgt ttgaagtgta 21240

cgagtttcaa gaaggagatt gtaccgctat atcacacgct aatattagta taaacaaaca 21300

tttagatgaa cattgtttcc ctatcaaagc attctatatc ttaaacgacg acctaactat 21360

gacgttaatt tggaaagatg gggagttggt agaatgatgt tgaaatttaa agcttgggat 21420

aaagataaaa aagttatgag tattattgac gaaatcgatt ttaatagtgg gtacattttg 21480

atttcaacag gttataaaag tttcaatgaa gtaaaactat tacaatacac aggatttaaa 21540

gatgtgcacg gtgtggagat ttatgaaggg gatattgttc aagattgtta ttcgagagaa 21600

gtaagtttta tcgagtttaa agaaggagcc ttttatataa cttttagcaa tgtaactgaa 21660

ttactaagtg aaaatgacga tattattgaa attgttggaa atatttttga aaatgagatg 21720

ctattggagg ttatgagatg acgttcacct tatcagatga acaatataaa aatctttgta 21780

ctaactctaa caagttatta gataaacttc acaaagcatt aaaagatcgt gaagagtaca 21840

agaagcaacg agatgagctt attggggata taggtaagtt aagagaacgt aacaaagatc 21900

tagagaagaa agcaagcgca tgggataggt attgcaagag cgttgaaaaa gatttaataa 21960

acgaattcgg caacgatgat gaaagagtta aatttgggat ggaattaaac aataaaattt 22020

ttacggagga tgacacaaat ggataaccgc gaacaaatag aacaatccgt aatcagtgct 22080

agtgcgtata acggtaatga cacagaggga ttgctaaaag agattgagga cgtgtataag 22140

aaagcgcaag cgtttgacag actacttgaa gacattaata actttactca aagtccgact 22200

aaaggagcgc tagaactaga tgaagcaata gggattatgg taagtcaagt tatctatgaa 22260

tacaaggagg aactggagaa tgaaaaaatt taatgttcaa atcacatata caggcatgat 22320

tgaagaggct atcgaggctg aaagtttaga agaagcagaa tttgaggctc atgatattgc 22380

gagaatggaa gtgccatttg attgtgatga atttgaaatt aatgtagagg tggaacagga 22440

aaatgaataa cacattaaca attgatcaat tacaagagtt attacaaata caaaaggagt 22500

tcgacgatag aataccaacc agaaatttaa atgacacagt agctagtatg attattgaat 22560

ttgcggagtg ggttaacaca cttgagtttt ttaaaaattg gaagaaacaa ccaggtaagc 22620

cattagatac acaattagat gagattgctg attacttagc tttcagtttg caattaactt 22680

tgactattgt tgatgaagaa gatttggaag aaactactga ggttatggtt gatttgattg 22740

aaaatgaagt tactttacct aaactacatt cagtttattt tgttcatgta atgcatacac 22800

taacagaaca atttgtaaaa ggtattgata atagtattgt acaagtttta ataatgcctt 22860

ttttgtacgc caatacttac tatacaatcg accaactcat tgacgcatac aaaaagaaaa 22920

tgaaaaggaa tcatgaaaga caagatggaa cagcagacgc aggaaaagga tacgtgtaaa 22980

gacatattag atagagtcaa ggaggttttg gtgaagtgac gcaatactta gtcacaacat 23040

ttaaagattc aacaggacgt aagcatacac acataactcg agctaagagc aatcaaaggt 23100

ttatagttgt tgaggcagag agtaaagaag aagcgaaaga gaagtacgag gcgcaagtta 23160

aaagaggtgc agttattaaa gtgggtcagt tgtttgaaaa tataagggag tgtgggaaat 23220

gattaagcaa atactaagat tattattcct actagcaatg tacgagttag gtaagtatgt 23280

aactgagcaa gtgtatatta tgatgacggc taatgatgat gtagaggcgc cgagtgatta 23340

cgaaaaaatc agagctgaag tttcatggta atagctatta tcatttttga attaattata 23400

ttaatgtgtt tagcaatagc actggaggtg ttgtaaatat gtggattgtc atttcaatcg 23460

ttttagctat atttttattg atcttgttaa gtagcatttc tcataagatg aaaaccatag 23520

aagcattgga gtatatgaat tcttatcttt tcaagcagtt agtaaaaaat aatggtgttg 23580

aaggtttaga agattatgaa aatgaagttg aacgaattag aaaaagattc aaaagctaaa 23640

gagggggcta aagccctctc aaggattaaa agaagcggat attttttatt tcgtttttgt 23700

aaattaaaac taatttatgt gaaattgtct ttgaagtctt gataattaag aaggttgaat 23760

tttctgagat tatattagtt acgggaaagg cttttcctga gtgcaataaa attaaagttc 23820

tcaagttttc attttcgtat atctaatggt gcagtttata cagatgatga agatgtaaga 23880

cttatacatg ataagaagtt agataagtta gaggaaatta tcgaagagtc acaaggtcaa 23940

ccaatactat tgttttataa cttcaaacac gataaagaaa gaatacttca aaggtttaag 24000

gaagcaacca cattagagga ttcaaactat aaagaacgtt ggaatagtgg cgacattaag 24060

atgcttatag cacatccagc aagtgcagga catggattaa acttacaaca aggtgggcac 24120

attattgttt ggtttggact tacgtggtca ttggaattat atcaacaagc aaatgctaga 24180

ttatacagac aaggacaaaa ccatacgact attattcatc atattatgac cgataacacg 24240

atagatcaaa gagtatataa agctttacag aataaagaac taacgcaaga agaattgatg 24300

aaagctatta aagcaagaat agctaagcat aagtaatgga ggtataagat gggaaaggca 24360

tcatacgata ttaagccagg tacatttaaa tatattgagt cagagatata taacctacaa 24420

gagaacaaga aagagataaa tagattgaga atggagatac ttaacccaac gaaagaggta 24480

gacactaaca ttgtgtatgg gccgttgcaa aaaggtgaac cagttagaac aactgaacta 24540

atggcaacaa ggttattgac taataagatg ttacgaaacc tagaagaaat ggtcgaagca 24600

gttgaaagtg aatacttaaa gttacctgaa gattataaga aagtaataag gttaaagtat 24660

tggaataaag ataagaagct aaagatagag caaataggag atgcatgtca catgcatcgt 24720

aatacagtta ctactatacg aaagaacttt gttaaagcgg tagcgtatca tgcaggtatc 24780

aaataacatt gtgcaaagat tgtgcaaaag gcctacaaat ctgtagtaat atgatagtat 24840

cggaaagatg tataaagtta tctaaaagtt atacgacata agtacacgag gcacatcgct 24900

atgcggtgtg tcttttgtta tgcaatcaaa gaggtgtaag agttgaccaa gcataataac 24960

atttataagc atggtcgtaa gtcatatcaa tacgattggt tctatcattc aaaagcatgg 25020

aagaaattaa gagagatagc attagataga gataattatc tttgtcaaat gtgtttacgc 25080

gaagatatta taacagatgc aaacattgtg catcacatta tttatgttga tgaagatttt 25140

aacaaagctt tagacttaga taatctaatg tcagtttgtt atagctgtca taacaaaatt 25200

catgcaaatg ataatgacaa aagtaatatt aagaaggtta gaattttaaa aatttaaata 25260

aaaaaattat ttaaataaaa tttcatagcc ccctgtccat cggcttaaaa tgttttttcg 25320

ccgggtaccg gcgggggccc ttcgcttgca acgcggataa acttttatga aagggggtct 25380

ttatatgaaa ttaacaaaaa aacagctaaa agaatatata gaggattaca aaaaatctga 25440

tgacatatta attaacttgt atatagaaac atatgaattt tattgtcggt taagagatga 25500

acttaaaaat agtgatttga tgatagagca tacaaacaag gctggtgcga gcaatattgt 25560

taagaatcca ttaagcatag aactgacaaa aacagttcaa acactaaata acttactcaa 25620

gtctatgggt ttaacagcag cacaaagaaa aaagatagtt caagaagaag gtggatttgg 25680

tgactattaa agttttaaat gaaccttcac caaaactatt aacaacatgg tatgcagagc 25740

aagtcactca agggaaaata aaaacaagca aatatgttag aaaagaatgt gataggcatc 25800

ttagatattt agaaaatgga ggtaaatggg tatttgatga agaattagcg catcgtccta 25860

ttcgatttat agaaaagttt tgtaaacctt ccaaaggatc taaacgtcaa cttgtattac 25920

aaccatggca acattttatt attggcagtt tgtttggttg ggttcataaa gaaacaaagc 25980

tgcgcaggtt taaagaagct ttgatattta tggggcgaaa aaatggtaaa acaacaacca 26040

tttctggtgt cgctaactat gctgtatcac aagatggaga aaatggtgca gaaattcatt 26100

tgttagcgaa tgtgatgaaa caagcgagaa ttctatttga tgaatctaag gcgatgatta 26160

aagctagccc aaagcttaga gaaaatttta gacctttgag agatgaaatt cattacgatg 26220

caacgatatc taaaattatg ccacaggctt cagacagtga taagttggat ggtttaaata 26280

cacatatggg tatttttgat gaaattcatg aatttaaaga ttataaattg atttcagtta 26340

taaaaaactc aagagcggca agattacagc cccttcttat atacattacg acagcagggt 26400

accaactaga tggaccactt gttgatatgg tagaagcggg aagagacacg ttagatcaaa 26460

tcatcgaaga tgaaagaact ttttactatt tagcatcttt agatgatgac gatgatataa 26520

atgattcgtc gaattggatt aaagcaaatc ctaacctagg cgtttctatc gatttagatg 26580

aaatgaaaga agagtgggaa aaagccaaac gcacaccagc tgaacgaggc gattttataa 26640

caaaaaggtt taatatattc gctaataacg acgaaatgag ttttattgat tatccaacac 26700

ttcaaaaaaa taatgaaatt atttctttag atgagttgga aggtagacca tgtactatag 26760

gttatgattt atcagaaaca gaggacttta cagccgcatg tgccactttt gcattagata 26820

atggcaaagt tgctgtctta acacattctt ggattcctaa gcataaagta gagtattcta 26880

acgaaaaaat accatataga gaatgggaag aagacggatt actaacaata caagataagc 26940

cttatataga ctaccaagat gttttaaatt ggataattaa gatgaatgag cattatgtag 27000

tagaaaaaat tacttatgat agagcgaacg cattcaaact aaatcaagag ttaaaaaaat 27060

acggttttga aactgaagaa acaagacaag gagctttgac cttgagccct gcattgaagg 27120

atctaaaaga aatgttttta gatggaaaaa taatatttaa taataatcca ttaatgaaat 27180

ggtatatcaa taatgttcag ttgaaactag acagaaacgg aaactggttg ccgtctaagc 27240

aaagcagata tcgtaaaata gatggctttg cagcattttt aaacacatat acagatatta 27300

tgaataaagt tgtttctgac aagggtgaag gaaacataga gtttattagt attaaagaca 27360

tcatgcgtta aggaggtgaa tgttatcgca aaagagaata ttgtcacacg cataaagaaa 27420

aaattgatag acaattggat tgatcaatca gcttctaagc tttatgactt tagcccatgg 27480

aaaaataaat ctttttgggg tgtaatcaat aatacgcttg aaactaatga aacgatattt 27540

tcagctatta caaagttatc taattcgatg gctagtttgc ccttgaaaat gtatgaagat 27600

tataaagtag ttaatacaga agtatctgat ttacttacag tgtcaccgaa taattctctg 27660

agcagttttg attttattaa tcaaattgaa acaatcagaa atgaaaaagg taatgcatat 27720

gtgctaattg aacgagacat ctatcatcaa ccatcaaagc ttttcttatt aaatccagat 27780

gttgttgaaa tgttaattga aaaccaatca cgtgaacttt attattccat tcatgctgca 27840

actggaaata aattgattgt tcataatatg gacatgttgc attttaaaca catcgtggca 27900

tctaatatgg tgcaaggcat tagtccgatt gatgtgttga agaatacaac tgattttgat 27960

aatgcagtaa gaacctttaa tcttacagaa atgcaaaaac ctgattcttt catgcttaaa 28020

tatggttcca atgtaggtaa agaaaaaagg cagcaagtgt tagaagattt caaacagtac 28080

tatgaagaaa acggtggaat attattccaa gagcctggtg ttgaaatcga accgttacct 28140

aaaaaatatg tctctgaaga tatagtggca agcgagaatt taacaagaga aagagtagct 28200

aacgtttttc aattgccctc agtattctta aatgcaagat caaatacaaa tttcgcgaaa 28260

aatgaagagt taaacagatt ttacttgcag cataccttat tgccaatcgt caaacagtat 28320

gaagaagaat ttaatcggaa actacttact aaaacagaca gagaaaaaaa taggtatttt 28380

aaatttaacg ttaaatctta tttaagggct gatagtgcaa cacaagcaga agtgtacttt 28440

aaagcagttc gtagtggtta ctacactata aatgacatta gagagtggga agatttacca 28500

ccagttgaag gtggagataa gccgctaata agcggtgatt tatacccaat tgacacgcca 28560

cttgaattaa gaaaatcttt gaaaggtggt gataaaaatg tcaatgaaag ctaagtattt 28620

tcaaatgaaa agaaaatcaa aaagtaaagg tgaaatattt atttatggtg atattgtaag 28680

tgataaatgg tttgaaagtg atgtaactgc tacagatttc aaaaataaac tagatgaact 28740

aggagacatc agtgaaatag atgttcatat aaattcatct ggaggcagtg tatttgaagg 28800

gcatgcaata tacaatatgc taaaaatgca tcctgcaaaa attaatatct atgtcgatgc 28860

cttagcggca tcaattgcta gtgttatcgc tatgagtggt gacactattt ttatgcacaa 28920

aaatagtttt ttaatgattc ataattcatg ggttatgact gtaggtaatg cagaagaatt 28980

aagaaagaca gcggatttac ttgaaaaaac agatgctgtt agtaattcag cttatttaga 29040

taaagcaaaa gatttagatc aagaacactt aaaacagatg ttagatgcag aaacttggct 29100

tactgcagaa gaagccttgt ctttcggctt gatagatgaa attttaggag ctaatgaaat 29160

agctgctagt atctctaaag agcaatataa gcgtttcgag aacgtcccag aagatttaaa 29220

gaaagatgta gacaaaatca ctaaaattga tgatgtagat acatctgaat tggttgaaac 29280

acctaaagaa agcatgtcac tagaagaaaa agaaaaaaga gaaaaaatta aacgcgaatg 29340

cgaaatttta aaaatgacaa tgaattatta ggaggaaatg aaatgccgac attatatgaa 29400

ttaaaacaat ccttaggtat gattggacaa caattaaaaa ataaaaatga tgaattgagt 29460

cagaaagcaa cagatccaaa tattgatatg gaagacatca aacaactaga aacagaaaaa 29520

gcaggtttac aacaaagatt taacattgtt gaaagacaag tacaagacat tgaagagaaa 29580

gaaaaagcga aagttaaaga cacaggagaa gcttatcaat ctttaagtga taatgagaag 29640

atggttaaag ctaaggcaga gttttatcgt cacgcgattt taccaaatga atttgaaaaa 29700

ccttcaatgg aggcacaacg tttattacac gctttaccaa caggaaatga ttcaggtgga 29760

gataagctct taccaaaaac actttctaaa gaaattgttt cagaaccatt tgctaaaaac 29820

caattacgtg aaaaagctcg tctaactaac attaaaggtt tagagattcc aagagtttca 29880

tacactttag acgatgatga tttcattaca gacgtagaaa cagcaaaaga attaaaagca 29940

aaaggtgata cagtcaagtt cactactaat aaattcaaag tatttgctgc aatttcagat 30000

actgtaattc atggatcaga tgtagattta gtaaactggg ttgaaaacgc actacaatca 30060

ggattagcag ctaaagagcg taaagatgcc ttagcagtaa gtcctaaatc tggattagaa 30120

cacatgtcat tttataatgg atctgttaaa gaagttgagg gagcagacat gtatgatgct 30180

attattaacg ctttagcaga tttacatgaa gattatcgtg ataacgcaac aatttatatg 30240

cgatatgcag attatgtcaa aattattagt gttctttcaa atggaacaac aaatttcttt 30300

gacacaccag cagaaaaagt atttggcaaa ccagtagtat ttacagatgc agcagttaaa 30360

cctattgtgg gagatttcaa ttattttgga attaactatg atggaacaac ttatgacact 30420

gataaagatg ttaaaaaagg cgaatatttg tttgtattaa ctgcatggta tgatcagcaa 30480

cgtacattag acagtgcatt cagaattgca aaagcaaaag aaaatacagg ttcattaccc 30540

agctaagccc caaaaggtta atgtaacagc taaggctaaa tcagctgtaa tatcagccga 30600

ataggggtga tgaaatgagt ttagaagaaa ttaaattgtg gttgagaatt gactataatt 30660

tcgaaaatga tttaattgaa ggtctcattc aatcggctaa gtctgaatta ctattaagtg 30720

gggttccaga ttatgacaaa gatgacttgg aatacccgct tttttgtaca gcgattaaat 30780

atatcattgc aagagattat gaaagtcgtg gatactcaaa tgaccaatct agaagcaagg 30840

tgtttaatga aaaaggattg caaaaaatga ttttgaaatt aaaaaagtgg taggtgattt 30900

ttaaatggaa tttaatgaat ttaaagatcg cgcgtatttt tttcaatata taaacaaagg 30960

accatatcca gatgaagagg aaaaaatgaa attgtatagt tgcttttgta aaatatataa 31020

tccttctatg aaagatagag aaattttaaa aacgactgaa tcaaaatcag gattaaccat 31080

aattgttagg tcttctaaaa ttgaatatct accacaaaca aatcacttag ttaaaattga 31140

cagtgcatta tattccgata aattattcaa cattgtagaa ataagaattg atacaccaga 31200

tattggctat aatacagtgg ttttatcaga aaaatgagtg tagaaattaa agggatacct 31260

gaagtgttga agaaattaga atcggtatac ggtaaacaag caatgcaagc taagagtgat 31320

agagctttaa ataaagcatc tgaatttttt ataaagactt taaagaaaga attcgagagt 31380

tttaaagata cgggtgctag tatagaagaa atgactaaat ctaaacctta tacaaaagtt 31440

ggcagtcaag aaagagctgt tttaattgaa tgggtaggtc ctatgaatcg caaaaacatt 31500

attcacttga atgaacatgg ttatacaaga gacggtaaaa aatatacacc aagaggtttt 31560

ggagttattg caaaaacatt agctgctagt gaacgtaagt atagagaaat tataaaaaag 31620

gagttggcca gataaatgaa tatattaaac actgtaaaag gaattttatt atctgatgca 31680

gagctccaaa catatataaa ttctagaata tactattata aagtcactga aaatgctgaa 31740

acttccaaac cttttgttgt tattacacct atttatgatt taccttcaga ctttatgtct 31800

gataaatatc ttagtgaaga atacttaatt caaatagatg tagaatcttc aaataatcag 31860

aaaacaattg atataacaaa acgaataaga tacctgttat atcaacaaaa tttaattcaa 31920

gcatctagtc agttagatgc ttattttgaa gaaactaaac gttatgtgat gtcgagacgt 31980

tatcaaggca taccaaaaaa tatatattat aaaaatcagc gcatcgaata ggtgtgcttt 32040

ttaattttta aggaggaaat aagcaatggc agaaggacaa ggttcttata aagtaggttt 32100

taaaagatta tacgttggag tttttaaccc agaagcaaca aaagtagtta aacgcatgac 32160

atgggaagat gaaaaaggtg gtacagttga cctaaatatc acaggtttag caccagattt 32220

agtagatatg tttgcatcta acaaacgtgt atggatgaaa aaacaaggta ctaatgaagt 32280

taagtctgac atgagtattt tcaatattcc aagtgatgat ttaaacacag ttattggacg 32340

tactaaagat aaaaatggta catcttgggt aggagagaat acaagagcac cgtatgtaac 32400

agtaattggc gaatcggaag atggtttaac aggtcagccg gtatatgtag ccttacttaa 32460

aggtactttt agtttagatt caattgaatt taaaacacga ggtgaaaaag cagaagcccc 32520

agaacctaca aaattaacag gtgactggat gaatagaaaa gttgatgttg atggaacgtc 32580

acaaggtatt gtatacggtt atcatgaagg taaagaagga gaagcagaat tcttcaaaaa 32640

agtattcgtt ggatacacgg acagtgaaga tcattcagag gattctgcag gttcgttacc 32700

cagctaatcc ccaaaatgtt gaagtagcag ttaattcaaa atctgcaaca gtttcagcag 32760

aataggggct ttcaaaataa atcaaaggag aataatttat gactaaaact ttaaaggttt 32820

ataaaggaga cgacgtcgta gcttctgaac aaggtgaagg taaagtgtca gtaactttat 32880

ctaatttaga agcggataca acttatccaa aaggtactta ccaagtggca tgggaagaaa 32940

atggtaaaga atctagtaaa gttgatgtac ctcaattcaa aaccaatcca attctagtct 33000

caggcgtatc atttacacca gaaactaaat caattatggt aaataccgat gacaatgttg 33060

agccaaacat tgcaccaagc acagcaacga ataaaatatt gaaatataca agtgaacatc 33120

cagaatttgt tactgtagat gaaaatacag gagcaattca cggtgtagct gaaggtactt 33180

cagtaatcac tgctacgtct actgatggaa gcgataagtc aggacaaatt tcagtgacag 33240

taacaaacgg atagggattt aaggcgcagt atatctgcgt cttttttatt tgaataaaag 33300

gagctaatac aatgattaaa tttgaaatta aagatcgtaa aacaggaaaa acagagagct 33360

atacaaaaga agatgtaaca atgggcgaag cagaaaaatg ctatgagtat ttagaattag 33420

taaatcaaga gaataaaaaa gaagcaccta acgcaacaaa aatgagacaa aaagagcgac 33480

agttattagt agatttattt aaagatgaag gattgactga agaagatgtt ctgaacaaga 33540

tgagtactaa aacttataca aaagccttac aagatatatt tcgagaaatc aatggtgaag 33600

atgaagaaga ttcagaaact gaaccagaag agatgggaaa gacagaagaa caatctcaat 33660

aaaagacatt ttatcgaaca ttaagaaaat acaacgtttc tgtatggagc agtatgggtg 33720

gacattaact gaagtcagaa aacagccgta tgtaaaactt ttagaaatac ttaatgaaga 33780

gaataaagaa gagactgaag aaaaacaaag tgaacaaaaa gtcattacag gtacggattt 33840

aagaaaactt tttggaagct agaaaggagg ttaatatgaa tgaaaaagta gaaggcatga 33900

ccttggagct gaaattagac catttaggtg tccaagaagg catgaaaggt ttaaagcgac 33960

aattaggtgt tgttaatagt gaaatgaaag ctaatctgtc agcatttgat aagtctgaaa 34020

aatcaatgga aaaatatcag gcgagaatta aggggttaaa tgataggctt aaagttcaaa 34080

aaaagatgta ttctcaagta gaagatgagc ttaaacaagt taacgctaat taccaaaaag 34140

ctaaatccag tgtaaaagat gttgagaaag catatttaaa gttagtagaa gccaataaaa 34200

aagaaaaatt agctcttgat aaatctaaag aagccttaaa atcatcgaat acagaactta 34260

aaaaagctga aaatcaatat aaacgtacaa atcaacgtaa acaagatgcg tatcaaaaac 34320

ttaaacagtt gagagatgca gaacaaaagc ttaagaatag taaccaagct actactgcac 34380

aactaaaaag agcaagtgac gcagtacaga agcagtccgc taagcataaa gcacttgttg 34440

aacaatataa acaagaaggc aatcaagttc aaaaactaaa agtgcaaaat gacaatcttt 34500

caaaatcaaa tgataaaatt gaaagttctt acgctaaaac taatactaaa ttaaagcaaa 34560

cagaaaaaga atttaatgat ttaaacaata ctattaagaa tcatagcgct aatgtcgcaa 34620

aagctgaaac agctgttaat aaagaaaaag ctgctttaaa taatttggag cgttcaatag 34680

ataaagcttc atccgaaatg aagactttta acaaagaaca aatgatagct caaagtcatt 34740

tcggtaaact tgcaagtcaa gcggatgtca tgtcaaagaa atttagttct attggaaaca 34800

aaatgacttc cctgggacgt acaatgacga tgggcgtatc tacaccgatt actttaggtt 34860

taggtgcagc attaaaaacg agtgcagact ttgaagggca aatgtctcga gttggagcga 34920

ttgcacaagc aagcagtaaa gacttaaaaa gcatgtctaa tcaagcggtt gacttaggag 34980

ctaaaacaag taaaagtgct aacgaagttg ctaaaggtat ggaagaattg gcagctttag 35040

gctttaatgc caaacaaaca atggaggcta tgccaggtgt tatcagcgca gcagaagcaa 35100

gtggtgcaga aatggctaca actgcaactg taatggcttc agcgattaac tctttcggtt 35160

taaaagcatc tgatgcaaat catgttgctg atttacttgc gagatcagca aatgatagtg 35220

ctgcagatat tcaatatatg ggagatgcat taaaatatgc aggtactcca gcaaaagcat 35280

taggagtttc aatagaggac acttctgcag caattgaagt tttatctaac tcaggtttag 35340

aggggtctca agcaggtact gcattaagag cttcgtttat taggctagct aatccaagta 35400

aaagtacagc taaggaaatg aaaaaattag gtattcattt gtctgatgct aaaggtgagt 35460

ttgttggaat gggcgaattg attagacagt tccaagataa catgaaaggc atgacgagag 35520

aacaaaaatt agcaacagtg gctacaatag ttggcactga agcagcaagt ggatttttag 35580

ccttgattga agcgggtcca gataaaatta atagctatag caaatcattg aagaactcta 35640

atggtgaaag taaaaaagca gctgatttga tgaaagataa cctcaaaggt gctctggaac 35700

aattaggtgg cgcttttgaa tcgttagcaa ttgaagttgg taaagattta acgcctatga 35760

ttagagcagg tgcggaagga ttaacaaaat tagttgatgg atttacacat cttcctggtt 35820

gggttagaaa ggcttcggta ggcttagcaa tttttggtgc atctattggt cctgctgttc 35880

ttgctggtgg cttattaata cgtgcagttg ggagcgcggc taaaggctat gcatcattaa 35940

atagacgcat tgctgaaaat acaattcttt ctaataccaa ttcaaaagca atgaaatctt 36000

taggtcttca aacattattt cttggttcta caacaggaaa aacgtcaaaa ggctttaaag 36060

gattagccgg agctatgttg tttaatttaa aacctataaa tgttttgaaa aattctgcaa 36120

agctagcaat tttaccgttc aaacttttga aaaacggttt aggattagcc gcaaaatcct 36180

tatttgcagt aagtggaggc gcaagatttg ctggtgtagc cttaaagttt ttaacaggac 36240

ctataggtgc tacaataact gctattacaa ttgcatataa agtttttaaa accgcatatg 36300

atcgtgtgga atggttcaga aacggtatta acggtttagg agaaactata aagttttttg 36360

gtggtaaaat tattggcggc gctgttagaa agctaggaga gtttaaaaac tatcttggaa 36420

gtatcggcaa aagcttcaaa gaaaagtttt caaaagatat gaaagatggt tataaatcat 36480

taagcgacga tgaccttctc aaagtaggag tcaacaagtt taaaggattt atgcaaacca 36540

tgggcacagc ttctaaaaaa gcgtctgata ctgtaaaagt gttagggaaa ggtgtttcaa 36600

aagaaacaga aaaagcttta gaaaaatatg tgcgttattc tgaagaaaat agcagaatca 36660

tggaaaaagt acgtttaaac tcgggtcaga tatcagaaga caaagcaaaa aaacttttga 36720

aaattgaaac ggatttatct aataacctta tagctgaaat agaaaaaaga aataaaaagg 36780

aactcgaaaa aactcaagaa cttattgata agtatagtgc attcgatgaa caagaaaagc 36840

aaaacatttt aactcgaact aaagaaaaaa atgacttgcg aattaaaaaa gagcaagaac 36900

tcaatcagaa aatcaaagaa ttgaaagaaa aagctttgag tgatggtcag atttcagaaa 36960

atgaaagaaa agaaattgaa aagcttgaaa atcaaagacg tgatatcact gttaaagaat 37020

tgagtaagac tgaaaaagag caagagcgta ttttagtaag aatgcaaaga aacagaaatg 37080

cttattcaat agacgaagcg agcaaagcaa ttaaagaagc agaaaaagca agaaaagcga 37140

gaaaaaaaga agtggacaag caatatgaag atgatgtcat tgctataaaa aataacgtca 37200

acctttctaa gtctgaaaaa gataaattgt tagctattgc tgatcaaaga cataaagatg 37260

aagtaagaaa agcaaaatct aaaaaagatg ctgtagtaga tgttgttaaa aagcaaaata 37320

aagatattga taaagaaatg gatttatcca gtggacgtgt atataaaaat actgaaaagt 37380

ggtggaatgg ccttaaaagt tggtggtcta actttagaga agaccaaaag aaaaaaagcg 37440

ataaatacgc taaagaacaa gaagaaacag ctcgtagaaa cagagaaaat ataaagaaat 37500

ggtttggaaa tgcttgggac ggcgtaaaaa gtaaaactgg cgaagccttt agtaaaatgg 37560

gcagaaatgc taatcatttt ggcggcgaaa tgaaaaaaat gtggagcgga atcaaaggga 37620

ttccaagcaa attaagttca ggttggagct cagccaaaag ttctgtagga taccacacta 37680

aggctatagc taatagtact ggtaaatggt ttggaaaagc ttggcaatct gttaaatcga 37740

caacaggaag tatttacaat caaactaagc aaaagtattc agatgcttca gataaagctt 37800

gggcgcattc aaaatctatt tggagaggca catcaaaatg gtttagcaat gcatataaaa 37860

gtgcaaaggg ctggctaacg gatatggcta ataaatctcg cgcgaaatgg gataatattt 37920

ctagtacagc ttggtcgaat gcaaaatccg tttggaaagg aacatcgaaa tggtttagta 37980

actcatacaa atctttaaaa gattggactg gggatatgta ttcaagagcc cacgatcgtt 38040

ttgatgcaat ttcaagttcg gcatggtcta acgctaaatc agtatttaat ggttttagaa 38100

aatggctatc aagaacatat gaatggatta gagatattgg taaagacatg ggaagagctg 38160

cagctgattt aggtaaaaat gttgctaata aagctattgg cggtttaaat agcatgattg 38220

gcggtattaa taaaatatct aaagccatta ctgataaaaa tctcatcaag ccaataccta 38280

cattgtctac tggtacttta gcaggaaagg gtgtagctac cgataattcg ggagcattaa 38340

cgcaaccgac atttgctgta ttaaatgata gaggttctgg aaacgcccca ggtggtggag 38400

ttcaagaagt aattcacagg gctgacggaa cattccatgc accccaagga cgagatgtgg 38460

ttgttccact aggagttgga gatagtgtaa taaatgccaa tgacactctg aagttacagc 38520

ggatgggtgt tttgccaaaa ttccatggtg gtacgaaaaa gaaagattgg ctagaccaac 38580

ttaaaggtaa tataggtaaa aaagcaggag aatttggagc tacagctaaa aacacagcgc 38640

ataatatcaa aaaaggtgca gaagaaatgg ttgaagcggc aggcgataaa atcaaagatg 38700

gtgcatcttg gttaggcgat aaaatcggcg atgtgtggga ttatgtacaa catccaggga 38760

aactagtaaa taaagtaatg tcaggtttaa atattaattt tggaggcgga gctaacgcta 38820

cagtaaaaat tgctaaaggc gcgtactcat tgctcaaaaa gaaattagta gacaaagtaa 38880

aatcgtggtt tgaagatttt ggtggtggag gcgatggaag ctatctattt gaatatccaa 38940

tctggcaaag atttggacgc tacacaggtg gacttaactt taatggcggt cgtcactatg 39000

gtatagactt tggtatgcct tctggaacaa acgtttatgc cgttaaaggt ggtatagcag 39060

ataaggtatg gactgattac ggtggcggta attctataca aattaagact ggtgctaatg 39120

aatggaactg gtatatgcat ttatctaagc aattagcaag acaaggccaa cgtattaaag 39180

ctggtcaact gatagggaaa tcaggtgcta caggtaattt cgttagagga gcacacttac 39240

atttccaatt gatgcaaggg tcgcatccag ggaatgatac agctaaagat ccagaaaaat 39300

ggttgaagtc acttaaaggt agtggcgttc gaagtggttc aggtgttaat aaggctgcat 39360

ctgcttgggc aggcgatata cgtcgtgcag caaaacgaat gggtgttaat gttacttcgg 39420

gtgatgtagg aaatatcatt agcttgattc aacacgaatc aggaggaaat gcaggtataa 39480

ctcaatctag ttcgcttaga gacatcaacg ttttacaggg caatccagca aaaggattgc 39540

ttcaatatat cccacaaaca tttagacatt atgctgttag aggtcacaac aatatatata 39600

gtggttacga tcagttatta gcgttcttta acaacagata ttggcgctca cagtttaacc 39660

caagaggtgg ttggtctcca agtggtccaa gaagatatgc gaatggtggt ttgattacaa 39720

agcatcaact tgctgaagtg ggtgaaggag ataaacagga gatggttatc cctttaacta 39780

gacgtaaacg agcaattcaa ttaactgaac aggttatgcg catcatcggt atggatggca 39840

agccaaataa catcactgta aataatgata cttctacagt tgaaaaattg ttgaaacaaa 39900

ttgttatgtt aagtgataaa ggaaataaat taacagatgc attgattcaa actgtttctt 39960

ctcaggataa taacttaggt tctaatgatg caattagagg tttagaaaaa atattgtcaa 40020

aacaaagtgg gcatagagca aatgcaaata attatatggg aggtttgact aattaatgca 40080

atcttttgta aaaatcatag atggttacaa ggaagaagta ataacagatt ttaatcagct 40140

tatattttta gatgcaaggg ctgaaagtcc aaacaccaat gataacagtg taactattaa 40200

cggagtagat ggtattttac cgggcgcaat tagttttgcg cctttttcat tagtattaag 40260

gtttggctat gatggtatag atgttataga tttaaattta tttgagcatt ggtttagatc 40320

tgtgtttaat cgcagacatc cttattatgt tattacttct caaatgcctg gtgttaaata 40380

tgcagtgaat acagctaatg ttacatctaa tttaaaagat ggttcttcaa ctgaaattga 40440

agtaagttta aatgtttata aagggtattc tgaatcagtt aattggaccg atagcgagtt 40500

cttattcgac tctaattgga tgtttgaaaa tggaattcct cttgatttca cacctaaata 40560

tactcataca tcaaatcaat ttactatttg gaacggttct actgatacga taaatccacg 40620

attcaagcac gatttgaaaa tattaattaa tttaaatgcg agtggaggat ttgaactggt 40680

taattataca acaggtgata tttttaagta caacaaaagt atagataaaa acactgattt 40740

tgttttagat ggtgtgtatg catatcgaga tataaacaga gtgggaattg atacaaatag 40800

aggtattata acattagcgc caggtaaaaa tgaatttaag attaaaggag acgtcagtga 40860

cattaaaact acatttaagt ttccttttat ttataggtag gtgatttaat ggattatcat 40920

gatcatttat cagtaatgga ttttaatgaa ttgatttgtg aaaatttact agatgtagat 40980

tatggttctt ttaaagaata ttatgaactg aatgaagcta ggtacatcac ctttacagtt 41040

tatagaacta ctcataatag ttttgttttt gatttattga tttgtgaaaa cttcataatt 41100

tatcatggtg aaaaatatac aattaagcag acagcaccga aggttgaggg agaaaaagtt 41160

tttattgaag ttacggcata tcatattatg tatgaatttc aaaatcactc tattgaatca 41220

aataaacttg atgataatag cgaatcttca aaaacgccag aatacacgtt agaagagtat 41280

ttgagatacg gatttgcaaa tcaaaaaact tcggtcaaaa tgacctataa aataattgga 41340

gattttaaac gaaaagtacc gattgacgaa ttaggtaaca aaaacggctt agaatactgt 41400

aaagaagcgg tagacctgtt tggctgtata atttacccaa atgatacaga gattggtttt 41460

tattctcctg aaacatttta tcaaagaagc gagaaagtga ttcgatatca atataatact 41520

gatactgtat ctgcaactgt cagtacattg gaattaagaa cagctataaa agtttttgga 41580

aaaaagtata cagctgagga aaagaaaaat tataatccta ttagaacaac tgacattaaa 41640

tattcaaatg gttttataaa agaaggtact tatcgtaccg aaacaattgg gtctaaagct 41700

actattaact ttgattgcaa gtatggtaat gaaacagtta gatttacaat aaaaaagggc 41760

tctcaaggtg gaatatataa gttgatttta gacggcaagc aaattaagca aatttcttgt 41820

tttgctaagt cggttcagtc tgaaacaata gatttaataa aaaatattga taaaggcaag 41880

cacgttttag aaatgatatt tttaggagaa gaccccaaaa atagaattga tatatcttca 41940

aataaaaaag ctaagccttg tatgtatgtt ggaactgaaa aatcaacagt cttaaattta 42000

attgctgata attcaggtcg caatcaatac aaagcaattg tcgactacgt cgcagatagt 42060

gcaaagcagt ttgggattcg atatgctaat acgcaaacaa atgaagatat cgaaacacag 42120

gataagctgt tagaatttgc aaaaaagcaa ataaatgata ctcctaagac tgaattagat 42180

gttaattata taggttatga aaaaatagag ccaagagata gcgtattttt tgttcatgaa 42240

ttaatggggt ataacactga attaaaggtt gttaaacttg ataggtcaca tccatttgta 42300

aacgcaatag atgaagtgtc tttcagcaat gaaataaaag atatggtaca aattcaacaa 42360

gcacttaaca gacgagttat tgcacaagat aatagatata actatcaagc aaatcgtata 42420

aatcatttat acactagtac tttgaattct cctttcgaga caatggatat agggagtgta 42480

ttaatataat ggcaacagaa gaagttaaaa tcaaagcgct acttgaaaac gataaacagt 42540

actttccagc tacacattgg aaagctataa atgggatacc ttatgcaggc agtagtgata 42600

ttgatggatt gcctcaagac ggtatcattt cggtagatga taaaaataaa ttagataatt 42660

taaaaatagg cgaagcagga attattcaaa atagcattgt acagaaatct ccaaacggta 42720

aattgtggaa aataacagtt gacgatagtg ggaaacttgg tacagtgcta ttttattaga 42780

aaggaaggtg cattatggaa aatttgtatt taataaagga tttgggagct ttagcaggtc 42840

gagattatag agctaaggaa atacaaaact tacaaagaat agagcaattt gcgcttggct 42900

tgacaacaga gtttaagttg catcagaaag ctaaaacaat tcaacacttc gctgagcaaa 42960

tttattataa tggtagatcg caagcagcag taaacaaatc tttacaaagt caaattaacg 43020

cacttgttgt ggcaccacgt aataacagtg ctaatgagat tgttcaagct cgagttaatg 43080

taaacggcga aacctttgac acattaaaag aacatttaga cgattgggaa accaaaactc 43140

aaattaataa agaggaaact ataagagaat taaataagac caaacaagaa attcttgata 43200

tcgagtatcg ttttgaacct gataagcaag aatttttatt tgtgacagaa cttgcacctc 43260

ttacaaatgc agtaatgcaa tccttctggt ttgataatag aacaggcata gtatacatga 43320

cacaagctag aaataatggc tacatgctaa gtcgtttaag acctaatggt caatttatag 43380

acagctcatt gattgtaggt gggggtcatg gtacacataa cggttataga tatattgatg 43440

atgagttatg gatttatagt tttatcttaa atggtaataa tgagaataca ttagttcgtt 43500

tcaagtatac gcctaatgtg gaaattagct atggcaagta tggtatgcaa gatgtattta 43560

caggacaccc agaaaaaccc tacatcaccc ctgtcataaa tgaaaaagaa aataaaattc 43620

tatacagaat tgagagacct agaagtcagt gggaacttga aaactcaatg aattatatag 43680

agataagaag tttagacgat gttgataaaa atattgataa agttttgcat aaaatcagta 43740

tccctatgag actaacaaac gaaacccaac caatgcaggg tgtgactttt gatgaaaaat 43800

acttgtattg gtatacagga gacagtaatc caaataatag aaactattta acggctttcg 43860

atttagaaac aggagaagaa gcgtatcagg ttaatgctga ctatggtgga acactagatt 43920

catttcctgg cgaatttgcg gaagcagaag gtttgcaaat atactatgac aaagatagtg 43980

gtaaaaaagc tttgatgcta ggtgttactg tcggtggtga tggaaataga acacatcgta 44040

ttttcatgat tgggcaaaga ggtattttag aaatactgca ctcaagaggc gttcctttta 44100

tcatgagtga cacaggtggt agagttaaac ctttaccaat gaggcctgat aaactcaaga 44160

atcttgggat gttaacagag ccaggtcttt actatttata cactgatcat acagttcaaa 44220

tcgatgattt cccattacca agagaatggc gtgatgcagg ttggttcttg gaagttaagc 44280

caccacaaac tggcggtgat gtaattcaga ttttgactcg taatagttat gcaaggaata 44340

tgatgacttt tgaaagggtg ctttctggaa gaactggaga catttcggac tggaattatg 44400

tgcctaaaaa tagtggtaaa tgggagagag taccttcatt catcacaaaa atgtcagata 44460

ttaacatagt aggcatgtcg ttttatttaa ctacggatga tacaaaacgt tttacagatt 44520

ttccaactga acgtaaaggg gtagctggtt ggaacttata tgtagaagct tcaaacacag 44580

gtggctttgt tcataggcta gttcgtaata gtgttacagc atctgctgag atactattga 44640

aaaattatga tagtaaaaca agttcagggc catggacttt acacgaaggg agaattataa 44700

gttaatgagt aatttagaga aatctgtagc tataaattta gaaaacacag cgcattatga 44760

aaatatttca aatctagata taacttttag aacaggagag agtgattctt ctgttcttct 44820

ttttaatatc actaaaaata atcaaccgtt attattgagt gaagaaaata tcaaagcacg 44880

aatagcgatt cgaggtaaag gagtcatggt agttgctcca ctagaaatat tagatccatt 44940

taaaggtatt ttaaaatttc aattacctaa tgatgtaatt aaacgagatg gaagttatca 45000

agctcaagtt tcggttgcag aattaggtaa ttcagacgtg gtagttgtcg agagaactat 45060

cacatttaac gttgaaaaaa gtttgtttag caagattccc tctgaaacaa aactacacta 45120

tattgttgag tttcaagaat tagaaaaaac tattatggat cgcgcgaaag caatggacga 45180

ggctataaaa aatggtgagg attatgcgag tctgattgaa aaagctaaag aaaaaggtct 45240

atcagatatt caaatagcaa aatcttcaag tattgatgaa ttaaagcaac ttgctaatag 45300

ccgtatatct gatt 45314

Claims (3)

1. Mutant staphylococcus aureus phage (Staphylococcus aureus bacteriophage) 4PHCISA25 is preserved in China Center for Type Culture Collection (CCTCC) NO: m20211354.

2. The use of the mutant staphylococcus aureus phage 4PHCISA25 of claim 1 for the preparation of a biofilm antimicrobial agent for controlling staphylococcus aureus.

3. The use of the mutant staphylococcus aureus phage 4PHCISA25 of claim 1 for preparing a bactericide for controlling staphylococcus aureus.