CN112063594B - High-temperature-resistant salmonella bacteriophage RDP-SA-18056 and preparation process of microcapsules thereof - Google Patents

Abstract

The invention discloses a high-temperature resistant salmonella bacteriophage RDP-SA-18056 and a preparation process of microcapsules thereof, wherein a host is salmonella S7, and the bacteriophage can form a plaque with the diameter of 2mm-4mm on a double-plate; observed by a transmission electron microscope, the bacteriophage has a polyhedral and three-dimensionally symmetrical head part, is wrapped by nucleic acid, has the diameter of about 50nm, has a tail part with the length of about 200nm, and is provided with a tail sheath, and the neck part is connected with the head part and the tail part; is of the order Narivulales Leptoviridae. Is preserved in the common microbiology center of China Committee for culture Collection of microorganisms, the preservation date is 2020, 01, 19 days, and the preservation number is CGMCC No. 19293. The bacteriophage has strong cracking effect on salmonella, and provides a bacteriophage source for industrial production of the bacteriophage and prevention and treatment of avian salmonella diseases and environmental disinfection. The bacteriophage has good temperature and acid-base tolerance; the preparation process of the phage microcapsule is simple, high in efficiency and low in cost, and the medicinal value of the phage is improved.

Description

High-temperature-resistant salmonella bacteriophage RDP-SA-18056 and preparation process of microcapsules thereof

Technical Field

The invention relates to the field of microorganisms, in particular to a high-temperature resistant salmonella bacteriophage RDP-SA-18056 and a preparation process of microcapsules thereof.

Background

Salmonella (Salmonella) is a species of bacillus-free bacillus parasitizing in the intestines of humans and animals, and its serotypes exceed 2500, many of which can cross-infect animals and humans, and most of which have strong pathogenicity and pose a great hazard to livestock and poultry husbandry and humans. Due to the abuse of antibiotics in poultry industry, the drug resistance of salmonella is stronger and stronger, multiple drug resistant strains are increased continuously, and the drug resistance spectrum of salmonella is changed continuously according to the difference of the types of the antibiotics. In recent years, drug-resistant salmonella has become the main pathogenic bacteria of salmonellosis of chickens,

the salmonella can infect people and animals through food pollution, can cause diseases such as gastroenteritis, bacteremia, extraintestinal infection and the like, is an important intestinal pathogenic bacterium, is an important infectious disease seriously harming poultry industry in China, and can cause the problems of reduction of hatching egg hatchability, chick survival rate, chick production performance and the like. Salmonella pullorum is one of the avian salmonella species and occurs only in chickens of various ages. The onset of chicks is acute septicemia, while the onset of adult chicks is mainly invisible infection.

The bacteriophage exists in natural environment widely, has various types, strong specificity, green and safety, and attracts people's attention to the bacteriophage again for the specificity and high efficiency of bacterial lysis.

According to the invention, through the separation of the salmonella bacteriophage, a high-temperature resistant salmonella bacteriophage is further separated, the biological characteristics of the salmonella bacteriophage are researched, and the bacteriophage stock library is expanded to lay a foundation for developing antibiotic substitutes for treating pullorum disease.

Disclosure of Invention

One of the purposes of the invention is to provide a safe and reliable high-temperature resistant salmonella bacteriophage RDP-SA-18056 for treating avian salmonellosis, provide a pathogenic bacterium S7 for producing the bacteriophage, and provide a separation method of the bacteriophage. The bacteriophage RDP-SA-18056 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of West Lu No.1 of Beijing, Chaoyang, with the preservation date of 2020, 1 month and 19 days and the preservation number of CGMCC No. 19293.

The invention also aims to provide the application of the bacteriophage in treating poultry salmonellosis, and the RDP-SA-18056 has higher temperature tolerance, wider acid-base tolerance range, higher titer, safety and effectiveness, is beneficial to industrial production, and provides a preparation process of the bacteriophage RDP-SA-18056 microcapsule by combining the characteristic of bacteriophage increment.

In order to achieve the purpose, the invention provides the following technical scheme:

the host of the high-temperature resistant salmonella bacteriophage RDP-SA-18056 is salmonella S7, and the bacteriophage can form plaques with the diameter of 2mm-4mm on a double-plate; observed by a transmission electron microscope, the bacteriophage has a polyhedral and three-dimensionally symmetrical head part, is wrapped by nucleic acid, has the diameter of about 50nm, has a tail part with the length of about 200nm, and is provided with a tail sheath, and the neck part is connected with the head part and the tail part; is a family of Flaviviridae of the order Narivulales, with a preservation number of CGMCC number 19293.

Furthermore, the length of the bacteriophage RDP-SA-18056 gene is 42960bp, and the bacteriophage RDP-SA-18056 gene has no virulence gene and lysogenic gene.

Further, the phage RDP-SA-18056 was treated at 65 deg.C for 60min, and the titer was still maintained at 10¹⁰pfu/mL or more.

Further, the phage RDP-SA-18056 was cultured at 37 deg.C for 1h at pH6-9, and the titer was still maintained at 10¹¹pfu/mL or more.

Furthermore, after the continuous 29 generations of the bacteriophage RDP-SA-18056, the titer is stabilized at 10¹¹pfu/mL or more.

Further, the bacteriophage RDP-SA-18056 is used in combination with antibiotics to improve the sensitivity of the antibiotics.

Furthermore, the bacteriophage RDP-SA-18056 has strong lysis effect on host bacteria, and the lysis rate reaches 98%.

Further, the bacteriophage RDP-SA-18056 is used for disinfection of the farm environment.

A preparation process of a high temperature resistant salmonella bacteriophage RDP-SA-18056 microcapsule is characterized by comprising the following steps:

(1) seed preparation:

a. preparing a host seed: selecting a single colony to inoculate in 5mL of LB liquid culture medium under an aseptic environment, culturing for 4-6h at 37 ℃ and 200rpm, inoculating the cultured host bacteria in 5% of inoculum size in 100mL of LB liquid culture medium, culturing for 4-6h at 37 ℃ and 200rpm, then inoculating in 5% of inoculum size in 1000mL of LB liquid culture medium, culturing for 4-6h at 37 ℃ and 150rpm, and placing the cultured host strain seeds in a refrigerator at 4 ℃ for later use;

b. preparing phage seeds, namely properly diluting the preserved phage seeds, putting 200 mu L and 200 mu L of host bacteria into an upper layer culture medium, pouring a double-layer plate, culturing for 8-12h at 37 ℃, selecting a plate with plaques presenting cloud, washing the upper layer culture medium with 5mL of LB liquid culture medium, then centrifuging at 12000rpm for 10min, filtering through a 0.22 mu m filter membrane, and collecting clear liquid;

respectively inoculating the clear liquid and the host into 5mL of LB culture medium according to the proportion of 2 percent, culturing at 37 ℃ and 200rpm for 4-8h (until a proliferation liquid becomes clear or floccules appear), simultaneously inoculating the cultured phage and the host into 100mL of LB culture medium according to the inoculation amount of 2 percent respectively, culturing at 37 ℃ and 200rpm for 4-8h, then simultaneously inoculating the cultured phage and the host into 1000mL of LB culture medium according to the inoculation amount of 2 percent respectively, culturing at 37 ℃ and 200rpm for 4-8h, and placing the cultured phage seeds into a refrigerator at 4 ℃ for later use;

(2) and (3) phage multiplication:

inoculating the prepared host bacteria into a seed fermentation tank in a proportion of 3%, culturing for 3-4h, wherein the OD600 value is 0.7-0.9, inoculating the host bacteria into the fermentation tank through a seed transfer pipeline, the inoculation amount is 5%, culturing the host in a seed tank for 3-4h, inoculating the phage into the seed tank in an inoculation amount of 5% when the OD is 0.7-0.9, culturing for 6-8h, and finishing proliferation after the dissolved oxygen is reversely increased and the pH tends to be stable;

wherein, the fermentation tank parameter is: the seeding tank is 200 plus 300rpm, the air ventilation is 1:0.6-0.8vvm at 37 ℃; fermentation tank: 120-150pm, 37 ℃, 1:0.6-0.8 vvm;

the components of the culture medium in the fermentation tank are as follows: 2.5% of yeast extract powder, 0.3% of glycerol, 0.8% of sodium chloride, 0.2% of monopotassium phosphate, 0.2% of dipotassium phosphate, 20ppm of calcium chloride and 20ppm of magnesium chloride;

(3) the phage post-treatment process comprises the following steps:

A. centrifuging: centrifuging the proliferated phage by using a tubular centrifuge at 14000rpm at a flow rate of less than or equal to 30L/H, and removing host, bacterial debris and the like which are not cracked;

B. further filtering and sterilizing by adopting a hollow fiber membrane with the thickness of 400 nm;

C. concentrating with 100KD roll-type membrane;

D. and (3) filtering and sterilizing: the concentrated phage is sterilized by three-stage filtration (polypropylene 0.45 μm, double-layer polypropylene 0.2 μm, polyethersulfone 0.22 μm);

(4) low-temperature spray drying of the phage:

adding the filtered and sterilized phage liquid into a carrier and uniformly stirring, wherein the carrier is as follows: 8% of porous starch, 2% of polyvinylpyrrolidone, 1% of lactose, 0.5% of modified chitosan and 0.2% of vitamin C; then carrying out warm spray drying, wherein the parameters are as follows: the air inlet temperature is 150 ℃, and the air outlet temperature is 75 ℃.

The preparation process of the high-temperature resistant salmonella bacteriophage RDP-SA-18056 microcapsule has the preferable scheme that the microstructure of the prepared bacteriophage RDP-SA-18056 microcapsule is a concave cavity.

The invention has the beneficial effects that:

(1) the invention discovers and separates salmonella avium S7, and obtains bacteriophage RDP-SA-18056 by taking salmonella S7 as a host, the bacteriophage RDP-SA-18056 has strong cracking effect on salmonella avium S7 in the culture environment, and provides a bacteriophage source for industrial production of bacteriophage for prevention and treatment of salmonella avium S7 in the culture environment;

(2) in the present invention, the bacteriophage RDP-SA-18056 is treated at 65 deg.C for 60min, and the titer is still maintained at 10¹⁰pfu/mL or more; the material has better tolerance to temperature; the phage RDP-SA-18056 is cultured at 37 deg.C for 1h at pH6-9, and the titer is still maintained at 10¹¹pfu/mL or more, and has good acid-base tolerance;

(3) in the invention, the lysis rate of the bacteriophage RDP-SA-18056 to the host bacteria is as high as 98 percent, and the bacteriophage RDP-SA-18056 is diluted by 10 percent⁷After doubling, the bacterial strain still has a good cracking effect on pathogenic bacteria;

(4) the bacteriophage RDP-SA-18056 can generate a synergistic effect with antibiotics, and on one hand, the bacteriophage can improve the sensitivity of the antibiotics; on the other hand antibiotics can improve the lytic property of the phage.

(5) Through the research on the biological characteristics of the phage obtained by separation, the invention provides theoretical guidance for the next step of research and development of new antibacterial drugs.

Drawings

The above and other features, properties and advantages of the present invention will become more apparent from the following description of the embodiments with reference to the accompanying drawings:

FIG. 1 is a schematic view of a plaque isolated by the present invention observed by an electron microscope;

FIG. 2 is a schematic diagram of a phage thermostability experiment according to the invention;

FIG. 3 is a schematic diagram of the experiment on the pH stability of the bacteriophage of the present invention;

FIG. 4 is a schematic representation of a genetic experiment for phage stability according to the present invention.

FIG. 5 is a schematic diagram of a one-step growth curve experiment for bacteriophage of the present invention;

FIG. 6 is a schematic representation of a phage-to-host lysis experiment of the present invention;

FIG. 7 is a schematic diagram showing the synergistic effect of the bacteriophage of the present invention and kanamycin monosulfate;

FIG. 8 is a schematic representation of the synergistic effect of bacteriophage of the present invention with fosfomycin sodium;

FIG. 9 is a scanning electron microscope image of the phage microcapsules of the present invention.

Detailed Description

The invention is further illustrated by the following figures and examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1 isolation and identification of pathogenic Salmonella S7

Sampling from a disease farm (a post-mountain farm behind Qingdao), taking diseased poultry livers by aseptic operation, streaking on a selective culture medium (SS agar), culturing for 18-24h at 37 ℃, forming red colonies which are round, flat, neat in edge and smooth and moist in surface on the culture medium, picking typical colonies, streaking and purifying for 3 times, picking single colonies, inoculating into 5mL of LB broth, and performing shaking culture at 37 ℃ and 200rpm for 8h to obtain a uniform and turbid bacterial suspension. And determining the salmonella as pathogenic salmonella by 16sRNA molecular identification and serotype identification, and storing one strain of the salmonella as S7 at-80 ℃.

Example 2 isolation and characterization of bacteriophage RDP-SA-18056:

(1) and (3) fecal treatment: collecting feces from a rear mountain farm of Qingdao, weighing 5g of chicken feces, adding 10mL of sterile water for soaking overnight, centrifuging the overnight leachate at 10000rpm for 5min, filtering the supernatant with a 0.22 μm filter, and keeping the filtrate for later use;

(2) preparing a mixed bacterial suspension: adding 0.2mL of the bacterial suspension and 0.1mL of the filtrate into 5mL of LB broth, performing shaking culture at 37 ℃ and 200rpm overnight, then centrifuging at 10000rpm for 5min, taking the supernatant, and filtering the supernatant through a 0.22-micron filter to obtain a filtrate for later use;

(3) phage separation: separating phage by double-plate method, mixing the filtrate 0.1mL of mixed bacterial suspension and 0.2mL of salmonella suspension, water bathing at 37 deg.C for 10min, spreading double-plate, culturing in 37 deg.C incubator for 6-8h, and observing the result. If there are phage, then there are transparent spots on the plate. Selecting the transparent spots, putting the transparent spots in 1mL of physiological saline, carrying out water bath at 37 ℃ for 30min, taking 0.1mL of leachate and 0.2mL of bacterial suspension, carrying out water bath at 37 ℃ for 10min, paving double plates, and culturing in an incubator at 37 ℃ for 4-6h for purification, wherein the purification is carried out for 3 times according to the steps.

The diameter of plaque formed by RDP-SA-18056 on the double plate is 2mm-4 mm.

Preservation of bacteriophage RDP-SA-17118: phage multiplication fluid was mixed with 60% glycerol according to 1:1, and storing in liquid nitrogen.

EXAMPLE 3 Electron microscopy of phages

Dropping 20 μ L of liquid containing crude phage particles on a copper net, settling naturally for 15 min, sucking off the excess liquid from the side with filter paper, adding a drop of 2% phosphotungstic acid (PTA) on the copper net to stain the phage for 10min, sucking off the staining solution from the side with filter paper, drying the sample, and observing the form of the phage with an electron microscope, as shown in FIG. 1.

The bacteriophage RDP-SA-18056 has a polyhedral head with a cubic symmetry, which is wrapped with nucleic acid, has a diameter of 50nm, a tail with a length of 200nm, and a tail sheath, wherein the head and the tail are connected with the neck. According to the ninth report of the organization of viral taxonomy of International taxonomy of viruses, this phage can be classified as the family of the order Nauroviridae.

Phage RDP-SA-18056 Whole genome sequencing and analysis: constructing a library by adopting an Illumina TruSeq ­ Nano DNA Sample Prep Kit method; the method comprises the following specific steps:

1. constructing a library by using the initial amount of 1 mu g of DNA;

2. the DNA is broken by Covaris M220 ultrasonic to 300-500 bp;

3. filling in, adding A at the 3' end, connecting with index adaptor (TruSeq. Nano DNA Sample Prep Kit);

4. enriching the library, and amplifying 8 cycles by PCR;

5.2% Agarose gel recovery of the target band (verified Low Range Ultra Agarose);

6. TBS380 (PicoGreen) is quantified and mixed according to the data proportion;

7. performing bridge PCR amplification on the cBot solid phase carrier to generate clusters;

8. the Illumina Hiseq sequencing platform carries out 2 × 150bp sequencing.

Example 4 measurement of the thermostability of phage

The phage stock solution was dispensed into 50 ml sterile centrifuge tubes, incubated at 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 80 deg.C and 90 deg.C for 30min and 60min, diluted with physiological saline 10 times in gradient, and then plated on double plates to determine the titer, the results of which are shown in FIG. 2. As can be seen from FIG. 2, the titer of the phage gradually decreased with the increase of temperature and the increase of time, and the titer of the phage decreased only 1 order of magnitude and remained at 10 after the treatment at 65 ℃ for 30min and 60min¹⁰And pfu/mL or more indicates that the phage has better high temperature resistance.

Example 5 determination of phage pH stability

Adjusting pH of normal saline with dilute hydrochloric acid and dilute NaOH solution to obtain buffer solution with pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and diluting phage with prepared buffer solution to 1 x 10¹²pfu/mL, the diluted solution was incubated at 37 ℃ for 1 hour in a water bath, diluted 10-fold with physiological saline and then plated on double plates to determine the titer. The statistics of the results after the culture at 37 ℃ for 4-6h in an inverted manner are shown in FIG. 3. As can be seen from FIG. 3, the pH was 6-9After being cultured for 1h at 37 ℃, the titer is only reduced by 1 order of magnitude, and the titer is still maintained at 10¹¹More than pfu/mL, which shows that RDP-SA-18056 has better acid and alkali resistance.

Example 6 genetic test for stability

Continuously passaging RDP-SA-18056 for 30 times, adding the same amount of RDP-SA-18056 and S7 into each generation of culture medium, culturing for 6h for each generation, spreading double plates, and measuring titer; the results are shown in FIG. 4. Continuously transmitting RDP-SA-18056 for 29 generations, measuring its potency, and stabilizing its potency at 10¹¹And pfu/mL or more indicates that the phage has better genetic stability and is suitable for industrial production.

Example 7 determination of phage growth curves

Inoculating salmonella S7 and bacteriophage RDP-SA-18056 to the salmonella suspension in logarithmic growth phase according to the optimal complex number of infection, carrying out water bath for 10min at 37 ℃, then centrifuging for 5min at 12000r/min at normal temperature, discarding the supernatant, removing the free bacteriophage which is not adsorbed on the host, and washing twice with LB broth culture medium. The pellet was resuspended in LB broth at 37 ℃ and quickly placed on a shaker at 37 ℃ and 200r/min for shaking culture and timing. And (3) sampling at regular time to measure the titer, drawing a growth curve by taking time as an abscissa and a bacteriophage titer logarithm value as an ordinate, obtaining the incubation period and the lysis period of the bacteriophage, and calculating the average lysis amount. Mean lysis = end-burst phage titer/initial host bacteria concentration. The results are shown in FIG. 5.

The titer is not obviously changed within 30min after the phage infects host bacteria, which shows that the incubation period is about 30min, the titer of the phage is obviously increased within 40-90min after infection, and then the titer is stable, which shows that the phage lysis period is about 50 min. Through calculation, the phage lysis amount is about 216 PFU/infected cell, and the phage RDP-SA-18056 has the characteristics of short latency, strong lytic capacity and high replication.

Example 8 phage vs host lysis curves

The bacteriophage RDP-SA-18056 is added into the suspension of the host S7, and the bacteriophage lysis curve of the host is obtained because the bacteriophage lysis effect on the host causes the change of the absorbance of OD 600. The host bacterial suspension cultured for 6 hours was inoculated into 100mL of LB medium at a ratio of 1:100, cultured at 37 ℃ for 2 hours with shaking (OD: about 0.15, about 5.0X 108 cfu/mL) at 200rpm, the bacterial suspension was divided into 4 sterilized Erlenmeyer flasks, to 3 of which 0.2mL (about 3.6X 1011pfu/mL, filtered through a 0.22 μm membrane) of phage-proliferated solution was added at the optimum multiplicity of infection, and the average was taken with a control without phage added. The results are shown in FIG. 6.

As can be seen from FIG. 6, the Salmonella phage RDP-SA-18056 is added into the suspension of the host bacteria S7, because the phage has a stronger lysis effect on the host, the OD600 absorbance increases slowly, and the difference between the OD600 increases with the time extension, and the difference between the OD600 and the host tends to be stable when 390-. The experiment further shows that the bacteriophage RDP-SA-18056 has stronger cracking effect, can crack pathogenic bacteria for a long time, effectively controls the production and propagation of host bacteria, and has important application value in animal disease treatment.

Example 8 synergistic Effect of phages and antibiotics

3% of the host S7 was inoculated into LB broth, kanamycin monosulfate and fosfomycin sodium were added to a final concentration of 30ppm, respectively, and 1X 10 was added⁷The change in OD600 absorbance was measured periodically for pfu/mL of phage RDP-SA-18056. The results are shown in FIGS. 7 and 8.

The OD600 value is gradually increased along with the time, but the combination of the phage and the antibiotic has the absorbance smaller than that of the other two groups, which shows that the antibiotic and the phage can more effectively inhibit the growth of host bacteria, the phage and the antibiotic have synergistic effect, the phage can improve the sensitivity of the antibiotic, and the antibiotic can improve the cracking property of the phage.

Example 9 RTD experiment

Dividing the region on the plate, dropping a drop of host bacteria (about 100 μ L) to the center of the region, air drying, dropping a drop of phage with different dilutions to the spot of host bacteria, air drying, and culturing at 37 deg.C for 16-24 h. According to the growth condition of the host bacteria, the CL concentration is called as the CL concentration (the CL concentration is the concentration of the host bacteria in the environment which can be completely lysed, and can be used for guiding the dilution scheme of the production practice). The result shows that the RDP-SA-18056 still has better lysis effect on the host bacteria after being diluted by 107 times.

EXAMPLE 10 cracking Rate experiments

Under the aseptic condition, 1mL of sample and 1mL of host bacterium liquid (1 × 105 CFU/mL) are respectively taken, incubated for 15 minutes at 37 ℃, uniformly mixed, diluted to 10-1-10-3 gradients by using physiological saline, 100 μ L of each gradient is taken and coated on an LB agar plate, the mixture is placed at 37 ℃, cultured for 24 hours, and each gradient is repeated twice. Simultaneously, 1mL of physiological saline and 1mL of host bacterium liquid (1X 105 CFU/mL) are taken as blank control, and the steps are repeated. Plates with 30-300 colonies were picked for enumeration. The test was repeated 3 times and the average was taken. Phage lysis rate = (1-number of treated group colonies/number of control group colonies) × 100%.

Finally, the experiment proves that the cracking rate of the RDP-SA-18056 reaches 98%, and the RDP-SA-18056 has a good cracking effect on a host and is suitable for being used in the culture process.

Example 11 determination of optimal multiplicity of infection

The phage multiplication solution and the host were added to the LB broth at a ratio of the multiplicity of infection of 100, 10, 1, 0.1, 0.01, and the total volume of the culture system was ensured to be the same. After shaking culture at 37 ℃ and 200rpm for 8h, centrifuging at 12000r/min at normal temperature for 5min, and spreading the supernatant on a double-plate to determine the titer. The results are shown in Table 1.

TABLE 1

As is clear from the data in Table 1, when the multiplicity of infection is 0.1, the growth medium is relatively clear and the titer is the highest after 8 hours of culture, indicating that the optimum multiplicity of infection of RDP-SA-18056 is 0.1.

Example 12 lysis general experiment

30 strains of Salmonella (including 10 strains of Salmonella typhi, 10 strains of Salmonella pullorum, and 10 strains of Salmonella dublin) were screened and subjected to the lysis-stacking experiment. The results show that RDP-SA-18056 can crack 26 strains of Salmonella.

Example 13 Process for the preparation of thermostable Salmonella bacteriophage RDP-SA-18056 microcapsules comprising the steps of:

(1) seed preparation

a. Preparing a host seed: selecting a single colony to inoculate in 5mL of LB liquid culture medium under an aseptic environment, culturing for 4-6h at 37 ℃ and 200rpm, inoculating the cultured host bacteria in 5% of inoculum size in 100mL of LB liquid culture medium, culturing for 4-6h at 37 ℃ and 200rpm, then inoculating in 5% of inoculum size in 1000mL of LB liquid culture medium, culturing for 4-6h at 37 ℃ and 150rpm, and placing the cultured host strain seeds in a refrigerator at 4 ℃ for later use;

b. preparing phage seeds, namely properly diluting the preserved phage seeds, putting 200 mu L and 200 mu L of host bacteria into an upper layer culture medium, pouring a double-layer plate, culturing for 8-12h at 37 ℃, selecting a plate with plaques presenting cloud, washing the upper layer culture medium with 5mL of LB liquid culture medium, then centrifuging at 12000rpm for 10min, filtering through a 0.22 mu m filter membrane, and collecting clear liquid;

respectively inoculating the clear liquid and the host into 5mL of LB culture medium according to the proportion of 2 percent, culturing at 37 ℃ and 200rpm for 4-8h (until a proliferation liquid becomes clear or floccules appear), simultaneously inoculating the cultured phage and the host into 100mL of LB culture medium according to the inoculation amount of 2 percent respectively, culturing at 37 ℃ and 200rpm for 4-8h, then simultaneously inoculating the cultured phage and the host into 1000mL of LB culture medium according to the inoculation amount of 2 percent respectively, culturing at 37 ℃ and 200rpm for 4-8h, and placing the cultured phage seeds into a refrigerator at 4 ℃ for later use;

(2) and (3) phage multiplication:

inoculating the prepared host bacteria into a seed fermentation tank in a proportion of 3%, culturing for 3-4h, wherein the OD600 value is 0.7-0.9, inoculating the host bacteria into the fermentation tank through a seed transfer pipeline, the inoculation amount is 5%, culturing the host in a seed tank for 3-4h, inoculating the phage into the seed tank in an inoculation amount of 5% when the OD is 0.7-0.9, culturing for 6-8h, and finishing proliferation after the dissolved oxygen is reversely increased and the pH tends to be stable;

wherein, the fermentation tank parameter is: the seeding tank is 200 plus 300rpm, the air ventilation is 1:0.6-0.8vvm at 37 ℃; fermentation tank: 120-150pm, 37 ℃, 1:0.6-0.8 vvm;

the components of the culture medium in the fermentation tank are as follows: 2.5% of yeast extract powder, 0.3% of glycerol, 0.8% of sodium chloride, 0.2% of monopotassium phosphate, 0.2% of dipotassium phosphate, 20ppm of calcium chloride and 20ppm of magnesium chloride;

(3) the phage post-treatment process comprises the following steps:

A. centrifuging: centrifuging the proliferated phage by using a tubular centrifuge at 14000rpm at a flow rate of less than or equal to 30L/H, and removing host, bacterial debris and the like which are not cracked;

B. further filtering and sterilizing by adopting a hollow fiber membrane with the thickness of 400 nm;

C. concentrating with 100KD roll-type membrane;

D. and (3) filtering and sterilizing: the concentrated phage is sterilized by three-stage filtration (polypropylene 0.45 μm, double-layer polypropylene 0.2 μm, polyethersulfone 0.22 μm);

(4) low-temperature spray drying of the phage:

adding the filtered and sterilized phage liquid into a carrier and uniformly stirring, wherein the carrier is as follows: 8% of porous starch, 2% of polyvinylpyrrolidone, 1% of lactose, 0.5% of modified chitosan and 0.2% of vitamin C; then carrying out warm spray drying, wherein the parameters are as follows: the air inlet temperature is 150 ℃, and the air outlet temperature is 75 ℃.

As can be seen from the scanning electron microscope picture (figure 9) of the microcapsule, the coating effect of the phage is good, and the microstructure of the microcapsule is a concave cavity and shows a flat-ball effect. The powder after spray drying is light yellow and has uniform texture. The phage can tolerate higher temperature, can be produced by adopting a conventional spray drying tower, improves the production efficiency and reduces the production cost.

It should be noted that the specific embodiments are merely representative examples of the present invention, and it is obvious that the technical solution of the present invention is not limited to the above-mentioned examples, and many variations are possible. Those skilled in the art, having the benefit of this disclosure and the benefit of this written description, will appreciate that other embodiments can be devised which do not depart from the specific details disclosed herein.

Sequence listing

<110> Ray union (Qingdao) bioengineering Co., Ltd

<120> high-temperature-resistant salmonella bacteriophage RDP-SA-18056 and preparation process of microcapsules thereof

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<170> SIPOSequenceListing 1.0

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ttccagcgca aagtcgtcct ccagttcttc cgccagtaca gagttaacgg tttttgtacg 1080

cgcggagcac tgttccgaga accgacacca ctgacaacca tcgacagacg gcttgaagtc 1140

cgacgctttc agattcttct tgccacggaa atatgcatca agagccaaca gtgcgcgttt 1200

ctgcgcgaac ttagcgaaca gttccagacc ttcaaccgag atgtcccact ccgacgcacc 1260

gccagcgtac ggctggaaga tgaccagacg aacaacggtt atgttatagc gtctcttgag 1320

tctgcgataa acaccgagag cgtaaagcat aagctgtttg ttttctttcg cttcaacacg 1380

atgccgtcct gttttcaggt cgccgataat gagcatgtgc tcgtctgtgt tcgccatctc 1440

ctgaacggca acaaggtcgg cggtcccgaa cgtctcaaca ccttcgtatc ctggatgtaa 1500

tacctcagta agattgacac gcatttccag cttggcgtaa gttgctacgt cgattatagc 1560

tttgcagtag tcggtgtact tgcgcacctg ctcaatcatg tctgccgtaa tcagtaccgc 1620

acctttcatc ggcttaatta atgctttaac agcgccttta ccttcctcca gcacatacgc 1680

acctacatcg cgttctaacg gcagctcagt tccttttatg tactggttaa gatgagtctc 1740

ggccagtgtg tgcatggctg tacccaatac cgcagcttta ccagatgtgt tgggtatatc 1800

tttttcacag gccagcgatg cactgcacgc cagccacttt ttagctcctg acggtgacag 1860

aagggcgtgc acgtcattat taccgccgcg ttcttttaga atcattattc atcctcctga 1920

aaggcgaccc gaaggccgcg ggataattat tcttcttcga aatacttgtt cttgattgct 1980

gtcagacgtt ccaggtattc agcaaggtct tcgtctttaa tcgcggcaat cttcattttc 2040

ttaccggtga actcttccag cagttcatcg gaatcatcgc acgcagcatc actagggcct 2100

tcgttaattg catcgtcgat agcctgaatc tggtcgcgaa gagattggta atcaatttct 2160

tccttctctt cttgtactgg ttcctctact tttgccttac gcggcttacg ttttggtttc 2220

tcctcttctg ccggtttagt gtcgacgatg tcctcacctt cgaccggaat ttcttttgct 2280

ttctcgactt cttccagtgc tttagccagt tcttttttaa ctgtctcgac accggcaacg 2340

gagacgtcaa cagtcatttc gcttacggat ttgttagacg tctgtttcgc gctgttcgca 2400

gcaatcagtt cgtgagcaac tacgaaacgt tcaaataatt taataaattg ttctaacatt 2460

gtgcattctc ctgtttagtt ggtgataaga actataatcg tactctatta tttatgcaag 2520

cactttttcg taaattgcac cgtgtatttt attactgtac tattactaca tatctaacta 2580

aggagtaaac atatgcagca atccgaatta ggcgcccgca tagagcgccg ccgcaaagaa 2640

atcggcatgg gtcaaactga gcttgctttc aaagctggcg tatcccaaag ccttattacc 2700

catctggcaa ccggtcgggt gtgtaaggta gactgtttta aaatcttcca cattgccgat 2760

gctctcggtg ttgaccctcg atggttaagc tttggtgata caggggcctg atggcccctt 2820

tctttttact ctaaatcccc ttctgttgcc tttatgttgt cgtttggttc atacctgttc 2880

tttggccgct tgcggtcatc tatacccgtc ggcaacctgt aagtattcgt tacaacttca 2940

ccattctcat cacaaccaag catcaattca ccctctcgta gcatcttctc gagcaccagg 3000

tttgttatgc tgtggtcccc agccctggcg actatttgcc gttgtgtgaa acctcgtccg 3060

gtatcatcgg cttgctgtaa atcctcgaga gcagacataa ccgattctcg tgcggaagca 3120

tctttagacc gtttaactgt atcctttacg taatctttac ctttaccgtc cagtccttcg 3180

ttgcgctctt tttcctcgtc cgtttcgaac ggttggaagc cccacggcat gagtacaaga 3240

gctttatgcg gttccggcag gtctaggtta acgatttccc cgtatccctc gcccccaacg 3300

aattctacag cctggaactc tttcggtggt ggagcttcgc gaaactgtac cggttccagc 3360

accataccta ttgttttctg ctgcataccg tttttgttct tggtatgtgc aacgtttatt 3420

tgtttctcgg tcgcccggac aagcgtaagt tcgacgtcga caccggcata tagcgcccca 3480

ctcccgcgcg ccttactccc acctttcggg gtgtggtgga caacccctac cgccccttta 3540

gtaccatcgc gtacttcttt gagtatcgcc acgacacgac ccataccgtc ctgcccggcg 3600

gagttttcat taaacttgtc tatccagtta ccgaaggtct ggttaagggt gtcgaatgca 3660

accatcccga cgggttcact accggctagc tggcgcattt tacgcaccag tttcttcgta 3720

tccgcgaact caccggcatc cagaacatgc atatatcgca taccttcatc gccgtattta 3780

gcggccaacg cagcgatacg ggtgtgcgtg aactccccac cctcaccatc aatatagaaa 3840

tgatgggctt tacgggtgtc cgcccccgcg aaccgatacc cggcagcact gatatacatc 3900

atgccgagtg tgtagaacga tttatacgtc ccggattcac cgacgatatc ccatatgcaa 3960

ttagacggca tgtacccttc gacgatgaag tccgcttgtg gtgctggcgg ttcttcgtcg 4020

gccaggtctt cttcgtcgca agtgaccccc tcggcccacc caagcgctac ctctacacgg 4080

tcgaacggca agccggtagc ggcgcaggca taagcccaca tatcacggcc gccgggcttc 4140

atcccgcccg tagctacaag gtcggtatcg tggtacaggg taacgttggg tcgctcgaag 4200

ccgtcgcgcg gccaacagaa caggaagtca tcctgcttag gttcaccggt agtgtattgg 4260

gcggcgtgct ccggcgtggc gggcatttgc aggccgcggt cggtcatacg gccgccgaac 4320

tcgaaggcga actcttcaaa caggtcagtt aattcggatt gctcaccctc gggcactttg 4380

taatcggaag cgcctgtaac gttaatttca ggaactccct ccataagctt actggccgta 4440

atcatgcggc tactttcgga cacgatgacc tgacttccca ctggcgggcg gtacatcggc 4500

tgagacaggg tgaagcccgc gctatctacg tcacggcctt taaggaaatg cgtcagcaga 4560

ccgtaccgga tgcggatgat gtctccacct gttacaggag tgcgtaccgg cataacgacg 4620

cgatagcgcg gcgcttcttc ggtgtgggac gccgtggtgt agagcatcat tgcgtaacgt 4680

gactctcgca ccatctcgca atcagcggcg aattcttccg gcgtagcact atccacgtcg 4740

gcgtaagcca gggacgaaga cgttacggac gcattgcagc gatagaacat accttcggcg 4800

gcttctttac ctgtagagct taccgtggcg gtacacgcgg cggtgatata tccggggtct 4860

gtcttggggt tccggcgcga acgtttaagc ggctgcataa gttcaacgaa ctcatcccag 4920

gtgccggaag tcgtggtgta aacgttaatg tcagcgcgtt cctcacggcg attgctacgc 4980

gaccatgagt atgctagatt acctgttgac atgtgctttt cctttgtagt gtttaaggcc 5040

ctgacgttcg cgcgttgggg cttttctttt attcaaggtc ttctggcgtc gcctgatagc 5100

ctttaacttc agctactgcc gaatatttac cggccttact tttaactgct ccgtctttaa 5160

ccagaacctt taacgtgttc tcgatgagag aggaactata gtactgaaaa taagtccggc 5220

gcaagtcaag gacactacaa gacccctttt taaatgtaag agcggctaca gccctgaata 5280

ctcttttctg gaaatcagtc acttcacatt ctccctaatc cacgcttcca ctttctccct 5340

gtcgaatgtt cccggcatcc ggcgacccat aatccggaca caacaatccg ggaacttgcc 5400

gtctcttaac cagttattca gcgtgcggcg ggtaacccca ataagctcag ctacttcatt 5460

ctgtgtcatt atcaaaccct ccatctcagt tagtaaagca agtataccca cggcataatg 5520

ggaaatcaac tataaacaca cctattgaca ttttcatttt tttcgtgtaa gttgtctctt 5580

gtaacttcaa gttacttaag tttctgtttc actttgtgag agcttagcgg gtccgcccta 5640

agcggacacg cgttaaggct tactgaaacc cgcaaactta aaactcagta acttccttcc 5700

ttagtatctg aactgccttg ggcgagctat attctcatcg gcagcatatc tcttaagtat 5760

ctgaattcct tgatgtgccg tagattcgca accgtaaaat gtaacgatta gtgggcaggt 5820

ctaaaacctg ccctttagtt accaggtaat aagaataaag gcacaaccct ggttgaagaa 5880

ccacaacggc aacgtaactg aaatgccgaa agattcttag tctactactc agggattaac 5940

gcacttgcct tacggcaagc gcttacctga cgagggacgg gagacaagaa tagaaaggca 6000

cttgcagaac caaatataat agagtactat tatcgcaggt taacgagaaa agaggatgag 6060

tcatgtttaa gaagggccag ttagtgcggt gtactatctc tcacgaatat tacatagttc 6120

ttgaccagat aagaaagatg ttagctgtca ggagtgtaag ttgggggcgt gccgggttta 6180

tggacgcgcg gtatgtaacg ttaatcggaa ataactacac ggcaaaagat tctaaagagt 6240

ctgcgtcggt gaatctcggg tcttaaagga ggttgccgtt tatgaagaac ttgcagttgg 6300

ccgtgaaaag tgaatatttc gatgccatga ttcgcgggga gaaaacggaa gagtatcgcc 6360

tggtaaatga ttattggtgg acgcgccttg ttggtcgtga attcgaccgt ttgattataa 6420

cgaagggtta ccctaaacgg gacgataaga gcaggagaat agagtgtaag taccgtgggt 6480

atgagattaa agaaataaca cacccgcatt tcggcagtga accagtggaa gtttttgcga 6540

taaaggtgga gttatgagca agtggattaa atgtagtgag cggatgcctc ctaaaatgac 6600

aggggtttta gtggctacgg aattcgggca taaaggcgac tggagaatga agtaggccac 6660

ttatgtaccg gagcatcctg gcgctgacga tgggtggctg attccggggg cataggggaa 6720

accgtcgcac tggatgccgt taccggaacc gccagaagtg cgatgttata ttgtaacaac 6780

tatttaggat tttactgagt gacttacgct gctgagtagc agcgtaccat ttttgttaat 6840

atttttctat ttcactagac gcaacaatca gcacaactat tgcaggagac gcaacaatga 6900

aaccgaacga cctcgtaacc tggaccgggc gtaacggaga gaccagacac ggcaaaataa 6960

cgtcgcttca cggcatctat gctcgcgtcg agtggtggcg ggcccatgct aagaagcctc 7020

gtcgcattct gacgcgtcag gatagattaa tcgcgaaata gtattgacgt aaccaaacat 7080

aatagtgtac tattacttta ttgaaacgaa aggataatca aatgacgagc atacttttca 7140

tatgggtcgt gtccgctggt cagctacaac tggccgggac agaaacgttt tacacgttag 7200

aggcgtgcca ggtagccgca cgcgctgcgg agaacgcgcc gttaactttc gtagagcgtc 7260

ctgttgacgc tcaggtacgc ggtatttgca cactgaaacg gttatctaag cagggggaga 7320

gatgagtctc gcaaccgata tactgaaaca cgccggcatc aacctggcac ctccttcatc 7380

ggcggtaaca aagaaggtta ctcgcgttaa agaaaaggtt aaacgcaaac ctaagccgcg 7440

tgtaaagccg gttaacgaga tgcctgacgt ctatccgcgc atccccggag tacaccagcc 7500

taaatattgt gtaggtaaag gtctgtggcg cgcgcactct tacgacggca agaaagtggt 7560

aaacctcggt gagttcagta gccaggcaag ggcgcatatg gcggttaaac tgtacaaact 7620

gtggcgtaaa cgtgggtatt ccgaaatccc gcacaagcca tctattcgac tttatacgtt 7680

caggtaattt atatgactac gatagctttc gacggcaaaa ccatggcctg cgatacatgt 7740

gtcacgggga attttaaata ctatacggat accaagattt acgagaacga ccactttgtt 7800

ataggggtat ccggggacgc cggggtgggt atgttgttgg ttgccgatga tgagatactg 7860

acgcctaagc actacgactt cgacttttcg gcgctggtgt ttgttaaaga ggataaccgt 7920

attttcaggg tagaattctt caagtcatgg gattcgccgt taagctcagt tataccaatt 7980

gcgggtaacg cagcggccgt tggctccggt gctccgtatg cacttacggc gatgttcatg 8040

ggcgcgacgg caacggaagc ggtagaggtt gcgaagaggt ttgaccctgg taccgatggg 8100

agagtggtaa cacatcgact aggataaatc cgaattatct gttactaaaa ttgtcgtata 8160

aatgttaatc tccgtaggta aactaacttt cggagatttt ttattgtgga cgataaatac 8220

ctttggctta gtgtggcggg cctcgccggg ggtgccgtat ctcagattaa gaagcgcgag 8280

gctatttcac cgtggttgag attgtgtcac ctggttgcct cagcatgttg tgcggtgtac 8340

gcatccccca tcattataag ttactatgaa ttatcacagt ctgagggtca gtacctggtt 8400

cctttcggcg taggtatgtt ctggcttaaa ttatttgagg ccgccgactc gtccctcagc 8460

aactttaagt taccgtgggg gaaataacat gctcaactca ccaatcggcg ccatttgcct 8520

ggtcgcaatc atcatcacct cgttaattaa tatctacgcg cactggattg aagacggtct 8580

tttcgggcgt ctgttgtata tggcgtcagt agtgaccgcc gccggaggtc tcgcccatct 8640

ctttaccggt gccattccac cgttcataat tacgactctc gtcgttatat tcgcgcttaa 8700

atcggtgcgt catatttgcg tgaaaggcgc ccgctactac aaataccgga ggatgtatgt 8760

caaaccgaaa catcagtgac aacgggttac acttcaccgc cgcgttcgag gggttccgag 8820

gaaccgcgta caaggcaacg aagaacgaga agtaccttac tattggttat ggtcactatg 8880

gtgccgatgt gaaagaaggt cagaagatta ccgaaggcca gggcctcctg cttctgcata 8940

aggatatggc taaggccgta gctgcggtag acgccgtcgc gcacccgtcg ctaaatcagt 9000

cacagttcga tgccgtgtgt gaccttgtgt ataacgctgg tgcaggtgtg attgccgctt 9060

ctaccggtac aggtcaggcg ctgcgcaaag gcgatgcatc tacactgcgt aataagttaa 9120

ctcagttcca ttatcagaac ggcaaatcac tcctcggatt gcggcgccga gctgctggtc 9180

gtgttgcgct gttcgatggc atgctgtggc aacaggccga agctatcggc cgcggcgcaa 9240

agtaggttga cacataggag gattcctaag atactaaacc tgctcctgct tattcatccc 9300

tctagctcct ttatcccggt aactgaccct accgggattt tttttatctg tatcctgaaa 9360

taatagttga ctagtagcat taaccctatt atatttagtt catcgacagc gagaacggag 9420

tagagaagat gaacttacaa agcgataaag ttttttacca ttgcaaaccg gtgcttgaca 9480

ccgaagcatt taaagacgcg caactgatgg cacgcattgc cgttaataac ctgagcacac 9540

gaattcccgc cgacgcgttc tggttcgccg cgatgcagac actgaaagca gcttatgcag 9600

gagaacaaaa atgaaagtat atatcgtatc cggatggcat tactacgagg acacttacgt 9660

tcttggtgtt tatctcagcg aagaaaaggc cgaagccgcc gccgcggtgg aacgaacctc 9720

ctatgattac gacggcgtag acgtatgcga atgggaggtg caggaatgag cgacaacgga 9780

caagtagcag taacgtttaa ggtgggcggt aaggtaagta acacgccctt cccgacacgc 9840

gaggaactga ttaaacgtaa cagtttcccg gggccggaca agaacaagta tctcaatcga 9900

atgtggggtg agaagaaatg agacgtaaac cgttgccaaa agttggcgac acccggctaa 9960

cccccgagtt gagaaggcta aaaataacag cagttgtcca tgaaagacac tcaatgccgc 10020

agttagatgt taaggctgtt tggtgtctgc attttaacga tggtacttac gagtacgttg 10080

atgaggatga gttttatgac taacaacgaa tacgaaaaga tgatggtaga agccgcagac 10140

ggcaaattca agattgaagc cgtagacgcg caaattaagg cacaccaggc tgcgcttgac 10200

taccttaacg aacatcgccg ggagcttatc aatcgttatg acctgaataa gtgtacgcac 10260

gattttgatt acaatcctaa agatatgtgc ggacagtgtg ttaagtgcgg ggcgattaaa 10320

cgatgaaacg agtatgtaaa gagacgttgc gcgagcgaat tatgtatctg gagtcggtaa 10380

gtaagttagg tctgtcaatg aacgaagaat tccatttaga agcatacaag atgctgctgg 10440

aacgacttga gaaggagagc gaacaggaat gattaccagt atcccaaaac taatagagac 10500

acacggcaca ttagcggata cgtgccggga gacgggactt aatgagatga cattgtccaa 10560

gtaccgccat gacacaaaat gcgagcaaca tgtcatctat aacaaccgac tgatgaccca 10620

tacgaaaacg tcaccggtca tctacacgaa acgtggatta tcgcgcaacg accgtatgaa 10680

gctggagggg tgatggatag ttttctcatc ctcatgggta taggattcac aacattactg 10740

cttgccggtg ctatatacct gatgtggatt ttgtttcttt ggccgtttgt tgaggcggta 10800

agtcttacaa ggatgatgag agagtattgg cgcattaatg ggtgcaagcg aggtgcctgg 10860

cgtacgatac gtttattctt tacgtggtat gccgaaacag tatttggtcg taggttcgaa 10920

gccatacata gtcgcgggtg gcgatgggaa gccgtaggta aatggtgggt ttataaagag 10980

gagagtggag aatgattgag attttgaagg ctaccgattc cactggcgac ctgatgtttt 11040

ggtttgcctt tctcagtgcg ctaataaaac aaccgactgt gtttaacagt cttccccgat 11100

atctgtggtg gcctatgttt ttaggcttgg gtgtatcagg agcggccggc atcctgctgt 11160

ggtttttcaa atgagattac taatcatccc gaacgcctgg gttatcgccg cagctaacga 11220

ccattacggc ggcgatggta gaagagcact acgacacggc ctatataact gacacaagcc 11280

cgcatatgcg ggcttttctg tacacaggcc tatctttccc tatacaatcc ttaatagacg 11340

ctcagggggt gtctgatgcg ctatgatggt catacgcatt taagagagga ttgcatgaaa 11400

ctcaaattaa aacagcctga cgccgaagta gtggctgccg cgcacgaaga agcggttaac 11460

gcaaaccgtc gccgtaaacg tccgcgtggt aaacaaagcc tgtatcagtc atccagacat 11520

tccgccgaac tatgggaccc ggactattgc gacgaattaa tccggttctt cgaccgcacg 11580

tcatgggaac tcgtacccac gtctaaaggt gacgaacgcc cgcttatcca ggataaaccg 11640

ccatcactgg cccgtttcgc actacacata ggcgtaaccg ttccgattat caagctgtgg 11700

ctgcgagagg ttcccgcatt cgccgaagca tacgagacgg cacaggccct ggaagaggcg 11760

tatttcactg agaccggtgc agccgggata tctgctacgt tcgccgcagc aaaacttggg 11820

cttaataaaa ctgttgtgga agaagcgcgc gacgaaccaa ttagcgaagt aactattaag 11880

gtggtgtccg gtgaacgttg atatcacagc tacgggaccg cagggcgcgt tccttaatct 11940

gcattgtaag ttcccggcct tcgtcgctgg cttcggcaca ggtaaatcgg aagtcatgtg 12000

caactccgcc ctgttagaca gcatggaagg tggtagtgat tcacttatcg ccatgtatga 12060

accgacatac gacctggtgc gccttatcct cgccccgcgt atggaagaga agttgtctga 12120

ttggggtatt cgttacaagt acaataaatc agacaacata atctatacat catccgggca 12180

attcggggat tttgtcctgc gtacattgga taacccggcg cgaatcgtcg gttacgaatc 12240

ttttcgcgca aaaatagacg aattggacac gttaaataaa gaccacgccg agcacgcctg 12300

gaacaaagtt atcgcccgta accgtcagtt gccgcgtaca tatcgtccga ttaccccgaa 12360

gcccgctaat acagtttcgg tatttacgac gccagaaggc ttccgttttg tgcacgacag 12420

atgggctgta aaaaagaaac caggctatga aatgattcag gcctcgacaa catccaatcc 12480

ctttctaccg gaagattatg tgcagtcgtt gcgggatacg tatccaggcc agttaattga 12540

tgcctacatc gatggtgaat ttgtcaacct gacatccggc agcgtgtatt atgcttacga 12600

ccgacgcaag aacagcagtc gagagactat acaacctggg gaaacgctgt atatcggtca 12660

ggacttcaac gtcgggcata tggctagcac cgtatacgtt cagcgtgagt atgtctggca 12720

cgcggtagcc gagctggtgg atatgttcga caccccggat gtggtcaggg agattaccga 12780

acgatggaag cgtaacgggc atcacattgt tatgtatccg gacgccagtg gtaagaaccg 12840

taaatcgact gatgcaagta cctcggatat tgcccaatta cagaacgcgg gtttcgagat 12900

acgtgcaaaa tcggttaacc ctgccgttaa agaccgtgta gcatcggtga ataaagcgct 12960

ggagtctggt agattaatgg tcaacgagca aacctgtccg gttactgctc gctgtcttga 13020

gcaacaggca tacgataaaa acggcatacc ggataagaca agcggcaacg accaccagaa 13080

cgacgcgaca ggatacccta tcgcctacga aatgccgttg gttaaacctg tttcccatat 13140

cccggttact tttgcacttt aagaggatta ttaaatgtta acagcaaacg gccagggttc 13200

tggcgtaaaa accaagcacc gcgaatggct gcactacgcg ccgaaatggc agaaggtgcg 13260

ccatgcgctt gccggggacc tggttggcta cttacgtaac gtcggcctta atgaaccaga 13320

taaagcatac ggcgaagcgc gtcaggcaga atacgaggcg ggcggcatcg tatacaactt 13380

cacccggcgc acgctgtcag ggatggttgg tagcgtcatg cgcaaagaac cggaaatcaa 13440

tatcccgaag gagttggaat atctgcttaa aaatgcagat gggtccggtg taggcctaat 13500

acagcatgcg caggacacgc tcatggagat tgactcagta ggtcgtggtg gtcttcttgt 13560

tgacgccccg gaaacaggcg cagctacggc tgccgaacag aacgcgggct tgcttaaccc 13620

tacaatcgct ttttacacga ctgagaacat cgttaactgg cgactcacgc gagtaggttc 13680

tgtaaaccgg gtcactatgg ttgtactgcg cgaaatatgg gagtaccacg aaccgggaaa 13740

cgagttcgaa actaaatacg gcgaacagta tcgcgttctg gacatcgata gcgacggcaa 13800

ctaccgtcaa cgtctttttc gtttcgatgc cgaaggtggt gcgcaggaag acgtcgtgga 13860

gatttacccg gacttagggg aatcattacg aggggtaatt ccgtttacct ttattggggc 13920

gaccaataac gacgccacta tcgacgacgc tccgctacta ccactcgcgg aacttaacat 13980

cgggcactac aggaacagcg cggacaacga agaatccagc ttcgttgttg ggcagccgac 14040

cctgtttatc tacccaggcg agaacttaac acctcaagca ttcaaggaag cgaaccctaa 14100

cggtatcaaa tttggtagtc ggcgtgggca taacctgggg tacggaggta gcgcacagct 14160

tatccaggca ggcgagaata acctggcccg ccagaatatg ctggacaaag agcagcaggc 14220

tatccagatt ggtgcgcagc ttatcacccc gactcagcaa atcaccgcac aatctgcccg 14280

aatccagcgc ggtgccgata catccgtcat ggcgacaatt gcgcgtaacg taagccaggc 14340

atatacagat gctttacgtt gggtggcggt aatgttgggt aaacccgaag ataccgaggt 14400

tgaattccgc cttaatatgg acttcttcct ggaacctatg acagctcaag acagggctgc 14460

gtggatggca gacattaatg ccggattact gcccgccact gcttattacg ctgcgttgcg 14520

taaggcgggg gtgactgact ggaccgacga ggatattctg aacgctattg aagatgcacc 14580

tttgccgtcg ggtgctgtta ctcaggtagc gggggagatt ccgcaagcgg cacaacaaca 14640

acaacaggag taaagaaaaa ggccccatta cggggcctta gttttagcgg atattagccg 14700

cgcatttacg cacaacccgc ggtatcgtct ctttcagctc atcccatgag ttgactgctt 14760

ccatatccaa caacgtcacc atagcctttt gaatatcact ccgtaattga ttagttttgg 14820

cgtcctgaga gtcccaagta tcaaaaccgg ttacttcacg gatatctata gactctttta 14880

tcgcattcgc cacatgtatg aagtgaccga tacgcccaat cctaccctcc gtacgctcaa 14940

tcatccggct aacggcggca ttaagttcta caaaactaac agcaccaatg ttacggagtt 15000

ctacctgccg ttgagttaag aaacggtcta taacgaggaa atggaaataa gtgctgtatt 15060

gctcagcaat atataccgcc aaatgaagac ttaaccacgt tttcccgttc ctccctctcg 15120

ttgttttcaa cggaacaacg ccagtctcct cttcacaaac acgcatgaag tctctgcagt 15180

cgtccgtttt cagtatctgc gcaagatttc ttggttgaag accattatca aatctccatt 15240

ggttcatttg aatggttagt gttgacgcat caatatactc atcgctggtg aactcaattt 15300

caccgtaggg gaactctaaa gtctgcgctt tcatatcttt ttcctcagtt aattgcctca 15360

cttaaattat attcctaatt tctttgttta tgcaaacatt aatacgcaaa attatagcaa 15420

attagacttt tttaggtgtt tgtgtaggaa taacctatgc catgctaaga tgcatcaaca 15480

acgcaaacag gagggatata tgccactact tacatcatta atcagccatc aaatatggct 15540

gcaacgcaac gccagtagcg aagtaaaaga cctggcgcca ttcatccagc agatgcgcga 15600

tgaggttaag cggcaggtgt tgttgtttgg tgatgacagc agaacggcgg cgagacttac 15660

caccatgctg cgggaacttg agcaagcact gaacgggatt acgtctgggt ggtatgagaa 15720

gctactggct gatgcccgtg agctttcaga ctatgaagtt aactggaacg taaaaaccct 15780

gtcgaccaac gttaacgcta atttcgtaac accggccgcc gaacaagtat gggccgccac 15840

tactttcgca ccacttgaac taagcgaaaa gccggttgat tttgtttcgt taatgggcgg 15900

atggcggcaa gccgaggtga atcgcttggt tatgggcgtt aagtctggat tcgtacaggg 15960

tatgaccaca cggcggatag ttaagaacgt tgtcgggccc ggcggactgg ctgatatctc 16020

cgagcgtaac gccgctaccg ttatccgtac cgccctggcg cacgtatcca acgaagcgcg 16080

gcaacaggtt tacgctcaga acgacgacat cattacaaaa tacgagtggg tatcaaccct 16140

tgattcccgt acttcggctg tttgtcgttc gcgagattct atgcagtacg aaataggcaa 16200

aggtccgcta ccaccagcgc atccgaactg tcggtcgagt acagcaccgg taataagttc 16260

cgaattcgac ttcctggata agggtgcaaa acgagcggcg aggggtgcag aaggaggcca 16320

gcaggtaagc gcagacacca cctattacga gttccttaaa caacaaccgg cgtggttcca 16380

ggacgaagcg ctcggtcctg tcaggggtaa gatttttcgt aatagtggga taaccccgga 16440

agaatttcgt gtaatatcgg tagacggatt cgggcgtccg ttaactctta aagagatggc 16500

ggaactcgat aggcgtgttg ccgattatct gaaagaggaa tagagatggg ctttttcaaa 16560

gttaaagatg tgccttcacg acgtgtagtt cagtactccc gtgtgtctgg tgcgggtgaa 16620

ggtgtcgtgt atatcaaaga tgaatcggtt ctcggtgaat cggtggccga aatgccgttt 16680

gcagataaga caggactgac cacaatctcg ggcggcgtcc tgtacgaagt gccgtatctg 16740

gacggagcag gtgatgtgta cttcgataca cagccagcag atgtagaatt gaaagacggt 16800

tccgcaaaat taaccgttgt cgtgaaaggt ggcaaagcgc cctacgattt gcaatggttt 16860

aaaaatggta aagaggtgat caacgtcccg tacgttgagg gtgaactaac tgttaaagac 16920

ccgggggagt atttcgtcag ggcggtagat gctgacgggg tatcggtggt aagtaaagcg 16980

gctaaggtct cagaacctga gtaacgaaag gccccgtcaa ggggccttaa ttttacacac 17040

cattattcca gttattcctt accaatgtaa attcaggaat actttattca aaaagtagac 17100

ttctattatt ttttatgtta cgctacctac gctttcagcg taccgcggtg cggacgcgtg 17160

aagcgggcgc cgcagcgccc tagcgtaacc gcgaacgcat tcggaagggc cgagctatat 17220

tgctttcagt atttaactaa gattcgccgt atgatgttta cagatatacg agtttaacgc 17280

ccggcttacg ccgggcttag cgtataaaga aaggggatac catgaattgg aaagtaaccg 17340

cagtaatcac ggcagccggc atcctttcct tgtggttata cggccagtac aactacagaa 17400

gcgggtgggt agaaggccga gagaatcttg tttcacagca acaaaagaag gcacaagctg 17460

ggctgacgaa gaaaacacaa cggcagcagc agaatgatac taaggccgcc tccgccgaat 17520

cggaaggcaa agaaaattcg gaggctatca cccatgaagt catcaaatat gttacccgtc 17580

ctggccgtac tgtctgtgag tttcctcctg aacgggtgtc aatcaaacga cgcgccgccg 17640

agaatgctaa ttccatcccc ggatatgaca ctaatgcaac cgccgtgcaa gcttattcca 17700

ccgagcagtg acgctgatga ggacctggcc gtcgatgtgc agaacgccga gtgcacacgg 17760

cagttacgtc tgaaagtgtt ccggttgcag gagtacataa ggaatattct ggaatagttg 17820

ccttagtaag tggaataatt tattcttgtt acagaaacac cgggtggccc ggtgtcctaa 17880

agtccagggg acatattgac tatgaatcgt tttttacgtt acccgcttca tgaagaagcc 17940

ggggtagaag ataaaccggg tgcaggtgac gcaccaaaaa tgtacaccgc cgaagaagtg 18000

caggccttga ttgagaaaga agtggccggg ctcaaggcaa atcaggaagc gctccttagc 18060

gagaaaaagg aagccgcgcg ccgggctaaa gaagccgaag aagaacggca acgcgcccac 18120

caggaagcac tgaaggctgc cggtaaaatg gacgaatttg aaaagacgat tcgtagtcag 18180

tatgaacccg tactgaaaga gaaagaagaa cgctatgctt ctttagccgc gcgaattctt 18240

ggtagtgaac gtaaggctgt tttaggttct ttcgctggtg attttatcac cccagaagcg 18300

gtagaaatcc tggcgccgtt cgttaagacc gagtttgaag gcgaagacgt agtgactaaa 18360

ttcatgggtg cggacggcaa tgttgttact accgaccctg agcagttccg caaatacctg 18420

cgcgaacata aagctttttc gcatttgatt aaagcaaatg cagcttccgg tggcggggct 18480

tccgggaata aaggcggcgg ggccgcacca gcgtttaaag atatgagtga aagtgagcgg 18540

ttggctctct ataaatctaa ccctgccgaa tttgaacggc aacttaaagc cctgaggaaa 18600

taaataatgg caatcaccac tattggtaac atcgtaactg gcaatatccc tgtactggcg 18660

tcctatatga cggaagaccc ggtagagaaa accgcgtttt tcaactccgg aattttgact 18720

ccaactccgt atgctgccga gattgcccga ggcccgtcta acatcgctaa cctgccgttc 18780

tggaaagcta tcgatacttc tatcgagcct aactactcga atgacgtata ccaggacatc 18840

gcgaccccgc gcgctattca gaccggtgaa atgatggccc gcgttgcata tctgaacgaa 18900

ggtttcggcc aggcagacct gactgtagaa cttaccagcc agaatccatt gcagtctgta 18960

gcctctcgcc tggataactt ctggcagcgc caggcacagc gtcgtctgat tgctaccgcc 19020

ctcggtctgt acaacgacaa cgtatccgat actgatgcat atcacaagca gaacgacatg 19080

gtggtcgacg tttctgcaac ttcaggcttc gacgcaggtg cattcatcga tgctacccag 19140

actatgggtg atgcattgat gggtaatggt ggcgaggttc tcggtgctat cgcgatgcac 19200

agcttcgtat atgcgcaggc gcgtaaagct cagcttatcg acttcatccg tgacgctgag 19260

aacaacacca tgttcgccac ctaccagggt taccgcgtca tcgtcgatga cagcatgacc 19320

gtagttggtc agggccctag ccgtaaattc atctccatca tcttcggcca gggtgctatc 19380

ggttacggcg aaggcaatcc tgagatgccg ctggaatacg agcgcgaagc gtcccgcggc 19440

aacggcggcg gtgttgaaac cctgtggacg cgtaaaacgt ggttgctgca tccgttcggc 19500

tacagcttca ctagcgctgt aatcaccggc aacggcaccg agactatcgc ccgttccgct 19560

agctggcagg acctggctaa cgccgccaac tggaaccgtg tagtcgaccg taagcatgtg 19620

ccaattgcgt tcctggtaac tggtgtaggt gcttaatcgt agggtatact cttgagggac 19680

ttcggtccct cttttcattt atacggagac tacataatgt caaagacagg taaagggctg 19740

ccccgcagtc tggttaacgc ggaattagat attcccgccg cgaccacaac ggcaatcgga 19800

ggggttaaaa agtccgctac ggtagcggca ccaccggtga ttagcgcggg tagcggtgca 19860

gccgccgccg ctgcacctac gaaagaagag ttcgacgcac tggtcaccaa ctacaataag 19920

ttgcgtacgg atgtgacttc attgcacacc gccgttacta atctgttaac cgcccttaaa 19980

aacgctggta ctgtatcctg aggaggataa aaatggttga tgtaattaaa cgtcgtattg 20040

ttggtgtatc tgatgatagt ccgcaggacg ggcaggtaga gattgatatg gaaaacgtga 20100

cgccgttgcg tttctctacc ggtctcaatg atactacctc ggtaacatct gggcaggcta 20160

tcaccctgac ggtggtactt gccgacggaa tggaccctaa aaccgttcaa tggtataagg 20220

ataataacgc tatcgctggt gcaaccgctt tgacttacac taaggccaac tccgcagcgg 20280

cggattccgg tacttacaaa gtcgtagcgc acgacggtta cggtaatatc atttcagata 20340

gtacggtagt taccgtaagt taaatacacg cggcctccgg gccgctttaa ggattagaca 20400

tgacagataa ttacgtagta cgggaacaat acaagggtgt ggtcgaggtt gacgggcggt 20460

tagtcccgat gcgcgaagaa gcgaacccgg aagcattaat cgaaactcag ccagtggtcg 20520

aagaaccgca ttacaacggt ggcggagagc ctaagcagcg tcgtcgccgc aaaagcgtag 20580

aggaataatt tatgccgctt atcgtggaaa ccggtcaagg catcccgaat gctgactctt 20640

acgtcagcct ggaagacggg cgcgcactgg ccgctaagta tggtctcgaa ctaccggaag 20700

atgacaccgc ggcggaggcc gctcttcgta atggcgcggt gtacgtcggc ctttttgagt 20760

ctcagatgtg cggacgtcga gtatccgcta gccaggcgct ggcgttcccg agaaccggta 20820

ttactctaca cgggttccct caaccgtcca acgttatccc tccgttaatt gttcaggctc 20880

aggtaatggc tgccgtagag tatggcgcgg gtaccgacgt tcgagggtct acagacgggc 20940

gagaggttca gaccgaacga gttgagggtg cggtaaccgt gtcctacttt aaaaatggct 21000

actcaggcag cacagtaagc atcacggcag ccgatgatgc gctacgtcct cttttatgcg 21060

gtagcaataa cgcctactcc tttaatgttt tcaggggtta atcatggcta agactaaatc 21120

agaaatattt tcccttatcg gtgccaactt ccctgataac cagtctggac taattacacc 21180

tgaaaagttg cgcgaagtta ccactcagat ggcggattct acgctgtacg gcgttaagga 21240

agtagaggta cttcgcgcgt cgtctacgga tattcaggcg ccaactacca caggtacagc 21300

gttaaccgta tcctttggtg gggcgcagaa aacaagcgct gaccctgtga tgattaatgc 21360

atccggggta gttacgttca acgccgctgg taactacgct atccgtgtta aattgcaggc 21420

tgggcgcacc ggggcgagcg gaacatccat ccttctgtcg cgtgttctgc tcggcggcgc 21480

ccaattcggt tcaccagccg ctactaaact ggcaagcgca gacgccacga ttcctattga 21540

atcgcgtgtg gtagttaacg ccgtcgctgg gcaaaacttc gcggtagaga ttatgcgaga 21600

cgcggccggg tctaactttg gtggattgta ccctcaaaca gcaacggtta cttcctgggg 21660

cgtagcccca tccgcattgc tggtcatctc aagactggag gccgcataat gagcaccgct 21720

ttcagtaaaa ggatgcaagg tgtaggaact cgcctgttaa caaaatacgg cagtaccgta 21780

atcttgattc agaaaggtca aaaaacatgg gatcctgttt taggagagta cgtttggggg 21840

gcggacacga cggtacctct taaatcagtt ccggtacctg ttaacgcggg gctggtaaac 21900

ggaactacca ttcaggcagg agatatggtg gttaaggccg attacagcgt aattcctaag 21960

atggatgata aagtacaatt caatggtgaa cagtggtctg tggtcaccat cgagaagaag 22020

atggttaacg atgacgttgt agcatacttt atccaggtga gaaaatgagt tttgcgcttg 22080

atgtgtccaa gttcgtggaa aaggctaaga agaatcctga aaaggtaatt cgtcaggtat 22140

ctatcaagtt gttttctgcg attatcaaag cgagtcctgt agatacaggt cgctttcgta 22200

tgaactggat ggcgtccggc ggtactcccg ccgacggaac tacggacgct accgataaag 22260

ctggcaccac ggcgaccagt aatgccgcta atttcgtgct aaatgccgct gactggcaca 22320

cgtttactct aactaacaat ttgccgtatg cgcagcgcct tgagtatggt tggtcgcaac 22380

aggcgcctca aggcttcgtt agggttaatg tcagcaggtt ccagcaatta ttaaatgaag 22440

aagcctctaa ggtgaaataa tggcaacata tttcgaggat ttaacaaaag catttgacac 22500

ggcactggta gcattcggcg ttaacaacgg tataaaggtt gcgttggaga acatcgacgc 22560

accgacgtct acggatgtac catatttggc gagttatatg ttgttggccg atacggaaca 22620

agctgacctg ttctggaccg agcaacgggc aggtatttat caggtggaca ttaacgttgg 22680

gtctaccttg ggtagcgccc ctctcaaccg gttagctgat aagctaaacg ccgccttcgc 22740

cgccggtaac tgttttagtc gtaacgaaat ctgtgctgag gtacaatcag taagcctcgg 22800

tcctcttatt gttgagaatg gatgggcgaa gaggcctctc tcaattaatt tcatagcatt 22860

tacagcgagg attagataat ggcgttacaa ccatataaag gcgcgatgac cgcgcagttt 22920

tacgttcttg agacgacgcc gggggtaaca cccactaacc cggtatggca accactgcgc 22980

aacactgggg gtattcccgc cgtaacccgc gacgccctca tctctaatga actggacggc 23040

agccgtgaaa catcatctat ccgcaccggt aaccgtcagg taactggtga atatgctatt 23100

gaacttagcg cggaaagcca ggatgagttg ctggccggcg caatgaccag ttcctgggta 23160

gcaggttcca ctaaatcggg aatcagcgtt accgtagacc cggtggcgaa aactttcaca 23220

cgcgcagacg gtagctttgt gacagacggc gttgaagtag gcgacctggt gcaattcgat 23280

ggtttatccg gaaataacga caaagcgttc atggtcacgg ctgtcacagc tacagttata 23340

accggtgcgg gtatccagca tacccttacc gccgaatccg acgtccaggc caatttgcgt 23400

atcgcggata aactggaaac tggtaacttg tgtaaaacct attcaatcct gacatggttg 23460

aaaggtaaat gcggaaatcc tgattcgtac atcataactc gtggtgttga atttaccggg 23520

ttcaccatcg aacaagccgt taacgcgatg gttacaggtt cattcccgtt cattggcttg 23580

aatcaggaaa tcctacaagc accgccgagt ggttcagatt tcacgaccaa ttttagcgcc 23640

cgtccgtttg catcggttga cgtatccgcc tatgacggcg ccgctccgct taaacttatc 23700

gacacgttca ccattactaa cgacaacagc gcgtccgcac agttcgagtt aggaaataac 23760

agcgtggcat ttgtcgaacg tggccgcgcg gctaacacct tctcgttggc gggtaagctg 23820

tacgacatga cgttattgaa taaattcctg aacgaaacgc aaatggaggt atcttctgtt 23880

ctgaacggcc cggacggtgc catgagtttc accttaaaac gcgcttcgtt gacatcagca 23940

actccggaaa tcggtggtcc cgaatctgtc accctgtctc ttgagggcca ggcaaccggc 24000

aaccagttcc agtcttcaat tgttatccag cgcattaagt acgcctaaga aaaaaaaaag 24060

gcccctaacg gggccttagt tttaaatcaa gccttctgct gttaacttac agataatcca 24120

catttcacct tctgcgtaga acatagcctg cgagtgccca gtttctgttt cacacataat 24180

gccgtaacct ttatccacaa accattgctg aaagacacgt cctcgcttaa cggaacgatt 24240

gtatacgcca aattgttcaa gttgtttatt catccacacg gcagattgac caaacttctg 24300

ggcaacctgc gtcgcgttat atagattatt gcggtcaact atacggtcgt agacatcggc 24360

ctttggtgcc atttccttgt tctcaagcgc aaggcgttct ttctcctccc acaagtccgc 24420

agccaacctt aaagcctccc ccattgtctt gggcaccggt aaggatggtt tgctcgcttg 24480

tgactccagt tcctgccaac ggcggatgat ggcagcgcgc attttgatgc tgtaccctga 24540

aatcagaatt aacgtttcct ctttatccaa aagaagcatt ttatacgtgc gccccttgct 24600

gtctttgtaa tcggctgact tttcagccga ttgaatctca gacaacattt tgcggatatc 24660

agccagaaca tttttatgct ccttacctgt cagttccgca atctcacggc tggacatggt 24720

ttgtgcttca ctaacattca ttaattcgtt catggtttca tctcctcttg ttggtatggt 24780

tatatttaac ttcgaacatg cttatcgttc aaatcagatt ctttatcgtg acgataaaaa 24840

aaaggcccct ttcggggcct ttttctcact catcaatatt actacggtat ttccgtaact 24900

tctccagact ttcaattgct tccgctaagt cggttcctgt gtccttgtgc ccccgcaagc 24960

ccatacacaa cagtttcttc aacgcatgct gtagtgccgg gtcgcggata tcaaatgccc 25020

tcagaacgtc gtacacatca caggtaatgc tatctccgtc agtatttgtc attgtgcgat 25080

tgtatttgtt agtcatcttt atgccctatt tatagccgtt tttgtagcta aaaacctcac 25140

tccaatccat cctttcacct acattttagt atgttgattt ggggtttatg tctcgcagca 25200

tttcaacagg tatacttatg cgggacacct ctccataacg tttatggtat gtaatcacat 25260

ttgcgcttct acctgaataa taaccgcctt tactagaata ctcatcttta gccgctaatg 25320

tctggtgttg ctcaacaatt agaagattag tctctttaac cgcccggtgg tggaggtgac 25380

ccgtatggac atatgtgaac ttacactgcc cgtacatctc ccgatactta cctattatag 25440

actgctctac tttagtaaag ttagaacaat gcccgtgatg tgctgaaatc atacaatccc 25500

cgaacttata cgcataatac ggcgacacct cccggttaac ctcgacgcga ggctcatttg 25560

tatacacctc gtagaacatc tctcggagcc acagggaaga actaaggtcg tggttaccct 25620

cacagataag aagtttaacc ttcttgtgtt ttcgcaagca cagcgagaca gccagcttga 25680

taaccttaat ggcggtacgt atcattttaa acaggcgact atccgcagaa agaatatgac 25740

cgctcatggg ggtcattgcc ttcatgccat cgtagtgcat ttggtcccct aagaagttaa 25800

tgatacattc gccgctctgc gggctcatac aaaccgcact gttgaaccag tcaactgcga 25860

gagattccgc gattgtggta tcgtaatcat ccccgccttc ttctcgacac gccagcattc 25920

caatgtgatg gtccgttatt gtgtagagat ttaataagtc gtcatctgtg ccatttttgg 25980

gcaaagtgac aggtttatat ttaggtaaat cttttttcat ctcatccaca acggctcgca 26040

ttaattctac ctgacgttca gcatcagtat ctgttttcac ccattgaagt ttcgtgttac 26100

cgaattcatc taccagtgac gacgtaccct ttattttata cccatccggc acaaggtgac 26160

ttacgtcacg cccgtgacca actcctttct ttgccagacg agctgacctt agctgaacat 26220

tgcggcgtga catgttgtac ttctttgcga tttcggtcgg cccgaggccc gcatttagct 26280

cttgttgcaa ctgttcatct gttattttgc gagtgaccat gcttatttcc tgattgttta 26340

agacaaatct gattctaata atgtaagttt aatttagcca gatcatttac cattaacatt 26400

agagttacag gcccagatga gagccccgac ccacggcaga agaatccacc ccagtaaaag 26460

attgcatacg aatattccta atttcgcttt atgattacgt agcaaggcta ccaggaacgg 26520

aatgaaatag ataaacgcga taataccgat aaaaacgctc atgttcttaa cctcatattg 26580

tttagttgac gtagagataa taggacacta ttaacggtta tgcaagctat tttgttattc 26640

ttatttggcg tctacggtcg caccggaaaa gcgggtggtt cccgcctggc gcaactacca 26700

accagtaacc gactaaccaa agggcattac tatgaaactt agcgattttt actacgaagc 26760

agaagccgag aaaggcgcgc gtatgccgat tcctttaaaa gatggtacag attcaggaga 26820

atggttgaac gttgtctccc cggaggccga tgtcgcggtt aaagctatgc gtgccttcac 26880

cctggcgtac cgagcggcgg taggtaaatt aaaaccgctt cgggataaat gcgaagagca 26940

aaaagacttc tcagaataca atttaaaaat ggaagacgcg gcgggagacc taaaccggca 27000

attggccctc gaattggtga atggctggag tcttgatgac gagttcacta aggaaaatct 27060

taagactctt ctcacccaat ataagcgcct ggcggaacat gtagtcgtat tccaccacga 27120

acagttgcgt caattgcagg aaaagtagac gcgttgtttc agtttgcccg ttggaacttc 27180

ataacccgcc acgaaaggcg caagtttgac agtatagccg acgggcacaa agccgcgctt 27240

atcgctatgg gggtaataga agacgcggaa gaaacaacgc aggacgccgg gcctgaatgc 27300

ccccctgaac tactaaccac ttttgagaag tatcgtgatg ttaaatttac ccgccgcgtc 27360

gatgacgacg gcgtaaagct atatccgaga gagcaactta gctggtcgga tttagtggca 27420

tatagcacta tttcaggtca gaatataggg atgtttgaat ctgaaattat catgggctta 27480

gacgccattt ttgagggcag aaacgatggc tgatgtagct agcttagtag taaaggtaac 27540

cgaacaaggc gcgaaggcca catcagaccg tcttgataac ctctctaaat ccgcaaaagt 27600

tgcgggcgcc gctgttaccg gcctggccgc cgttgtagcc gctacagcct ataaggcggc 27660

tcaggaacta gtcgagtcac aacggcaact ggataagatg tcggccagcc tgaaaacact 27720

aaccggaagc acacaaggtg caaaacaggc cctgagtata ttacaggact tcgctcgtga 27780

cacaccttac ggccttgaac aggcggtaga agggttccgt aaactggtag ctctaggcct 27840

caccccatca gaagaagcgc tacgctctta cggcaatact gcatcagcga tgggtaaaga 27900

ccttaaccag atgattgagg ccgtcgcaga tgcgagcacc tttgaattcg aacgtctgaa 27960

agaattcggc attaaagcca agcagaacca aagtgatatc gaatttacct tccaggggac 28020

gactactgta gttaagaaaa acgctgccga tattgagcaa tatctcctca acataggcaa 28080

cgttaacttc gccggggcga tggctgacca ggctaatacc ctgaacggag ctatcgctag 28140

tgcggaggac tcatggtccc agttgaagat gactctcgct actagtctgg atgtagggtc 28200

gctggcggaa cctttacgtt atattgacga cctgatacag gagataaacg ctcaggtcgc 28260

atccggtgag ttcgtagccg agatgcggat gtggggtgac atggcatccg atgttggggg 28320

tgcgatagag gcttcattcg acgcggcgtt tgggatggtt gcagacgcgt taaacgcttt 28380

gaactccgcc tggacttaca ccagtgaaag cattaccgga agcggagagg agaccgcatc 28440

tacgatagct gagtcagcgg cggatgcgct ggacttcatt gcccaggaat ttacggcaat 28500

ggagcggttt tttgaagata tggttaaagg tgctcaggat gcgggacgtc ttgtaaaggc 28560

cgcattgacg ccgggagagt cggtagctga ggccaagaac cttaacttcc agttagcgct 28620

tgctatggat acccaaaggg gtgttaccga cctgacgagg aaaagtttcc gggaacaggt 28680

agaagcccag gaagacctca tcgcgttaaa gcgcgctgct tacgacattg ataaagaagc 28740

ggctaaggcc gaaggtctgg gtaagtttaa ggtgtccggt acgggaggta gttcggttga 28800

tgattccgcg gccaaagctg ctaaaaaagc cgccgacgca ttcgaacgcc agaagaaagc 28860

tgcggaagat ttttattacc agtcggttca ccttaacgat gacgtattcc agaagataca 28920

agctaaccag gaagagcaac ttactaagtt acaagagttt tacggcaacc gtctccttag 28980

cgaccaacag tacgaaactg ctaaaacgca gattatgctt gaggcggata cggcccgcca 29040

gactgaatta gacaaacgcg agaaagagcg cctggaaaaa cagttctccg cggatgcgta 29100

tgtcgctcag atgcaggccc ttgctgaagg agaatttgcg gaattagacc ggcagtacga 29160

ggtcaagcta caaaaactta acgactttca tgcgcagggt ttgattgccg aagaaactta 29220

ccaacaaacg ttaagcgcca tggatgaatc ttactccctt gaccgagcga aggcgaccgg 29280

cgcggctttc ggcaatatgg ctagtaacat cggggccgcg ctaggagagg cttccacggc 29340

atacaaagca tttgccatcg cacaggcgac aatcgctacg tacacatcag cggtcgaagc 29400

gtataaatca acagcagcca taccggtagt cggcccgtat ctggcgcctg ttgctgcggc 29460

tgctgcggtg gcggctggtt tagcaaacgt aggtaagatt cgctccgccc gagaacaggg 29520

tggtaacctg gccgcagggc agatatccac tattgcagaa cgtggtaaac cagaagtaat 29580

catgcccgct agcgcctccc gtgtccggac ggcggagcag atgcgacaga ttatgggtga 29640

gagcgacgct aaatccggcg gggataatgt tactattgtg aacaatacca ccggaagaat 29700

tgattctgct gcaacagaac gcgacgatga aggccggttg cgtattataa tcagtgaaac 29760

cgtaagttca gcgttgctgg atagtaacag cgccatttct aaatcacgtc gcgctacacg 29820

cggccaacca ggatattgat atgagcgatt accatttccc ggcctctttg aggccgatag 29880

tatcgaaagg ctactcgatg acacgcggta acaacgtgtg gcgggtagac ctggccggag 29940

gcggagttcg ccaggggcga gacacctatt ttgacgtgtt tccgattaac gttaccctgg 30000

tcgtatcgcc gttgggacgg caggcattcc tcagcttcat ggagaaagta gacggtggtg 30060

cgtccagttt ctggatgaag cacgacctgg gccagggtat cgatgactac caggtaacgc 30120

taacatcaac gtggaatgag tccaccgacg acggtaaaaa ctgggtaatc actttcacgg 30180

ccaccgccga gaagtcacca ttccaggaag ccagcaacgc ttgtctgaac cagaacctac 30240

cagatttgta cgggtgctat ggtgattgcc ttggcgaatt ccttaaaacc tacggagtgt 30300

atcagactac attccctcga atctgggacc ctatgcaatg agtcaggaat cagtagaagc 30360

ggcataccgg cgtaaactgg catccaatcc agacggtgaa atggatttta ttactcttga 30420

gatataccac ccacttcttt cgaaacggtg gttactcgtg cgcggggctg acgatttaac 30480

cgcaactctt gagacagggg aggtcgttac gttcgaaggt acgccgatgg aggccaagaa 30540

cgccgctaac aataacgata tggaccagac cgcctctttc tctttgccgg atgtgcttaa 30600

tatactggac gaggaaatgg accgcatccc ttatgataat aaggaattgc ccaaattcat 30660

cttccggcgt tacgtgagca cggacctgtc atacccatgc gatgggccgg tggtatatga 30720

attgcaaaca ctcacgcaag agaaaggagt gttcacagcg gaaacaggta cacccatgct 30780

taaccaacga gctaccggaa tcctgatgac gccggaagaa atacccttac tccgcgggat 30840

actgacgtca tgaatattaa cgattacact ggcctgccgt atgacttccg ccgccgtaat 30900

tgctggcatc atgtacgcaa cgtccgggct gacgcggggt tatcaactcc aatgttcgat 30960

gttacaagcc caacggcgat agatgcagct ttcgatgacg gccacgctaa cccgaaagga 31020

ctaacccgcg cgtttatccc gcagaatttt gacgcggttc tactaggagt gaaacatagg 31080

gggcgaatag tgtggcatgc tggggtatat tacgaaggaa tggtaagcca ctgtgagctg 31140

gcgtccagac aagttagact ggatagtctg gaagacctta aagatactta ttcggagatt 31200

gaattttggc gctagtaatc cactacacac gaaacgaaga cggcacgttt gatgtcaagc 31260

gctaccgcga taatccgatg aacttcgtcg tgaaccatgt tccggacggg gttccggtac 31320

gtgttttcat tgacgaaatc ggagaagata acgacgtaac agaagacttc gaagcactga 31380

aagaaaacgc gactttccac attgtggaat cagctggtgg tggcgccatt aaaggcgtca 31440

tgaagatttt tagcgttatc cttaaaccgc tggcgaaact attatcgccg tccgtgaaag 31500

gtgcgtcctc gaaccttgcg aactcgcagg cggattcccc aaacaacagt ctcactgacc 31560

gtaacaacaa ggcgcgcccg tacgagcgca gctacgacat ctgcggaacg gtgcaaacta 31620

tcccaaataa ccttatgtct acttataagg tgtttaacgc cgctggtaaa attgtagagt 31680

acggctatta cgacgccggg cgtggttacc tagacataca ccctgaaggt ataactgacg 31740

gggacacccg cgtatcagac ataacaggca cgtcggttgc cgtgtacgca ccatatacat 31800

cgcccaataa cacatctaca ccgcaggtca tggttgggga ccccatagag caaggcctat 31860

acattaccgt agaatctaac gaagttgacg gcgtggttct taaagcaccg aacggtctgg 31920

gcatttcttt ctcttacatg tccgggtatc cgtctttgtc tggaaacatt ggcacgatat 31980

atgacccaac aggtggttcg gatttttctg gggtgttggt gcctaatgat acattttcgc 32040

tggtgtcagc gtggacaaat acggacgtcg acctatccgg cggcgggtat caggtgttaa 32100

gcgtatccga agggaccgtt acctttatag tgcccggtgg cctcattggt aggtggcaag 32160

aaataagacc cggttcattt tttcgaggtg acggagaggc gtcgctgcaa ccggataaca 32220

catacgagaa aaccttaacc gattgggtgt caataaaccg taccgaggtt gagcgcatag 32280

ttgccaatat cgccgccgcc aatggtatgt ataaagacaa cggcaaatct aaaacactag 32340

cgtcggttac cgcggagata cagtaccagc tgcttgatga aaatagcacc ccttatgggc 32400

cgatatacac tgcacaagga accgtgtccg ggcgaacccc agactacaac ggtgtcacta 32460

tttacgccga cctgccggtt gtgtcgcggg tgcgggtgcg cgccagaagg gtgacagacc 32520

tcgactttaa tttcgaaggc tcagtggtag acgaaataac ctacgttaac ttgtacgggc 32580

aaacacgcga caacaccccg cactacggca accgaaccac tgtgcactca atgcgtaagc 32640

agacaccacg ggctgccgaa gttaagcaac ctcagttgcg catgattgcg accgaaatgg 32700

tatacaaata ccttggtaat ggcgtttttg aggatacgat gactcccaat acccaggccg 32760

tgcaatctct catccgcctg gcacgtgacc cggatgtggg gggtttaaac ctgacggtac 32820

gcaacatgga taagttactt gctgtccaaa aggaaataga ggattacttc ggcgacaaac 32880

aggcggggga attttgctac acgtttgatg actataaaac cactatgcag gacatagtta 32940

gtactatagc agacgccata ttctgcaccc catatcggcg tggggcggat attcttctcg 33000

attttgaacg cccccgcatg ggtcctgaga tggtgttcac ccaccgaagc aaggccggta 33060

cttctgaaaa gtggaccaga acatttaacg attctcaggt ttttgacagc cttaaattct 33120

cgtacataga ccctaagacg aacgtcaaag agactataac catacccgaa accgggggcc 33180

ttaaaacgga gacttacgac tcaaaaggaa tccgcaacta taagcaggct ttctgggcgg 33240

caaaccgtcg ccaccagaag aacattttaa agaaaatttc ggtgtcgttt accgccactg 33300

aagagggtat ttttgccctt ccgaatcgtg ccgttagtgt ggttaagggt tcgcgtatgt 33360

ctacttacga cggctacgta accgcggtta acggtcttac cgtagagcta tcccagccgg 33420

ttaagttcac atccggagat gaccattatt tggttctgaa gttacgtgat ggcggagtcc 33480

aaagtgttcg tgttgtccct ggcgcacatg accgacaagt aattatgacg tctgtgccgc 33540

aagaagccat ttacactggt aatagcgctt tgaaaactga attttcattc ggcaacgaag 33600

caaggcataa tgctcagatg attcttgttt ctacggtaga ccctggcgat gacagaacag 33660

tcaaaataac cgggtttaac tatgacaagg atttctataa gtttgacaac gtgcctcctt 33720

tcggtcgtgc gttctccaac ggattcgata acggttttaa ctaagaggat agccatatgt 33780

ctagtggttg cggtgaggta atgtcactta atgatttaca gatagctaaa aaacaccaga 33840

ttttcgaagc cgaggttatc accggtaaac aaggcggtgt agccggcggt gcagatatcg 33900

actacgccac taacccggta accgggcaga cgcagaagac gcttcccgca gtcttacgtg 33960

gtgctggttt ctccccggca tcttttaatt ttacgaccgg gggtactctc ggcgtaaacg 34020

acgcagataa agctgttctt tggtcgaaag aagatggtgg agacggtaac tactacgcat 34080

ggcgtggctc cctgccgaaa gttatccctg cggcgtcgac acctcttgcg acaggcggca 34140

tttcggattc cgcttgggac gcgttcggag atatcacctt ccgtgcggaa gcggataaga 34200

aatttaagta ctccgttaag ctgtctgact ttactacgtt acaacaattg gcagacgccg 34260

ccgttgatag tgttcttatc gaccgcgatt acaacttcag caacaatgag accgttaact 34320

tcggcgggaa gaccctgacc atcgactgta aagcgaagtt tatcggcgac ggaaacctgg 34380

tatttacgca attaggtaaa ggttccattg taatagcccc ctttatggag agtgctacaa 34440

cgccgtgggt gattaaaccg tggaccgacg ataatcagtg gataaccgac cccgcggcaa 34500

tcgtggccac acttaaacag tctaaaacag atggatacca gccgacagta aacgattatg 34560

ccaagtttcc tggtatagaa tcccttctcc ctccggaagc taaagggcaa agcatatctt 34620

ctaccctgga aattcgggaa tgtacaggcg tcgaggttca ccgggcgagt ggtcttatgg 34680

cgtgtttcct gttccgcgga tgccatttct gtaagatggt agacgctgac aacccgagcg 34740

gcggaaaaga tggcgtaatt acctttgaaa acctgagcgg cgattggggc aagggtaact 34800

atgttattgg cgggcgcaca agttacggtt cggtaagtag cgctcaattc ttacgaaaca 34860

atggcggttt cgcgcgcgat ggcggggtca tcgggtttac ttcgtatcgt gcgggggaaa 34920

gtggtgttaa gacgtggcaa ggtacggtag gttctacgac ctctcgtaac tacaacctgc 34980

aattccggga ttcagcagtg ctataccctg tatgggacgg cttcgattta ggcgcagata 35040

ctgacatgaa ccccgaagat gaccgcccag gggatttccc catttctcag tacccggtac 35100

atatgctccc attaaaccat ttgatagaca atctatttgt tagaggttcg ctgggggtag 35160

gtttcggcat ggatgggaaa ggtctgtatg tctctaacat aaccgtcgag gattgcgctg 35220

gttctggggc ttatattctt gcccacgaaa cagtattcac taatatcgca ataatcgaca 35280

ccaatactaa aaacttcccc gcgaaccaga tatatatctc aggggcctgc cgtgtaaacg 35340

gccttcgttt ggtcggcatc cgttcaacta ccgaacaggg tcttacgata gacgcgccta 35400

actccacggt aagtgggata accggcttcg tagacccctc aaggattaac gtagccaatt 35460

tgatggagga aggtcttggt aactctcgca taaacagttt caataatgat tctgcggcgc 35520

tgcggttacg gatacataaa ctgtcaaaaa ctcttgatag cggttctgtg tactcccaca 35580

ttaacggcgg gccaggttct ggttcagcat ggaccgaaat taccgctatt gcggggtcct 35640

tgcctgatgc cgtgtcatta aaaataaaca ggggcgatta tcgtgctgtt gagataccgg 35700

tagcggtgac cgtcctacca gacaacgctg tcagggataa cggggctata tcactgtatc 35760

tggaaggcga tagccttaag gcgttagtta agcgggccga tggaagctat acaagattaa 35820

ctttggcata aatagtaaag gccctgtaag gggccttaat ttatgataac agaaccacag 35880

cagcgataag taaagcgacc gcagccaatc cgtagcctat taaaaagcat ttcgcaccaa 35940

ttgagtattt catttagcgc ccctctcttt gtcaacttcc tgttgttcta gtagccagtc 36000

aagttgcgcg ttagcagcgt ctctttgctg ccgtagccgt aaaacctctt cttcgagttc 36060

cctgatacgt tttatttctc gctttaattg catagctaga agttgctcgc gggaattaat 36120

ggcgtctttt atcaattcgt tgcaagcgtc gatatcaaac tcattattca tctcttacct 36180

cgtctcttca tataattaag caactcttcc tggacagatt ttttctcgtc cgtacgcgcg 36240

gcaacgacct catccagcgt gtctttagcg actatgtgat acaggaacac tggacgctcg 36300

tggccagcct gtttctggcg tacaggacct atacgctcga caacctgcaa atagtgctcc 36360

aggttccagc cttgcgaaat aaacgccaga tgatgcccgc cgtcctgtaa attcaaacca 36420

tggcccgccg atgcagggtg cacgcacaaa atctcgattt ccccgcggtt ccaagattcc 36480

atctgcttat tacccttagc acctttcgca aacgcctgcg cctgggggaa tcgcctaaga 36540

atgcgctcaa gttcgtgctt gaactgatag gccaccagca gcggcgcacc ctgcaactcc 36600

tcgacaatgg actctaacgc atcgagtttc gcgtcgtgca ctttctccca gtctttcgtt 36660

gcttcaccat ccggccccga cacatacacg gcaccggaag caatctgcaa gcacttagcc 36720

gttttagcgg cggcgttagc cgcttcaact tctccgctct ccagttccgc gaataacttc 36780

tcctccatat cgatgtaggc ttgacgcgct ttcttcggca ggtcgatttc aaccggcact 36840

ataataggcg cttcacaacc gaaccactca gcggcgtcaa tcgtgagact aatgtccttc 36900

atcctctggt gaatctcgtt atccgcgcct gggcgagcat ggtactcccg cgccatcgca 36960

gatttgcctt tctgtaccga gttaaaccat ctatcggtaa aggccgtgta tgaagaccca 37020

aggcgttcac ctgcgtcgat gaaccagttc tgaccccaca agtccttgag gccgttaggt 37080

gatggtgtac cggtcaggtt aatgaaacgc ttaactttac cgaacgccac cttactaagc 37140

gccctcgccc tcttgctccc gccagaacgg ctacggaacg atttcagttt cgtgctctca 37200

tcggcaacga taacggtaaa aggccagtcg tctttgccgt agtaatcaat aagccactcg 37260

ataacttcgt agttagtgca caccacatta gcgtccgact ccagcgccgc tatgcggcgc 37320

ttctccgaac ccgtagcatc gatgacacga aggcacggga aaccccattt ctcttgttcc 37380

gccggccacg tacccgacgc gacgcgcaac ggggcgagga ttaacacgcg gtcgtcatca 37440

ttaagctgac cattacggaa taaccggtta agcacccaca ttgtcgagct tgtgttgtgc 37500

gttacggtga agtcacctaa tagaaaccgg tggtcaccgt cgatggtgaa gccgtaataa 37560

tcatctacac cgaccggggt tatagattct atcccgacgt taagtacgtt tttattaata 37620

ttacgcttcg gtaaattctg gtggcggccg cgcacgaaag gcacctcaga aaaatcgcct 37680

gataccgata cgcggaagta atccccccaa acatcagtgt ttgcgcaacg cttacgagtt 37740

ttcttcttgt aagcggcgaa accaagagaa cggcatagat agcaaaaatc atcagcgagt 37800

cgctcgctta ccgaaatcca gtcaaaacct gctttagaca aatcacaata accgtcacta 37860

tcaagaagcc ccgccagcaa ttctaaacgt tgttgccggt cgccgcattt gtaattgtgc 37920

ggtatatgct tattgttcag gacacccgcc gatttcagcg cgtgcgtaaa cccgtgcttc 37980

ttatgacctg tgttgccgtg agatatactc catgtaagtc cttcttttcg tatttgcatc 38040

ccattacgtg ctgcgtacga ctcaagatac gcccttattt ccttttcgtt ctctcctgat 38100

gttatggccc cagacgacga agtaccgtcg cccagccata gccccataag gtacggtggc 38160

aataaagcct cgtcctgctc tttccttgta aaatcgacag gtacccgcca acccttaaga 38220

tacccattag gcccggttac atatttcggc aacttaagcc agtcgcggac gcttatgtcg 38280

aaaacggtat tatctggcca tgaacctttt gctatccctg tagtagttcg cagggatagt 38340

atgtggcttt cgttgacagt ataagactct ccttttcgcg gtttaacctc atacatcatc 38400

tcccggcctc taccgagaga aagtacattt ctcggcgttg aatccgggcc cataagaacg 38460

tcaccgacta taacgtcttc tactttcttg gttgtgccgt caaacataat aacttcagtc 38520

ccgcgtttca ggcacttgcc gccgcccata ccgcaccaga tattgcaacg ttggtgctgt 38580

agcataaacg acgtcattat tttctgatat tccctgcgcc taaacttact catgccttga 38640

actccccgaa gtatatttct gcggctcttt ggtatacgcg cgacgcttct tctatagtgt 38700

caaaatagcc aaggtggatg tatctgttat tgacgcctat tctggcgcgc cacctgccag 38760

atggtatttg gtaaacccct atcgcgccgc tggtgttatg cgaggtgcag tcgcggttct 38820

gaatctgttg gctccttgtt gcaggcctta tattatttat ggcgttattg gccctgttac 38880

ggtctttatg gtcaagctca ccattgtgat attccccgta gacgaaagcc catgccagat 38940

ggtgggcttt tattcttaca ccggagactg taataactat gtatcctttg tcgtttggcc 39000

ctccggctac ctccccagca tttctccctt ttatgcttac ttttcgtgtg aatatacctg 39060

tttcggggtc atagttaaga agacgtttcg cctcctcaat atcgggtctt ctagtgaatt 39120

tactcatttc atttcaccac cagcaccaat tccttccgcc cgaacgccgt aacgttaccg 39180

gttacgtctt cgataaccag tttaccgttc gactcgacga acaccgtatc aacggcaaca 39240

gggctacgtg tcctaacgtt gaaaatcatg tcacccggta cgatgtcacg tgctggtttg 39300

cggtcatatt cgtatttcat ttctcaattc cttatttgtg tttggtgtga actaaatata 39360

atagtgttct attaatttat caacctgttt cttagaccca acaacaaaaa catttgcacc 39420

tcgcttacgc atcctctcgt gctcccgtaa ctggtgcggg tctggcttcg tgctttcgtc 39480

tttcttcacc tcgacgaacc agatgacacc accgggaaga acaaccaaca ggtcaggagc 39540

accggagcgc ccctcatagg aaagtttccg aacgaggcca cccagggcct caaatcgctc 39600

ctttgcgtat ttttgcacgc gcccttcagg agtactcatc tatgcacccc tcacgtttcg 39660

tgtgttcaat accgcagcga ggacagattc gacagtcttc ttcccggaaa taataaataa 39720

tcagtttact tagcataccg ttttaactcc gcaccttccg ctacaagagg aaaaccatca 39780

gcccactccg gcaatgcaca cattagtttt tccagttcag ctaccgtgta atctgatgta 39840

tccggagtct cgcataccag ttcatcgtga acagaaagaa ctattggata cccaccggct 39900

tccacattaa gcatcgcata ggccaataag tcacggcaca acgcctgaac aatgttttca 39960

caagcctttc cgccgtgtgt gtacagggtg gtccattgcc gtgttaactg gttctcaccc 40020

tggtacttaa ttcttacatt ggtgtttatc cgtccgtctt cgtctgtttc ctttgtcaca 40080

ctaacgccga ttcccggata cgataggata cggcctgacg gcaactccat acacaaccac 40140

cagccaggaa ccttcctgcc cgacgaatca aattctacgg tacgccatat acggatagcc 40200

ctttcaccat tccggcgcaa gtgtgcccca gcccaaaatt cacgaccagg attacgaaca 40260

gcagctaaaa ttccgtcttt aaggtcgcgc cagaacgcta ctgtttcagg gtgtgactca 40320

cgccacatac gcttgatcgc gtcacatgtg cgccacacct tcttgtccag aatatacgac 40380

ggcctgtcat ccttttcacc gggatgcggt ggtctcttgg cttcctgaat acgcgcccac 40440

tcatacccac gtgcagtagc agcccagatg tggtcgggga aagtcccgtc cattgttttt 40500

gccatctcaa taaggtcaag acccaggttt ttagcaaacg taacgaacgc tccgacacct 40560

ccctcgtagc cgaggcccag ttcgcagtta tgcgccacaa gaaagcccga gtctgaggct 40620

ataagaaaac ggttgccgtc ttccacatca atcaggtcgt aaactggctc gcagtaggtt 40680

aatttcccgt tcaagctcag caatgcgggc aacggctgca tcatactttc gttgaagctc 40740

aggtattcga cgtctgttac ggcagttttc agaacggctg acaaaccgaa tgtttccagg 40800

ttcgtagtta ccgtctgtgt cgattcggtc catttcgagg tttgggtcat cccagccttc 40860

cagagtttga acatatctaa gaaacgacct tttatcgacc ctccactggt caaataccgt 40920

gatacccctg gccccgtagt ttttatagct cttgtttcca gggattgttg ttcgtgatat 40980

cgccgctgat aatcggttga gtaatcgcgt tctgtgctcg tcgtccggca ttgcgtcttc 41040

gtagcagaaa tattgtttac gatatttttt tgttgcttca agggcgcaac ggttgcattg 41100

agtggtttta cctgccctga cattgtcata atagacccgc cccggcatct ctccgcaatc 41160

acattggacg agtagctgtc tttggccttg tcgttttggt aactcgcgca ggataaccag 41220

gcgtccatac ctatctccag ggcgcggtcc atcatatatt tgtttgaaac gagttgcttt 41280

gcctgcttcc aggaatgcgt caagattttg tggtcttcgg tcattagtac cccgtccaca 41340

tttatcaccg gtttccaccc cataaggtgc gcgccttttg tattcaccca tttttcacca 41400

ctccatactt tatctgctga tgtaaccgcc ataatatcct taaatccacc atcggtcaaa 41460

acttgggtat gacggtgaag gcaagcctta cctatctggc gtaagtcttt acgttctttc 41520

ttaatatcgt cggggtccat gccaaacatc ttacccgccg ttacgcagta aatatccagc 41580

ccggcgcgga acgtatcaag cgcggtttct tcgcccgcca gccacgctaa accacggcct 41640

tctacgttgg aatagtccgc aacaacaaac ttacacccgt cttccgggat tatacagctg 41700

cggacagtgg atgccgttag cttggccaca tcaaaacggc ggtgtgcacg gcctttaagt 41760

aatgcggaaa tacccttatc aagttcatcg tcgtgataat atccgcgcgc caggttctga 41820

ggctgaaaac ctttcccggc ccatcgcagt gttcgcttcg ctccgccgta ttgcagacaa 41880

cctcgacggc ggtcatcgga agaacgacct aacagcaacg gcgcgtattt cgtcgatgcg 41940

gtggaagcag ccccgagacg catctcgata atagtgcgcg catcgtccgg taaatcctca 42000

tctgccagca agtcgttaag cgtcgacttc tgtgcgttgt ggatgcggtg tgccggtgct 42060

agttcctgta aaataggcaa gaagtctttg ccggttagtg aaccgccata tttacgttgg 42120

gcttcttcct gtaactcttc tttatggcgc gtgactgctt caatcgctgc ttccgccaat 42180

gccacatcaa ccttaaatcc tcggtcatta atcacctggt ctaagtgcag cacgcggtct 42240

tcgaactcag aattccccca cttaggcatt ttcttataca cttcacgcat ggacgtaatg 42300

tcgctttttg cgtatgcaat aaattctgcc cactcttttg ggtgggtatc ggcagtataa 42360

cgtcgaatct tatagttctt gggcgtaggc ttactgaatc gctgtatcag cgccttaccg 42420

cgtttatcct tagccatgct ggcgtcaatg tttaaaacct cacacagcgc ggccagtgaa 42480

ccagggaggg cgtgccggaa tgcaactatc atagtgtcga tgatgttttc tacgagaagc 42540

tcgaaacccc agcaatgctt aataacaggt cggtcgaaca taaggaagtt ttggcctacc 42600

agtttgaggt tgctgtctgg tttttgcagc tttagcatcg cccggcgcaa atcacgcggc 42660

atatcactcc cgtcggtggc atcccatacc tgcacggggc cttcatcgaa agcgtaggtg 42720

caaataacaa tttcggtagt gggatgttca gcgtaggcat aggagccgac ttttttcaaa 42780

tcagcttcgg agaatgtttc agtatcaaga taaagcaagt tcattcattt gacccttata 42840

gaaaaggcga ccgaagccgc cttaattgaa aagaaaaatg tattaacggt tacggcgacg 42900

ttcacgacgc ggcgtttcgt cttcatcatc ttccaggtcg tcaacgctgg cagaaaccgc 42960

Claims (3)

1. A high-temperature resistant Salmonella phage (Salmonella Gallinarum phase) RDP-SA-18056, wherein the phage can form a plaque with the diameter of 2mm-4mm on a double-plate; observed by a transmission electron microscope, the bacteriophage has a polyhedral and three-dimensionally symmetrical head part, is wrapped by nucleic acid, has the diameter of about 50nm, has a tail part with the length of about 200nm, and is provided with a tail sheath, and the neck part is connected with the head part and the tail part; is a family of Flaviviridae (Siphonviridae) of the order Narivavirales (Caudovirales) with a accession number of CGMCC No. 19293.

2. A process for the preparation of the thermostable salmonella bacteriophage RDP-SA-18056 microcapsules according to claim 1, comprising the following steps:

(1) seed preparation:

a. preparing a host seed: selecting a single colony to inoculate in 5mL of LB liquid culture medium under an aseptic environment, culturing for 4-6h at 37 ℃ and 200rpm, inoculating the cultured host bacteria in 5% of inoculum size in 100mL of LB liquid culture medium, culturing for 4-6h at 37 ℃ and 200rpm, then inoculating in 5% of inoculum size in 1000mL of LB liquid culture medium, culturing for 4-6h at 37 ℃ and 150rpm, and placing the cultured host strain seeds in a refrigerator at 4 ℃ for later use;

b. preparing a phage seed: b, properly diluting the stored phage seeds, putting 200 mu L of host bacteria cultured in the step a and 200 mu L of host bacteria cultured in the step a into an upper layer culture medium, pouring a double-layer plate, culturing for 8-12h at 37 ℃, selecting a plate with a cloudy plaque, washing the upper layer culture medium with 5mL of LB liquid culture medium, then centrifuging at 12000rpm for 10min, filtering through a 0.22 mu m filter membrane, and collecting clear liquid;

respectively inoculating the clear liquid and the host bacteria cultured in the step a in a ratio of 2% to 5mL of LB culture medium, culturing at 37 ℃ and 200rpm for 4-8h, simultaneously inoculating the cultured phage and the host bacteria in an inoculation amount of 2% to 100mL of LB liquid culture medium, culturing at 37 ℃ and 200rpm for 4-8h, simultaneously inoculating the cultured phage and the host bacteria in an inoculation amount of 2% to 1000mL of LB liquid culture medium, culturing at 37 ℃ and 200rpm for 4-8h, and placing the cultured phage seeds in a refrigerator at 4 ℃ for later use;

(2) and (3) phage multiplication:

b, inoculating the host bacteria cultured in the step a into a seed fermentation tank in a proportion of 3%, culturing for 3-4h, then inoculating the host bacteria into the fermentation tank through a seed transfer pipeline, wherein the OD600 value is 0.7-0.9, the inoculation amount is 5%, culturing for 3-4h in the fermentation tank, inoculating phage into the host bacteria in an inoculation amount of 5% when the OD is 0.7-0.9, culturing for 6-8h, and finishing proliferation after the dissolved oxygen is reversely increased and the pH tends to be stable;

wherein, the fermentation tank parameter is: the seeding tank is 200 plus 300rpm, the air ventilation is 1:0.6-0.8vvm at 37 ℃; fermentation tank: 120-150pm, 37 ℃, 1:0.6-0.8 vvm;

the components of the culture medium in the fermentation tank are as follows: 2.5% of yeast extract powder, 0.3% of glycerol, 0.8% of sodium chloride, 0.2% of monopotassium phosphate, 0.2% of dipotassium phosphate, 20ppm of calcium chloride and 20ppm of magnesium chloride;

(3) the phage post-treatment process comprises the following steps:

A. centrifuging: centrifuging the proliferated phage by using a tubular centrifuge at 14000rpm at a flow rate of less than or equal to 30L/H, and removing host bacteria, bacterial debris and the like which are not cracked in the step (2);

B. further filtering and sterilizing by adopting a hollow fiber membrane with the thickness of 400 nm;

C. concentrating with 100KD roll-type membrane;

D. and (3) filtering and sterilizing: performing three-stage filtration sterilization on the concentrated phage;

(4) low-temperature spray drying of the phage:

adding the filtered and sterilized phage liquid into a carrier and uniformly stirring, wherein the carrier is as follows: 8% of porous starch, 2% of polyvinylpyrrolidone, 1% of lactose, 0.5% of modified chitosan and 0.2% of vitamin C; then carrying out warm spray drying, wherein the parameters are as follows: the air inlet temperature is 150 ℃, and the air outlet temperature is 75 ℃.

3. The process for preparing the high temperature resistant salmonella bacteriophage RDP-SA-18056 microcapsule according to claim 2, wherein the microstructure of the prepared bacteriophage RDP-SA-18056 microcapsule is a concave cavity.

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