CN111647567B - Acid-resistant salmonella bacteriophage and composition, kit and application thereof - Google Patents

Abstract

The invention belongs to the technical field of salmonella bacteriophage, and particularly relates to an acid-resistant bacteriophage isolate capable of specifically cracking salmonella and application thereof in sterilization and bacteria prevention. The invention mainly discloses a salmonella pullorum bacteriophage SG4P1 (S)almonella pullorum phase SG4P1) with the preservation number of CCTCC NO: M2018765. The salmonella bacteriophage in the invention is a virulent bacteriophage separated from nature, and toxicological experiments prove that the salmonella bacteriophage is safe and has no side effect. Experiments show that the bacteriophage can resist extremely strong acidity, so that the bacteriophage can have a strong lysis effect on salmonella in intestinal tracts through gastric acid. The bacteriophage is expected to be developed into a biological bactericide for preventing and treating salmonella infection and pollution.

Description

Acid-resistant salmonella bacteriophage and composition, kit and application thereof

Technical Field

The invention belongs to the technical field of salmonella phages, and particularly relates to an acid-resistant phage capable of specifically cracking salmonella, and a composition, a kit and application thereof.

Background

Salmonella generally colonizes in animal intestinal tracts, and if the salmonella is not properly treated in food processing, food is easily polluted, so that potential food safety hazards are caused, and human health is harmed. In recent years, the abuse of antibiotics by farmers and feed producers has led to the emergence of more and more resistant strains of the pathogen, rendering many antibiotics for the treatment of salmonella infection disorders ineffective. The resistance of salmonella china to sulphonamide antibiotics has been reported to approach 100%, and other effective methods of combating the pathogen infection have to be sought.

Bacteriophages are a class of obligate parasitic or bacteria-infecting viruses that are widely found in nature. The virulent phage can be proliferated in sensitive host bacteria cells, and finally the host bacteria are cracked to release progeny phage, so that the effect of destroying the host bacteria is achieved. At present, the virulent bacteriophage is widely applied, and particularly has good effect on treating infection caused by drug-resistant 'super bacteria'.

The Chinese patent with the prior art publication number of CN106497888B discloses a salmonella bacteriophage and a bacteriophage antibacterial composition and application thereof. The bacteriophage provided by the invention is Salmonella enteritidis bacteriophage LPSE1 and Salmonella typhimurium bacteriophage LPST10, and both bacteriophage LPSE1 and LPST10 have good inhibitory effect on Salmonella. The phage LPSE1 disclosed in the prior art is stable at pH of 10-12⁸PFU/mL, phage titer dropped to 0 at pH < 3, 13PFU/mL at pH 13, titer dropped significantly, and 0 at pH 14.

The prior art described above has the following drawbacks: when the pH value of the Salmonella enteritidis phage LPSE1 is less than 3, the phage titer is reduced to 0, which shows that the acid resistance of the Salmonella phage of the strain is weaker. The salmonella can live in the acidic intestinal digestive system environment, even other severer acidic environments, so that an acid-resistant phage isolate capable of specifically cracking the salmonella needs to be found urgently.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the acid-resistant salmonella bacteriophage which has extremely strong acid resistance and can specifically and partially or completely inactivate salmonella.

The above object of the present invention is achieved by the following technical solutions: an acid-resistant Salmonella bacteriophage, wherein the Salmonella bacteriophage is Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1), and the preservation number is CCTCC NO: M2018765.

By adopting the technical scheme, the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phage SG4P1) is provided, has extremely strong acid resistance, can specifically and partially or completely inactivate Salmonella, and has broad-spectrum bactericidal capability on Salmonella.

The present invention in a preferred example may be further configured to: the Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) has a nucleotide sequence shown in SEQ ID No. 1.

The present invention in a preferred example may be further configured to: the Salmonella pullorum phage SG4P1(Salmonella pullorumphage SG4P1) has acid resistance under the condition that the pH value is 1-4.

The present invention in a preferred example may be further configured to: culturing for 8-10 h under the condition that MOI is 0.001, and the titer is 3 multiplied by 10¹⁰PFU/mL。

The second purpose of the invention is to provide a composition containing acid-resistant Salmonella bacteriophage, i.e. the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) has extremely strong acid-resistant capability, and can be mixed with other substances to meet the requirements of specific applications.

The second aim of the invention is realized by the following technical scheme: a composition containing acid-resistant Salmonella phage at least comprises a Salmonella pullorum phage SG4P1(Salmonella pullorum phage SG4P 1).

Through the technical scheme, the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) and other bacteriophages are used in combination to obtain a wider lysis spectrum range. As an illustrative illustration, the ratio of the Salmonella pullorum bacteriophage SG4P1 to other bacteriophages can be determined by one skilled in the art in connection with the present invention and the field of practical use and general knowledge in the field.

The present invention in a preferred example may be further configured to: the composition also comprises one or more of myotailed phage BP-66(Myoviridae sp.BP-66), myotailed phage BP-63(Myoviridae sp.BP-63), and long-tailed phage BP-12(Chilikevirus sp.BP-12).

The present invention in a preferred example may be further configured to: the composition also included chlorine dioxide at a concentration of 0.0002%.

The Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) and other antibacterial agents are mixed for use to obtain broad antibacterial spectrum and specific killing effect on Salmonella. Antimicrobial agents that can be used in conjunction with the bacteriophage of the present protocol herein include, but are not limited to, antibiotics and chemical antimicrobial agents. By way of illustration, the ratio of Salmonella pullorum phage SG4P1 to other antibacterial agents can be determined by one skilled in the art in view of the present invention and the field of practical use and general knowledge in the art.

It is a further object of the invention to provide a reagent or kit comprising a composition of acid-resistant Salmonella bacteriophage.

The third object of the invention is realized by the following technical scheme: a reagent or kit comprising an acid-tolerant Salmonella phage composition, comprising said Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) or Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) composition.

The fourth purpose of the invention is to provide an application of acid-resistant salmonella bacteriophage.

The fourth object of the invention is realized by the following technical scheme: the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) is used as a biological bactericide.

By adopting the technical scheme, the novel safe environmental biological bactericide of the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) can specifically kill Salmonella in the environment and improve the distribution of microorganisms in the environment.

The Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) is used as a biological bactericide for livestock and poultry product culture, transportation and preservation, and can prevent and treat pathogenic Salmonella pollution in the livestock and poultry culture, transportation and preservation processes.

The Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) is used alone or in combination with other antibacterial substances, and the surface of food contaminated by Salmonella is sprayed with 10⁵～10⁶The phage with PFU/mL can completely inactivate the salmonella within 12h, and can be applied to preventing and treating the salmonella pollution on the surface of food.

The Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) is used as a bactericide and is sprayed in a food production workshop, so that Salmonella in the environment can be specifically and continuously prevented and treated, and meanwhile, the Salmonella pollution in the food processing process can be prevented and treated.

The fifth purpose of the invention is to provide application of the acid-resistant salmonella bacteriophage.

The fifth invention object of the present invention is achieved by the following technical solutions: the application of the compound in bacterial infection diseases caused by salmonella can be used as preventive medicines, therapeutic medicines or medical devices.

By adopting the technical scheme, the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) is used as a potential drug to treat and prevent bacterial infection caused by Salmonella.

Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) is used for treating and preventing bacterial infection caused by Salmonella.

The invention further aims to provide application of the acid-resistant salmonella bacteriophage.

The sixth object of the present invention is achieved by the following technical solutions: the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) is used as a feed additive.

By adopting the technical scheme, the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) can be used singly or in a mixed way, and can be used as a potential feed additive to be added into feed, so that the Salmonella pollution in feed production, storage and animal breeding can be prevented and treated specifically and continuously.

In summary, the invention includes at least one of the following beneficial technical effects:

1. after a long measurement time, for example 6 hours, the Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) still has extremely strong acid resistance, can specifically and partially or completely inactivate Salmonella, has an MOI of 0.001, can complete a large amount of proliferation only by a small amount of initial phage, and provides a high-quality phage strain source for industrial production of phage bactericides; according to the description of the invention and the common knowledge in the field, the technicians in the field can prepare the salmonella pullorum phage SG4P1 or the composition thereof into various products applied to the aspects of medical treatment, detection, disinfection, food protection and the like for industrial application.

2. The Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) is a virulent phage separated from nature, and toxicological experiments prove that the Salmonella pullorum phage is safe and has no side effect; the test phage does not contain virulence genes or undesirable genes; the present invention does not subject the test phage to any genetic modification.

3. The Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) can be used singly or in a mixture at a concentration of 10²～10³PFU/mL Salmonella culture Medium 10⁵～10⁶The sterilization rate of the phage of PFU/mL to the salmonella of the concentration reaches more than 99 percent, and the phage has broad-spectrum sterilization capability to the salmonella. The Salmonella pullorum bacteriophage SG4P1 can be used as an effective component of various products for environmental disinfection, including but not limited to liquid soaking, spraying, and combining with aqueous carrier to disinfect and decontaminate water distribution system, medical facilities, breeding facilities, public and private facilities, or other environmental surfaces, and can effectively control the growth and activity of target bacteria.The liquid soaking, spraying forms include but are not limited to detergents, disinfectants, detergents, etc.; the aqueous carrier includes but is not limited to phosphate buffer, LB medium, chloride free water, etc.

4. The Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) can effectively kill Salmonella on the surface of chicken and prevent and control the contamination of the Salmonella in the storage process of the chicken, and can be used as an effective component of various products for food protection. The present invention includes, but is not limited to, the prevention of food spoilage caused by Salmonella infestation in liquid form, by spraying, by use in combination with synthetic ingredients, and the like, particularly for cooked or unpasteurized foods. The liquid soaking and spraying forms comprise but are not limited to food degerming agents, food disinfectants, food preservatives and the like; synthetic components of the present invention include, but are not limited to, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, calcium propionate, and the like.

5. The Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) can be used for preparing a composition, a reagent or a kit, can be applied to the rapid detection of Salmonella, and comprises but is not limited to detection of Salmonella in a target sample in the form of test paper, a kit and the like, or screening of target pathogenic bacteria in a clinical sample, and can effectively ensure the detection sensitivity.

6. The Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) can be used singly or in a mixed way, can be prepared into a biological bactericide, a feed additive and a therapeutic drug by the technical personnel in the field according to the record of the invention and the common knowledge in the field, and is applied to treating or preventing infectious diseases caused by Salmonella. Hosts that can be infected by Salmonella include humans, livestock (pigs, cattle, sheep, etc.), poultry (chickens, ducks, geese, etc.), and various beasts, fishes, rats, etc.

7. The Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) can be applied to industrial production, can be specifically amplified by host bacteria, can be highly purified by a standard virus purification method, can be developed into a biological bactericide, and is used for preventing and treating Salmonella infection. The product form can include, but is not limited to, the carrier carrying, the concentrated injection or the medicament soaking and the like, and the product is applied to the controlled host body surface, the oral part, the rectum, the inner pleura and other parts; as one embodiment, the carrier-borne form includes, but is not limited to, oral aqueous carriers, oral anhydrous carriers, cream formulations, and the like; concentrated injection forms include, but are not limited to, vaccine injections, pleural cavity injections, transvenous injections, etc.; dosage soaking forms include, but are not limited to, aerosols, rinses, and the like.

Drawings

FIG. 1 is a photograph of the plaque morphology of the bacteriophage SG4P 1.

FIG. 2 is a schematic representation of the single electron micrograph of phage SG4P 1.

FIG. 3 is a bar graph of the results of the acid resistance test of bacteriophage SG4P 1.

Detailed Description

The following examples are further illustrative of the present invention in detail with reference to the accompanying drawings, but do not limit the effective scope of the invention in any way.

The Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) has a preservation number of CCTCCNO: M2018765, a preservation unit of China center for type culture Collection, and a preservation time of 11 months and 09 days in 2018.

Myotailed phage BP-66(Myoviridae sp.BP-66), with preservation number of M2015146, preservation unit of China center for type culture Collection, address of Wuhan university at Lodoku mountain Wuhan university, Wuchang, Hubei, and preservation date of 2015, 3 months and 23 days.

The myotailed phage BP-63(Myoviridae sp.BP-63) has the preservation number of CCTCC NO: M2015145, the preservation unit is China center for type culture Collection, the address is Wuhan university at Lojia mountain of Wuchang, Wuhan city, Hubei, and the preservation date is 2015, 3 months and 23 days.

The long-tail phage BP-12(Chilikevirus sp. BP-12) has a preservation number of CCTCC NO: M2015141, the preservation unit is China center for type culture Collection, the address is Wuhan university at Lojia mountain of Wuchang, Wuhan, Hubei, and the preservation date is 2015, 3 months and 23 days.

In the following examples, the following examples are given,

the LB liquid culture medium has the formula: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 1000mL of distilled water and pH 7.0.

The LB solid culture medium has the formula: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar and 1000mL of distilled water, and the pH value is 7.0.

The semi-solid agar culture medium comprises the following components: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 7g of agar and 1000mL of distilled water, and the pH value is 7.0.

The SM buffer solution formula is as follows: 8.5g of sodium chloride, 2g of magnesium sulfate, 50mL of 1mol/L Tris-HCl, 0.25g of gelatin and 1000mL of distilled water.

Compositions 1-7 of Salmonella pullorum phage SG4P1(Salmonella pullormugage SG4P1), Myoviridae BP-63(Myoviridae sp. BP-63), Myoviridae BP-66(Myoviridae sp. BP-636), and Long-tailed phage BP-12(Chilikevirus sp. BP-12) were prepared as described in example 5.

A composition of Salmonella pullorum phage SG4P1(Salmonella pullorumphage SG4P1) and chlorine dioxide at a final concentration of 0.0002% (composition 8) was prepared as described in example 6.

Example 1: screening and purification of phages

Sample collection and treatment the samples in this example were collected from farm effluents from Shandong, Henan, Jiangsu and from soil in nearby farmlands.

The collected sample is centrifuged for 10min at 5000r/min and filtered through a 0.22 mu m filter membrane to obtain filtrate.

And (II) aiming at the enrichment of the target salmonella bacteriophage in the sample, mixing the filtrate with 2 times of LB liquid culture medium in a ratio of 1:1, inoculating 100 mu L of the target salmonella strain, and carrying out overnight enrichment culture to obtain an enrichment solution.

And (III) centrifuging the enrichment solution, filtering the supernatant through a 0.22-micron membrane, taking 1mL of filtrate, performing gradient dilution by using SM solution, respectively mixing each gradient dilution with 5mL of LB semisolid culture medium containing target salmonella, pouring the mixture onto a culture dish containing the LB semisolid culture medium, after the semisolid culture medium is solidified, performing overnight culture at 37 ℃, observing whether plaque exists on the next day, and recording an experimental result.

And (IV) purifying and picking the single plaques in 1mL of SM buffer solution, shaking at 150rpm for 15min, passing through a 0.22-micron membrane, performing gradient dilution on the filtrate by using SM solution, uniformly mixing each gradient dilution with 5mL of LB semisolid culture medium containing the target salmonella, pouring the mixture on a culture dish containing the LB semisolid culture medium, and after the semisolid culture medium is solidified, performing overnight culture at 37 ℃. Repeating the step for 3-5 times to obtain a phage monoclonal sample, and naming the sample as Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P 1). The plaque morphology of Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) is shown in FIG. 1; FIG. 2 shows a TEM micrograph of Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P 1). The salmonella pullorum phage SG4P1 has the following biological characteristics: observing the form of the SG4P1 bacteriophage under an electron microscope, wherein the head of the bacteriophage is an icosahedron, the length of the bacteriophage is about 90-100 nm, and the transverse diameter of the bacteriophage is about 60-70 nm; is a tailed bacteriophage, non-folded, with a collapsible tail, a tail length of about 95-115 nm, and a diameter of about 20-30 nm, and is systematically classified as Myoviridae (Myoviridae) based on its unique size and morphology, as defined by the International Committee for Virus Classification (ICTV).

Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) with preservation number of CCTCC NO: M2018765.

(V) phage particle preparation

Amplifying Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) by using host bacteria, centrifuging for 10min at 8000g after a culture medium is clarified, removing impurities, adding solid polyethylene glycol (PEG8000) to a final concentration of 10% (w/v), stirring for dissolving, and standing overnight at 4 ℃. The pellet was resuspended in SM buffer after centrifugation at 8000g for 20min at 4 ℃. The resulting phage suspension was stored at 4 ℃ for future use.

Example 2 determination of Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) on the optimum multiplicity of infection (MOI) of Salmonella

And picking a single salmonella colony, inoculating the single salmonella colony into a test tube containing 3mL of LB culture solution, and performing shaking culture at 37 ℃ and 150rpm for 12h to obtain a host bacteria suspension. The bacterial suspension was transferred to l0mL LB medium at a ratio of 1:100, and cultured at 37 ℃ with shaking at 150rpm until the pre-log phase. Pure culture solution (prepared from example 1) of salmonella pullorum phage SG4P1 and host bacteria (MOI ═ number of phage/number of bacteria) were added in a complex infection ratio, and LB liquid medium was added to make the total volume of each tube the same. Shaking overnight at 150rpm in a shaker at 37 ℃. After the culture, centrifugation is carried out for l0min at 5000g, and the supernatant is collected to determine the titer of the phage. Each point was subjected to duplicate multi-tube culture and averaged to obtain the MOI producing the highest phage titer as the optimal multiplicity of infection. The experiment was repeated 3 times.

TABLE 1 titer of Salmonella pullorum phage SG4P1 at different multiplicity of infection

As shown in Table 1, the titer of Salmonella pullorum phage SG4P1 was highest (2.0X 10)¹⁰PFU/mL), the MOI is 1: 1000. The method has the advantages of small initial input amount and high propagation speed, and can be applied to industrial production.

Example 3: the virulence genes or the undesirable genes of the salmonella pullorum bacteriophage SG4P1 are deleted and tested, 65 virulence genes (shown in Table 2) which are identified to be originated from lysogenic bacteriophages in pathogenic bacteria are selected, and the whole genome of the salmonella pullorum bacteriophage SG4P1 is determined and subjected to bioinformatics analysis, so that whether the salmonella pullorum bacteriophage SG4P1 contains the virulence genes or not is determined. The results showed that the Salmonella pullorum phage SG4P1 did not contain the following virulence genes. The test phage had no undesirable genes.

TABLE 2 major known virulence genes of lysogenic phages in pathogenic bacteria

The salmonella pullorum phage SG4P1 does not contain a virulence gene or an undesirable gene, wherein the virulence gene or the undesirable gene is described in a table 2.

Example 3: toxicological experiments

The experimental mice are divided into two groups (phage group and control group) at random after 20 mice with half male and female are bred adaptively for three days, each group comprises 10 mice (5 mice for male and female), and the dose of the phage group is 10¹⁰PFU/kg Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1), control group was given equal amount of physiological saline and administered for 15 days continuously, and the experimental mice were sacrificed by neck-cutting and checked for visceral condition.

The experimental results show that this dose of Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) had no effect on the daily behavior of mice. The viscera were examined by dissection without abnormality. Has biological safety, and can be used as feed additive.

Example 4: salmonella pullorum phage SG4P1 acid resistance test

1. Phage counting method (potency)

And (3) carrying out gradient dilution on the obtained Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) sample according to a proportion of 10 times, taking 100 mu L of the sample with a certain dilution proportion, paving a double-layer plate according to the method in the step (IV) described in the example 1, taking the plate with a proper proportion, and calculating the number of plaques, wherein one plaque represents a phage monomer.

2. The titer was 1x10⁹PFU/mL samples of Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) (prepared in example 1) were adjusted to pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0, respectively, and the titers were determined after 4h of treatment. The test results are shown in FIG. 3.

The results show that: the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) can maintain high titer between pH2.0 and 9.0, and starts to grow at high speed when the pH is 1.0, and has the highest titer when the pH is 7.0. The resistance to acid environment is extremely strong, which proves that the composition can resist the low pH environment of animal gastrointestinal tract, and has extremely strong potential in the aspect of treating salmonella infection of animal gastrointestinal tract.

Example 5: acid resistance test of the salmonella pullorum phage SG4P1 phage composition respectively takes titer of 1x10⁸PFU/mL of the stock solutions of Salmonella pullorum phage SG4P1(Salmonella pullormula SG4P1) and Myoviridae BP-63(Myoviridae sp.BP-63), 2 strains of phage were mixed uniformly in equal volumes to make a 1:1 composition of Salmonella pullorum phage SG4P1(Salmonella pullormula SG4P1) and Myoviridae BP-63(Myoviridae sp.BP-663) (composition 1).

Respectively taking the titer of 1x10⁸PFU/mL of the stock solutions of Salmonella pullorum phage SG4P1(Salmonella pullormula SG4P1) and Myoviridae BP-66(Myoviridae sp. BP-66), 2 phage were mixed uniformly in equal volumes to make a 1:1 composition of Salmonella pullorum phage SG4P1(Salmonella pullormula SG4P1) and Myoviridae BP-66(Myoviridae sp. BP-66) (composition 2).

Respectively taking the titer of 1x10⁸PFU/mL of Salmonella pullorum phage SG4P1(Salmonella pullorumphage SG4P1) and Long-tailed phage BP-12(Chilikevirus sp. BP-12) stock solutions, and 2 phage were mixed uniformly in equal volumes to make a 1:1 composition of Salmonella pullorum phage SG4P1(Salmonella pullorumphage SG4P1) and Long-tailed phage BP-12(Chilikevirus sp. BP-12) (composition 3).

Respectively taking the titer of 1x10⁸PFU/mL Salmonella pullorum phage SG4P1(Salmonella pullormula SG4P1), myotailed phage BP-63(Myoviridae sp.BP-63) and myotailed phage BP-66(Myoviridae sp.BP-66) stock solutions, 3 strains of phage were mixed uniformly in equal volumes to make a 1:1:1 composition of Salmonella pullorum phage SG4P1(Salmonella pullormula SG4P1), myotailed phage BP-63(Myoviridae sp.BP-63) and myotailed phage BP-66(Myoviridae sp.BP-66) (composition 4).

Respectively taking the titer of 1x10⁸PFU/mL Salmonella pullorum phage SG4P1(Salmonella pullorumphage SG4P1),The stock solutions of Myoviridae BP-63(Myoviridae sp.BP-63) and Long-tailed phage BP-12 (Chilikuirus sp.BP-12) were mixed uniformly in equal volumes with 3 strains of phage to prepare a 1:1:1 composition of Salmonella pullorum phage SG4P1(Salmonella pulorumhage SG4P1), Myoviridae BP-63(Myoviridae sp.BP-63) and Long-tailed phage BP-12 (Chilikuirus sp.BP-12) (composition 5).

Respectively taking the titer of 1x10⁸PFU/mL Salmonella pullorum phage SG4P1(Salmonella pullormula SG4P1), myotailed phage BP-66(Myoviridae sp. BP-66) and long-tailed phage BP-12 (Chilikuirus sp. BP-12) stock solutions, 3 strains of phage were mixed uniformly in equal volumes to make a 1:1:1 composition of Salmonella pullorum phage SG4P1(Salmonella pullormula SG4P1), myotailed phage BP-66(Myoviridae sp. BP-66) and long-tailed phage BP-12 (Chilikuirus sp. BP-12) (composition 6).

Respectively taking the titer of 1x10⁸PFU/mL Salmonella pullorum phage SG4P1(Salmonella pullorumphage SG4P1), Myoviridae BP-63(Myoviridae sp.BP-63), Myoviridae BP-66(Myoviridae sp.BP-636), and Long-tailed phage BP-12 (Chilikrus sp.BP-12) stock solutions were mixed uniformly in equal volumes to 4 phage to make a 1:1:1:1 composition of Salmonella pullorum phage SG4P1(Salmonella pullorumphage SG4P1), Myoviridae BP-63(Myoviridae sp.BP-63), Myoviridae BP-66(Myoviridae sp.BP-66), and Long-tailed phage BP-12 (Chilikus sp.BP-12) (composition 7).

Samples of composition 1, composition 2, composition 3, composition 4, composition 5, composition 6, and composition 7 were adjusted to pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, and 14.0, respectively, and after 6 hours of treatment, the titers were determined, respectively, as in example 4. See table 3 for results.

TABLE 3 pH stability of the compositions after different times of reaction

As shown in Table 3, the potency of each composition was relatively stable within a pH range of 5 to 8, and each composition still had a high activity within a pH range of 1 to 4.

Example 6: salmonella pullorum phage SG4P1 and bactericidal effect of composition thereof in liquid 1. salmonella counting method

Counting by adopting a BS salmonella selective medium and applying a dilution coating method, and culturing for 24h at 37 ℃, wherein the black colony with metallic luster is a positive colony.

2. Salmonella pullorum phage SG4P1 is cultured to logarithmic growth phase in liquid sterilization effect experiment, subpackaged into different test tubes, diluted with liquid LB culture medium with the same volume until the final concentration of Salmonella is 1000cfu/mL, and inoculated with Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) with different concentrations to make the concentrations of Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) in samples respectively 1x10²PFU/mL、1x10³PFU/mL、1x10⁴PFU/mL、1x10⁵PFU/mL，1x10⁶PFU/mL. A control group and a blank group (CK) were simultaneously prepared, and the control group was administered at a final concentration of 1X10³cfu/mL Salmonella; the blank group was given an equal amount of physiological saline. And detecting the residual quantity of the salmonella after 4 hours. See table 4 for results.

TABLE 4 Bactericidal Effect of Salmonella pullorum bacteriophage SG4P1 at various concentrations in liquids

The test results in table 4 show: the Salmonella pullorum bacteriophage SG4P1 concentration is 10⁵～10⁶At PFU/mL, the phage SG4P1 has the best bactericidal effect on salmonella in liquid LB medium, and the killing rate is more than 99%. Shows that the salmonella pullorum bacteriophage SG4P1 has the application of being applied as a biological insecticideThe potential of the microbial inoculum.

3. The experiment of the bactericidal effect of the composition of the high-concentration salmonella pullorum bacteriophage SG4P1 in liquid shows that the titer is 1x10⁸PFU/mL Salmonella pullorum phage SG4P1(Salmonella pullorumphage SG4P1) stock solution and chlorine dioxide at a final concentration of 0.0002% were mixed together in equal volumes to form a 1:1 composition (composition 8).

Culturing Salmonella to logarithmic phase, subpackaging into different test tubes, and diluting with liquid LB medium to obtain final concentration of Salmonella of 1x10³cfu/mL, inoculated with a final concentration of 1X10⁶ Compositions 1 to 8 of PFU/mL Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1), and methods for formulating compositions 1 to 7 were as described in example 5. Control and blank (CK) groups were set, and the control group was given a final concentration of 1X10³cfu/mL Salmonella; the blank group was given an equal amount of physiological saline. After 4h, the residual amount of Salmonella was determined as described in example 6. See table 5 for results.

TABLE 5 Bactericidal Effect of a composition of high concentration Salmonella pullorum bacteriophage SG4P1 in liquids

Table 5 the test results show: the Salmonella pullorum bacteriophage SG4P1 has a concentration of 10⁶When PFU/mL is adopted, the composition with the PFU/mL has good bactericidal effect and no antagonistic effect on other composition components. The composition of the salmonella pullorum bacteriophage SG4P1 has the potential of being applied as a biological bactericide.

Example 7: control of salmonella infection during chick farming by bacteriophage SG4P1 and compositions thereof compositions 1, 2, 3, 4, 5, 6, 7 were prepared as described in example 5. Composition 8 was prepared as described in example 6.

The test chicks 550 were adaptively bred for 3 days and then randomly divided into 11 groups (phage SG4P1 group, composition 1, composition 2, composition 3, composition 4, group A, group B, group C),Composition 5, composition 6, composition 7, composition 8, control and blank), 50 per group, each treatment experiment group was administered at a dose of 1x10⁸PFU/kg of phage tested and 1X10⁸PFU/kg Salmonella; control group was given 1x10⁸PFU/kg Salmonella; the blank group was given an equal amount of physiological saline and continued for 15 days. The daily growth and survival rate of the chicks are counted, and the results are shown in table 6.

TABLE 6 influence of Salmonella pullorum bacteriophage SG4P1 and compositions thereof on chick survival

Table 6 the results show: chicks in the control group all died on day 7; the blank composition has a survival rate of 96 percent, and the chicks have a large amount of diarrhea and white diarrhea in life; the survival rate of single phage SG4P1 group chicks is 80%; the survival rate of 8 chicks of the composition of the phage SG4P1 and the chlorine dioxide is 82%, which shows that the phage SG4P1 can be used with other components in a coordinated way without affecting the performance of the components; wherein, the survival rate of the chicks of the composition 7 is the highest and is 94 percent. The result shows that the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) and the composition thereof can be used as a biological bactericide or a feed additive to effectively prevent and treat Salmonella infection in the chicken breeding process.

Example 8: salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) and composition thereof for preventing and treating Salmonella pollution in chicken sample preservation process

1. The phage counting method comprises the following steps: the procedure is as described in example 4.

2. The salmonella counting method comprises the following steps: the same procedure as described in example 6.

3. Methods for preparation of compositions 1 to 7: the same procedure as described in example 5; composition 8 was formulated as described in example 7.

4. Cutting autoclaved chicken breast into small blocks 55 of 1cm square, and dividing into 11 groups (single phage SG4P1 group, composition 1, composition 2, composition 3, composition 4, composition 5, group)Compound 6, composition 7, composition 8, control and blank), 5 pieces per group, placed in sterile plates. Experimental groups Each block was inoculated with 10⁵PFU Salmonella and dose 10⁸PFU/kg of test phage; control group was inoculated with 10 inoculations per block⁵PFU salmonella and equivalent sterile water; the blank group was given equal amount of sterile physiological saline. Each treatment was incubated in a 37 ℃ incubator. A piece of chicken was taken every 2h and placed in 10mL of sterile water, shaken well and the salmonella content of the liquid was determined, and the results are shown in Table 7.

TABLE 7 Salmonella pullorum bacteriophage SG4P1 and control results of the composition thereof on Salmonella contamination during the storage of chicken samples

Table 7 the results show: after 8h, a large amount of salmonella grows on the surface of the chicken of the control group; and the Salmonella on the surface of chicken is always controlled at an extremely low level by adding Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) and a composition thereof in each experimental group. The Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) and the composition thereof can be used as a biological bactericide to effectively kill Salmonella on the surface of chicken and prevent the contamination of the chicken by the Salmonella in the storage process. Of course, the Salmonella pullorum phage SG4P1 can also be used as a pharmaceutical agent, and example 3 also proves safe.

Example 11: preparation of kit of Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) and composition thereof, and use of kit containing 5-10 mL of titer of 1 × 10⁷The composition of Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) or Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1), 1L LB semisolid medium, 1L LB solid medium.

The using method of the kit comprises the following steps: taking out the titer of 1 × 10⁷PFU/mL Salmonella pullorum phage SG4P1(Salmonella pullorum phase SG4P1) phage liquid orA liquid of a composition of Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) was used to determine the lysis profile of the test phage by double-plate titration. And (3) selecting a single bacterial colony to be detected, inoculating the single bacterial colony to a target liquid culture medium, and performing shake culture at a target temperature in combination with the growth characteristics of the bacterial strain to be detected to obtain a bacterial liquid of the bacterial strain to be detected. And mixing 300 mu L of bacterial suspension of the strain to be detected with 5mL of LB semisolid culture medium respectively, spreading the mixture on an LB solid plate, and dripping 10 mu L of Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) liquid or a composition of Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) on the plate. And after natural air drying, culturing at a target temperature according to the growth characteristics of the strain to be detected, and observing the result.

The embodiments of the present embodiment are all preferred embodiments of the present embodiment, and the protection scope of the present embodiment is not limited by this, so: all equivalent changes made in the structure, shape and principle of the present embodiment are intended to be covered by the protection scope of the present embodiment.

Sequence listing

<110> Philippinecaceae (Nanjing) Biotech Ltd

<120> acid-resistant salmonella bacteriophage, and composition, kit and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 35258

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gccgttgttg aatgtcgtca gtccatgatt tatgtggaac ctattagcca tagacatgta 60

aattctttcc gcgaaagcgt cctctattct gtcaaatgtt ttactaatct gcttaccgtt 120

gacaacggca taagctctcc acttagcctt tcctttccgc gccatctgtt attttacgag 180

ttaccatgct tatttcctga ttgtttaaga caaatctgat tctaataatg taagtttaat 240

ttagccagat tatttaccat taacattaga gttacaggcc cagatgagag ccccaaccca 300

cggcagaaga atccacccca gtaaaagatt acatacgaat attcctaatt tagctttatg 360

attacgtagc aaggctacca ggaacgggat gaaatagata aacgcgataa taccgacaaa 420

aacgcccatg ttcttaacct catattgttt agttgacgta gagataatag gacactatta 480

acggttatgc aagctatttt gttattctta tttggcgtct acggtcgcac cggaaaagcg 540

ggtggttccc gcctggcgca actaccaacc agtaaccgac taaccaaagg gcattactat 600

gaaacttagc gatttttact acgaagccga agccgagaaa ggcgcgcgca tgccgacgct 660

gttaccggcc tggccgccgt cgtagccgct gcagcctata aggcggctca ggaactagtc 720

gagtcacaac ggcaactgga taagatgtcg gccagcctga aaacactaac cggaagcaca 780

caaggtgcaa aacaggccct gagtatatta caggacttcg ctcgtgacac accttacggc 840

cttgaacagg cggtagaagg gttccgtaaa ctggtagctc taggcctcac cccatcagaa 900

gaagcgctac gctcttacgg caatactgca tcagcgatgg gtaaagacct taaccagatg 960

attgaggccg tcgcagatgc gagcaccttt gaattcgaac gtctgaaaga attcggcatt 1020

aaagccaagc agaaccaaag tgatatcgaa tttaccttcc aggggacgac tactgtagtt 1080

aagaaaaacg ctgccgatat tgagcaatat ctcctcaaca taggcaacgt taacttcgcc 1140

ggggcgatgg ctgaccaggc taataccctg aacggagcta tcgctagtgc ggaggactca 1200

tggtcccagt tgaagatgac tctcgctact agtctggatg tagggtcgct ggcggaacct 1260

ttacgttata ttgacgacct gatacaggag ataaacgctc aggtcgcatc cggtgagttc 1320

gtagccgaga tgcggatgtg gggtgacatg gcatccgatg ttgggggtgc gatagaggct 1380

tcattcgacg cggcgtttgg gatggttgca gacgcgttaa acgctttgaa ctccgcctgg 1440

acttacacca gtgaaagcat taccggaagc ggagaggaga ccgcatctac gatagctgag 1500

tcagcggcgg atgcgctgga cttcattgcc caggaattta cggcaatgga gcggtttttt 1560

gaagatatgg ttaaaggtgc tcaggatgca ggacgtcttg taaaggccgc attgacgccg 1620

ggagagtcgg tagccgaggc caagaacctt aacttccagt tagcgcttgc tatggatacc 1680

caaagagctg tgactgacct gacacgtaaa agtttccgtg aacaggtaga agctcaggaa 1740

gaccttatcg cattgaagcg cgcggcttac gacaccgata aagaagcggc caaggccgag 1800

ggtcttggta agtttaaagt gtcgggtaaa gacaacggtt ctacaggaga ttccgcggac 1860

aatgccgcta agaaatccgt cgacgcattc gaacgtcaga agaaagccgc tgaggatttc 1920

tattatcagt caatccacct taacgatgac gtattccaga agatacaagc taaccaagaa 1980

gagcaactta ctaagctaca ggagttctac agcaaccgtc tccttagtga ccagcaatac 2040

gaaaccgcta agacgcagat tatgctcgag gcggatacgg cccgccaggc tgaattagat 2100

aaacgcgaga aagagcgcct ggaaaaacaa ttctccgcag atgcctacgt cgctcagatg 2160

caggcccttg ctgaagggga attcgccgag ttagatcgtc agtacgaggt caagctacaa 2220

aaacttaacg actttcatgc gcagggttta atcgcggaag aaacatacca gcaggcccta 2280

aacgcaatga atgatactta cgcattagac cgagcgaagg cgacaggcac cgcatttggc 2340

aatatggcta gtaacatcgg ggccgcgcta ggagaggctt ccacggcata caaagcattt 2400

gcaattgctc aggcgaccat agctacgtac acatcagcgg tcgaagcgta taaatcaaca 2460

gcagccatac cggtagtcgg cccgtatctg gcgcctgttg ctgcggctgc tgcggtggcg 2520

gctggtttag caaacgtagg tattccttta aaagatggta cagattcagg agaatggttg 2580

aacgttgtct ccccggaggc cgatgtcgcg gttaaagcta tgcgtgcctt caccctggcg 2640

taccgagcgg cggtaggcaa attaaaaccg cttcgggata aatgcgaaga gcaaaaagac 2700

ttctcagaat acaatttaaa aatggaagac gcggcaggag acctaaaccg acaattggct 2760

ctcgaattgg tgaatggctg gagtctcgat gatgagttca ctaaggaaaa tcttaagact 2820

cttctcaccc aatataagcg cctggcggaa catgtagtcg tattccacca cgaacagttg 2880

cgtcaattgc aggaaaagta gacgcgttgt ttcagtttgc ccgttggaac ttcataaccc 2940

gccacgaaag gcgcaagttt gacagtatcg ccgatgggca caaagccgcg cttatctcta 3000

tgggggtaat aaaagacgcg ggagaaacaa cgcaggacgc cgggcctgaa tgcccccctg 3060

aactactcac cacttttgag aagtatcgtg atgttaaatt cacccgccgc gttgatgacg 3120

acggcgtaaa gctataccca agagggcaac ttagttggtc agatttagtg gcgtatagca 3180

ctatttcagg tcagaatata gggatgtttg aatctgaaat tatcatgggc ttagacgcca 3240

tttttgaggg tagaaacgat ggctgatgta gctagcttag tagtaaaggt aaccgaacaa 3300

ggcgcgaagg ccacatcaga ccgtcttgat aacctctcta aatctgcaaa agttgcgggt 3360

gcagattcgc tccgcccgag aacagggtgg taacctggcc gcagggcaga tatccactat 3420

tgcagaacgt ggtaaaccag aagtaatcat gcctgctagc gcctcccgtg tccggacggc 3480

ggagcagatg cgacagatta tgggcgagaa cgacgctaaa tccggcgggg ataatgttac 3540

tattgtgaac aataccaccg gaagaattga ttctgctgca acagaacgtg acgatgaagg 3600

acggttgcgt attataatca gtgaaaccgt aagttcagcg ttgctggata gtaacagcgc 3660

catttctaag tcacgtcgcg ctacacgcgg ccaaccagga tattgatatg agcgattacc 3720

atttcccggc ctctttgagg ccgatagtat cgaaaggcta ctcgatgacg cgaggtaaca 3780

acgtgtggcg agtaaacctg gccggtggcg gggttcgcca ggggcgtgat acatactttg 3840

atgtgttccc gattagcgtt acgctggtcg tgtcgccact gggtagacaa gcattcctca 3900

gtttcatgga gaaggtagac ggaggggctt ccagtttctg gatgaaacac gacttgggtc 3960

agggtatcga ggattaccag gttactctaa catctacgtg gaacgagtcc accgacgacg 4020

ggaagaactg ggtaataact ttcacggcca cagccgagaa atcaccattc caggaagtcg 4080

gcagcgcctg tcttaaccag aatctgcccg acttatatgg atgctacggc gattgtcttg 4140

gcgaatttct aaaaacttac ggagtgtacc aaactacatt ccctcgaatt tgggacccaa 4200

tgcaatgagt caggaatcag tagaagcggc ataccggcgt aagctggcgt ccaatcccga 4260

tggcgagatg gattttatta ccctggagat atcccaccct cttctttcta agcgctggtt 4320

gcttgtgcgc ggggttaacg acttgaccgc tactctagag actggggagg ttgttacatt 4380

cgagggtacg ccgatggagg ccaagaacgc cgccaacaat aacgacatgg accagaccgc 4440

ctctttctct ttgccggatg tgctcaacat tctcgatgaa gaaatggaca gaatccctta 4500

cgacaataag gaattaccta aattcatctt ccggcgttac gtaagcaccg acctgtcgta 4560

cccatgcgac ggcccggtgg tatatgaatt gcaaacactc acgcaagaga aaggtgtatt 4620

caccgcggaa actggtacgc cgatgcttaa ccaacgggct accggaatcc tgatgacacc 4680

agaagaaata cctttactcc gcgggatact gacatcgtga atattaatga ttacactggc 4740

ctgccatatg acttccgtcg tcgtaattgc tggcatcacg tccgcaacgt ccgcgcggac 4800

gctgggttat cgaccccgat gttcgatgta acaagcccaa cggcaataga cgccgccttc 4860

gacgatggcc actctaatcc gaaaggatta acccgcgcgt ttatccctca gaatttcgac 4920

gctgtcttgc tgggggtgaa acatcgaggg cgaatagtgt ggcacgctgg ggtatattac 4980

gaaggaatgg ttagccactg tgagctggcg tccagacagg ttagactgga tagtctggaa 5040

gaccttaaag atacttattc ggagattgaa ttttggcgct agtaatccac tatacccgta 5100

acgaagacgg cacatttgac gttaaacgtt atcgcgataa tccgatgaac ttcgtcgtga 5160

accacgttcc cgatggggtg ccggtgcgtg ttttcattga cgaaatcgga gaagataacg 5220

acgtaacaga agacttcgaa gcactgaaag aaaacgcgac tttccacatt gtggaatcag 5280

ctggtggtgg cgccattaaa ggcgtcatga agatttttag cgttatcctt aaaccgctgg 5340

cgaaactatt atcgccgtcc gtgaaaggtg cgtcctcgaa ccttgcgaac tcgcaggcgg 5400

attccccgaa caacagtctc accgaccgta acaacaaggc ccgcccatac gagcgcagct 5460

acgacatctg cggaacggtg caaactatcc caaataacct tatgtctact tataaggtgt 5520

ttaacgccgc tggtaaaatt gtagagtacg gctattacga cgccgggcgt ggttacctag 5580

acatacaccc tgaaggtata actgacgggg acacccgcgt atcagacata acaggcacgt 5640

cggttgccgt gtacgcaccc tatacatcac ccaataacac atctacaccg caggtcatgg 5700

ttggggaccc catagagcaa ggcctataca ttaccgtaga atctaacgaa gttgacggcg 5760

tggttcttaa agcaccgaac ggtctgggca tttctttctc ttacatgtcc gggtatccgt 5820

ctttgtctgg aaacattggc acgatatatg acccaacagg tggttcggat ttttctgggg 5880

tgttggtgcc taatgataca ttttcgctgg tgtcagcgtg gacaaatacg gacgtcgacc 5940

tatccggcgg cgggtatcag gtgttaagcg tatccgaagg gaccgttact aggtgtaggt 6000

ttaggcatgg atggtcgtga tggttatgtg tcgaatgtta ccgtgcagga ttgtgcaggc 6060

gctggtatgc ttgcttacac attcaaccgt accttctcta acattacggt gattgactgc 6120

aactatatga actttgattc agaccagata atcatcattg gtgactgtat tgtaaatggc 6180

atccgagccg caggcattaa gcctcagcca tccaaaggta tgatcatcag cgcacctaac 6240

tcgaccctaa gtggtgttgt aggtaacgtg ccaatagacc gtattcttgc tggtaacatt 6300

attgactctg cgttaggaca gacaaggatt aatgggttta atggtgactc ggtggaaatg 6360

ggtttaagag tccacaagct taccaaaacc ttggatagtg gtgctattcg ctctacgctg 6420

aatgggggcc ccggttctgg ttccgcgtgg acagaagtca cggctatttc tgggtcacta 6480

ccggatgccg tgtcgctaaa aattaaccgc ggcgactatc acgcggtaga aataccagta 6540

gccgttaccg tgctacccga tgctgccgtt atagataatg gcagcatcgc actataccta 6600

gaaggcgata gtcttaaagc gttagttaag cgcgcaaatg gtaaccttta tagccccgca 6660

agcccataca caacagtttc ttcaacgcat gctgtagtgc cgggtcgcgg atatcaaatg 6720

ccctcagaac gtcgtacaca tcacaggtaa tgctatctcc gtcagtattt gtcattgtgc 6780

gattgtattt gttagtcatc tttatgccct atttatatgc ccggtggcct cattggtagg 6840

tggcaagaaa taagacccgg ttcatttttt cgaggtgacg gagaggcgtc gctgcaaccg 6900

gataacacat acgagaaaac cttaaccgat tgggtgtcaa taaaccgtac cgaggttgag 6960

cgcatagttg ccaatatcgc cgccgccaat ggtatgtata aagacaacgg caaatctaaa 7020

acactagcgt cggttaccgc ggagatacag taccagctgc ttgatgaaaa tagcacccct 7080

tatgggccga tatacactgc acaaggaacc gtgtccgggc gaaccccaga ctacaacggt 7140

gtcactattt acgccgacct gccggttgtg tcgcgggtgc gggtgcgcgc cagaagggtg 7200

acagacctcg actttaattt cgaggggtct gtggtcgatg aagtaacgta cgttaacttg 7260

tacgggcaaa cacgcgataa cactccacac tacggcaaca ggactaccgt acactcaatg 7320

cgcaagcaga caccacgcgc cgcggaagtt aagcaacctc agttgcgcat gattgcgacc 7380

gaaatggtat acaaatacct cggtaatggt gtttttgaag acacgatgac ccccaatacg 7440

caagccgtgc aatctcttat ccgcctggcg cgtgatccgg atgtgggggg tttaaacctg 7500

acggtacgca acatggataa gttacttgct gtgcagaacg aggtcgaagc gtattttggc 7560

gacaaacagg ctggagaatt ttgttacacg tttgatgact ataaaaccac catgcaggac 7620

atagttagta ctatagcaga cgccatattc tgcaccccat atcggcgtgg ggcggatatc 7680

cttctcgatt ttgagcgccc tcgcatgggc cccgagatgg tgttcaccca ccgaagcaag 7740

gccggtactt ccgaaaaatg gacaaggacg tttaacgata ctcaggtgtt cgatagtctt 7800

aaattctcgt acatagaccc taaaacaaac gttaaagaaa ccataacaat acctgaaacc 7860

gggggcctta aaacggagac ttatgactca aaaggaatcc gcaactataa gcaggctttc 7920

tgggcggcaa accgtcgtca tcagaagaac attttaaaga aaatttcggt gtcgtttacc 7980

gccactgaag agggtatttt tgcccttccg aatcgtgccg ttagtgtggt taagggttca 8040

cgtatggcga cctacgacgg ctacgtaacc gcggtaaacg ggctcaccgt agaactgtca 8100

caaccagtta agttcacctc gggagatgac cattctttga ttctcaagtt acgtgacggc 8160

ggcgtgcaaa gtgttaatgt agtccccgga gcgcacgacc ggcaggtaat catgacgtca 8220

ataccgcaag aagcaattta cacaggtaat agtgctttga aaactgaatt ttcattcggc 8280

aacgaagcaa ggcataatgc tcagatgatt cttgtttcta cggtagaccc tggcgatgac 8340

cgaacagtca aaataaccgg gtttaactat gacaaggatt tctataagtt tgacaacgta 8400

ccgcctttcg gtcgtgcgtt ctctaacgga ttcgataacg gttttagcta cacaagatta 8460

actttggcat aaaaagtaaa ggccccgaaa ggggcctttt tcatactgcc agataaagtg 8520

catacgcagt gacaaacccg gcaacaacaa gtcctgcgat gaaacatttt gcgccaattg 8580

agtatttcac ttaactctcc tgtatttttg cggccaagcc attacttact tccggttgct 8640

caagtagcca gtctaattgc gcgttcgccg cgtctcgctg ctgccgtagt cgtagaactt 8700

cctcttctag ttccgcgata cgcgctaaca acggcagccg cacttcttct atagcttttt 8760

cgatgctact catttattac ctcgtctctt catataatta agcaactctt cctggacaga 8820

ttttttctcg tccgtacgcg cggcaacgac ctcatccagc gtgtctttag cgactatgtg 8880

atacaggaac actggacgct cgtggcccgc ctgtttctgg cgtacaggac ctatacgctc 8940

gacaacctgc aaatagtgct ccaggttcca gccttgcgaa ataaacgcca gatgatgccc 9000

gccgtcctgt aaattcaaac catggcccgc cgatgcaggg tgcacgcaca aaatctcgat 9060

ttccccgcgg ttccaagatt ccatctgctt attaccctta gcacctttcg caaacgcctg 9120

cgcctggggg aatcgcctaa gaatgcgctc aagttcgtgc ttgaactgat aggccaccag 9180

cagcggcgca ccctgcaact cctcgacaat ggactctaac gcatcgagtt tcgcgtcgtg 9240

cactttctcc cagtctttcg ttgcttcacc atccggcccc gacacataca cggcaccgga 9300

agcaatctgc aagcacttag ccgttttagc ggcggcgtta gccgcttcaa cttctccgct 9360

ctccagttcc gcgaataact tctcctccat atcgatgtag gcttgacgcg ctttcttcgg 9420

caggtcaatc tcaaccggta caataaccgg tgcttcacaa ccgaaccact cggcggcatc 9480

aatggtaagg ctgatatcct tcatcttctg gtgaatctcg ttatccgcgc ctgggcgagc 9540

atggtactcc cgtgccatcg cagatttgcc tttctgtacc gagttaaacc atctatcggt 9600

aaaggccgtg tatgaagacc caaggcgttc acccgcgtcg atgaaccagt tctgacccca 9660

caagtctttg aggccgtttg gcgacggtgt cccggtaagg ttaatgaaac gcttaacctt 9720

accgaacgcc actttgctga gggccttcgc cctcttgctc ccgccagaac ggctacgaaa 9780

cgatttcagc ttcgtgcttt catcggcaac gataaccgta aaaggccagt cgtctttgcc 9840

gtagtaatca ataagccact cgataacttc gtagtttgta cacaccacgt tagcgtccga 9900

ttccagcgcc gcgatgcggc gcttctccga accagttgca tcgacgacac gcagacacgg 9960

gaaccgccac ttcgtttgtt ccgccggcca tgtgccggac gcaacgcgca acggggcgag 10020

gattaacaca cggtcgtcgt ccgtaagttg cccgttgcgt aacaggcggt ttaaagccca 10080

cagtacgctg ccagtcttcc cggcgcccat gcttgcccat atgttgcatc gatggtgtcg 10140

cagcatgaac gaggtcatga gcttctggta ctcgcgtctt tgaaacttac acatgattag 10200

cctcgtaaac tgcttttgcg aaacctcgcg gagtaaggct gcggattgtt tttgtccgca 10260

ctgatttacc gccaagtttg ttgtgctggt cggagtaccc atcaacaacg gcaacacatg 10320

atttaaccgg cataacaaaa ccgttacccg cccaaataca tgtcttctta gggtacgcgt 10380

cccgtggttt tatgtactct gggaacattg gatgtgtgtc gttttccggt agataaccgc 10440

cgtagtcgta aggatcgaac acgtaatcgg gtttacgcca taaagacgaa agaacgctaa 10500

cagggttctc aatcatatac ggtacgttgt actttttagc cagacgcgct gccactttgc 10560

atgtaagcac agcctctacc tgaaacgttg ggttcttctt gcgcttcgtt tcgaaatgcg 10620

ccgcgccgct aactgctaaa tctgtgcaag gagggaacgc aaaaataata tccggcgcaa 10680

cctcgaagtc aaacttagta tcaatccacg cgtttatata gcgtatgttc tcgtgttcaa 10740

cttttgcacc caatcgtgcg tagtcgccgt ggtcagcgcc atcgtagttg aaacaatagc 10800

aagtatggcc cgcttctgcc cacggttggg ccataaggcc cgaaccgtca aacaatgacc 10860

agacaatcat tttgccacca gaactaactc tttacgcccg aacgccgtaa cgttaccggt 10920

cacatcttcg ataaccagtt tgccattcga ctcgacgaac acagtatcaa cggcaacagg 10980

ctggcgtgtc ttaacgttga aaatcatgtc acccggtacg atgtcacgtg ctggtttgcg 11040

gtcatattca tatttcattt ctcaattcct tatttctgtt ttgtgtgaac taaatataat 11100

agtgttctat taatttatca acctgtttct tagacccaac gacaaaaaca ttcgcaccgc 11160

gtttgcgcat ccgctcgtgc tctcgtaact ggtgcgggtc tggcttcgtg ttttcgtctt 11220

tcttcacctc gacgaaccag acgatgccgc cagggagaat tatcaacagg tcgggagcgc 11280

ccactctatt ttcgtacgac aatttgcgta cgaggccccc cagggcctcg aatcgctctt 11340

ttgcgtattt ttggacgcgc ccctctttag tcgccatggt tacaaaactc cccgtgaagc 11400

ttttccctaa attctctcag cgatacctcc gcgtcattta tgttctggaa atacccaatc 11460

tggtagcgtg tacctttaaa agtaactcga gcacaccaag cactatggtg tttactccag 11520

ttaacccctt tcaccccgct ggtgttcgta actggggtct tgcggttatg gttctgttga 11580

aattcatccg caagccgtag gttacaaatc cggttatcgt cccgtatgcc gttaatatgg 11640

tctatttgct cagttggcat cactccgtac acccaccacc acgccagcct gtgtgcccgg 11700

taatacaccc tattccatcc taacaatatg taccctttac ggtccttgta ggtaacaaca 11760

tctccgggtt tggcggtctg gcatctgtgc ttgacccatt taaatatccc cgtatccggg 11820

tcgtatgaca gaaaatcttt tatatcggcg cttggggctt tcataataca cacccctcac 11880

gtttcgtgtg ttcaatcccg cagcgcgggc agattcggca gtcttcttca cggaaataat 11940

agataatcat tcaagcaccc acagataaat tgcgactaac atacacaata cggcggccat 12000

catgccgtat tggccctcgt ggtagtaaac accggcggcg aatcccgcca gtaccgcgat 12060

aatcggttta cttagcatac cgttttaact ccgcaccttc cgctacaaga ggaaaaccat 12120

cagcccactc cggcaatgca cacattagtt tttccagttc agctaccgtg taatctgatg 12180

tatccggagt ctcgcatacc agttcatcgt gaacagaaag aactattgga tacccaccgg 12240

cttccacatt aagcatcgca taggccaata agtcacggca caacgcctga acaatgtttt 12300

cacaagcctt tccgccgtgt gtgtacaggg tggtccattg ccgtgttaac tggttctcac 12360

cctggtactt aattcttaca ttggtgttta tccatccatc ttcgtctgtt tcctttatca 12420

cactaacgcc gattcctgga tacgagagga tacgtcctga cggcaactcc atacacaacc 12480

accagccagg aaccttcctg cccgacgaat caaattctac ggtacgccat atgcggatag 12540

ccctttcacc attccggcgc aagtgtgccc cagcccaaaa ttcacgacca ggattacgaa 12600

cagcagctaa aattccgtct ttaaggtcgc gccagaacgc tactgtttca gggtgtgact 12660

cacgccacat acgcttgatc gcgtcacatg tgcgccacac cttcttgtcc agaatatacg 12720

acggcctgtc atccttttca ccgggatgcg gtggtctctt ggcttcctga atacgtaccc 12780

actcataccc acgtgcagta gcagcccaga tgtggtcggg gaaagtcccg tccattgttt 12840

ttgccatctc aataaggtcg agacctaagt ttttagcgaa cgtaacgaac gctccgacac 12900

ctccctcgta gccgaggccc agttcgcaag ccttgcctat ctgtctgatt tccttgaagt 12960

ttttcttaat atcgtcaggg tccatgccaa acatcttacc cgccgttacg cagtaaatat 13020

ccagcccggc gcggaacgta tcaagcgcgg tttcttcgcc cgccagccac gctaaaccac 13080

gaccttctac gttagagtaa tcggcaacta caaacttatg tcctgcttcc gggatgatgc 13140

agctacgcac cgtcgatgcc gttagtttag ctacatcgaa gcggcgatgc gcccggcctt 13200

taagtaacgc ggcaataccc ctatccagtt catcgtcgtg ataatacccg cgcgccaggt 13260

tttgcggctg aaaacctttc cccgcccacc gcaatgttcg cttcgctcct ccgtattgca 13320

ggcaaccacg gcggcggtca tctgaagaac ggcctaacag caacggcgcg tatttcgtcg 13380

atgcggtaga agcagccccg agacgcattt cgataatagt gcgggcgtcg tccggtaaat 13440

cctcatccgc cagcaaatcg tttagtgttg acttctgtgc gttgtgaatg cggtgcgccg 13500

gcgccagttc ctgtaaaata ggcaagaagt ctttgccggt tagtgaaccg ccgtatttgc 13560

gttgggcttc ttcctgtagc tcttccttgt ggcgcgtcac tgcttcaatc gcggcttccg 13620

ccagcgcaac gtcaacctta aatccgcggt cgttaattac ttggtctaac cacagcacgc 13680

ggtcttcgaa ctcagaattc ccccacttcg gcattttctt atacacttca cgcatagacg 13740

taatgtcgct ttttgcgtat acgataaatt ctgcccactc ttttgggtgg gtatcggcgg 13800

tatatcgtcg aatcttatag ttcttaggcg taggcttact gaatcgctgt atcagcgcct 13860

taccgcgttt atccttagcc atgcttgcgt caatgtttaa aacctcacac agcgcggcca 13920

gtgaacccgg gagagcgtgc cggaatgcaa ctatcatagt gtcaataatg ttttctacca 13980

ggagttcgaa tccccagcaa tgcttaataa ctggtcggtc gaacataagg aagttttggc 14040

ctaccagttt gaggtcgctg tctggttttt gcagctttag catcgcccga cgcaaatcac 14100

gcggcatatc gctgccgtct gtggcgtccc atacttgcac aggaccttca tcgaaagcgt 14160

aggtgcagat aacaatttcg gtagtcggat gttcggcgta ggcataggaa ccgacttttt 14220

tcaaatcggc ttctgaaaat gtttcagtat cgagataaag cagattcatt catttgaccc 14280

ttatagaaaa ggcgaccgaa gccgccttaa atgaaatgag aagatattaa cggttacggc 14340

ggcgttcgcg ggtttgtact ttacgaagat taagtttgat tcccatttta gttttctcgc 14400

attttagagt ttatccggga aactgcccgg tcagtgatta agactataat agagtactat 14460

taacttgtca agcttctaaa tcgtcttccg taacattttt ccactctggc cgtttgtcgt 14520

ctgctgttgc tacgcatggc gcgcccggct tacggattac gagtttagct agctcggccc 14580

aaagttctgg gtcttccttg ccgatggtct tttccgcttc ggtcggcgat acaggaacag 14640

tcttatccag cagatagccg tttacatggt tttccctaaa tttatttaca gcatcttcat 14700

ctttccacat gcgattacct ggacgaccct caaccaactt gtaccccggc acttttttac 14760

cggaatgaag cgcggccgcc atcgcttttt ctactttgtc gatgtgctga cgcaaaaacg 14820

gcaacttctc atactcagcc actagttgtt tagtggtaag ttctaacgca aagtcatcct 14880

ctctaactgg cttcttgggt aaatctgtct cgtcgggcag caacttaagg atatcgctcg 14940

gttgaacatc ctcccaaacg ctcatatagt ccgcctcgtt gcagaactta gccacccgtt 15000

ctacggcccg ccggtataca tctgcatcgt tctccagttc tttggccaga acggcattgg 15060

atgctctagt ccttgcctgg cactgttccg agaaccgaca ccactgacaa ccatcgacag 15120

acggcttgaa gtccgacgct ttcagattct tcttgcctcg gaaatatgca tcaagagcca 15180

acagtgcgcg tttctgcgcg aacttagcga acagttccag accttcaacc gagatgtccc 15240

actccgacgc accgccagcg tacggctgga agatgaccag acgaacaacg gttatgttat 15300

agcgtctctt gagtctgcga taaacaccga gagcgtaaag cataagctgt ttgttttctt 15360

tcgcttcaac acgatgccgt cctgttttca ggtcgccgat aatgagcatg tgctcgtctg 15420

tgttcgccat ctcctggata gcaacaaggt cggcggtccc gaacgtctca acgccttcgt 15480

aacccggatg caacacctca gtaagattga cgcgcatttc aagtttagcg taagttgcta 15540

cgtcgattat agctttgcag tagtcagtgt actttcgcac ttgctcaacc atatcctccg 15600

taataagtac cgcgcctttc attggcttaa ttaatgcttt aactgcgcct ttaccttctt 15660

ccagcacata tgcccctaca tctcgctcta acggcaacgc agtgccgcgt atatacgcgt 15720

ttaaatgata ttcagaaatg ctatgcattg ccgtacctaa cactgcagcc ttgccggaag 15780

tgttagggat atctttttcg catatcagcg acgcagggca tgttaaccat ttcttagcgc 15840

ctgatggtga cagtaaggcg tgtacatcat tattaccgcc gcgttctttt agaatcatta 15900

ttcatcctcc tgaaaggcgg cccgaaggcc gctggatatt tattcttctt cgaaatattt 15960

gttcttgatt gcggtcaggc gttccaggta ttcggccagg tcttcatctt taatcgcggc 16020

aatcttcatc ttcttgccag tgaactcttc cagcagttca tctgaatcgt cgcacgcggc 16080

atcgctcgga ccttcgttaa tcgcatcgtc gatagcctga atctggtcgc gaagagactg 16140

gtaacctact tcttccttct cttcttcagg ttccggtaca ggttcctcta cttttgattt 16200

acgcgtctta cgtttcggtt tctcttcttc tgtgggttta gtgtcgacga cgtcttcacc 16260

ttcgacaggg agttcttttt ctgacgcctt gcatactgct gaactagccc gatactcgtc 16320

cggcgccgcc atttctaccg atttttctat caactctttg tttactgcgg tgttatcact 16380

tttaagttgg tacacagtct gtttcgcact gttcgcagca atcagttcgt gagcaactac 16440

gaaacgttca aataatttaa taaattgttc taacattgtg cattctcctg tttagttggt 16500

gataagaact ataatcgtac tctattattt atgcaagcac tttttcgtaa attgcgtcgt 16560

gtattttatt actgtactat tactacatat ctaactaagg agtaaacata tgcagcaatc 16620

cgaattaggc gctcgcgtag aacgtcgtcg taaagaaatc ggcatgggcc agaccgagct 16680

tgccttcaaa gctggtgtat cccaaagcct tattacccat ctggcaactg gtcgggtgtg 16740

taaggtagac tgttttaaaa tcttccacat tgcggatgct ctcggcgtcg accctcgatg 16800

gttaagcttt ggtgataagg gggcctgatg gcccctttct ttttactcta aatccccttc 16860

tgttgtcttt atgttgtcgt taggttcgta cctgttcttc ggccgctttc gatcatctat 16920

tccagtcggc aacctgtaag tattcgttac aacttcaccg ttttcgtcgc aaccaagcat 16980

caattcgcct tcacgtagca tcttctcaag taccaggttt gttatactgt ggtctccagc 17040

tcttgcaact atttgccgtt gtgtgaagcc ccgtcctgta tcatcggctt gctgtaaatc 17100

ctcgagagcg gacataaccg actcacgggc cgaagcgtct ttagaccgtt taaccgtatc 17160

ctttacggaa tctttacctt taccatccaa tccttcgttt cgctcttttt cctcgtccgt 17220

ttcgaacggc tggaagcccc acggcataag tacgagagct ttatgtggct ccggaaggtc 17280

gaggttgact atctccccgt atccctcacc cccagcgaat tctacagcct ggaactcttt 17340

cggcggtgga gcttcacgaa actgtaccgg ttcaagcacc atgcctattg ttttctgctg 17400

catgccgttt ttgttcttgg tgtgcgcaac gtttatttgt ttctcagtgg cccggacaag 17460

cgtaagttcg acgtcgacac cggcatatag cgccccactc ccgcgcgcct tactcccacc 17520

tttaggagta tgatgaacga cacccacagc gcctttagta ccgtcgcgta cttctttaag 17580

catagccacg acacgaccca taccgtcctg cccggcggag ttttcattaa acttgtctat 17640

ccagttaccg aaggtctggt taagggtgtc gaatgcaacc atcccgacgg gttcactacc 17700

ggctatctgg cgtattttac gcaccagttt cttcgtatct gcgaattccc cggcgtcaag 17760

cacatgcaca taccgcatac cttcatcgcc gtatttagcg gccaacgcag cgatacgggt 17820

gtgcgtaaac tccccaccct caccatcaat atagaaatga tgggctttac gggtgtccgc 17880

ccccgcgaac cgatatccgg cggcgctgat atacatcatg ccgagtgtgt aaaaagactt 17940

atacgtcccg gattcaccga taatatccca gatgcaattg gacggcatgt acccttctac 18000

gacgaaatcg gcctgcggcg ccggaggttc ttcgtcgtcc aggtcttcct cgtcgcaagt 18060

aacgcccggc gcccagccaa gagctacctc tacgcggtca aacggcaaac cggtagcggc 18120

gcaagcgtaa gcccacatat cccgtccgcc aggggtcatc cagcccgtgg ctaccaggtc 18180

ggtgtcgtga tacatggtta cgttggggcg ctcgaagcca tcgcggggcc aacagaaaag 18240

gaagtcgtcc tgtttcggtt cccccgtcga gtattgggcg gcatgttctg gcgtcgccgg 18300

catttgcagg ccacggtcag tcatacggcc gccgaactcg aaagcgaact cttcaaacag 18360

gtcagttaac tcagattgct cgccctccgg cactttataa tcagaagcac cggtaacgtt 18420

aatttctggc accccctcca taagcttgct tgctgtaatc atgcggctac tttcagacac 18480

gatgacctgg cttcccactg gcgggcggta catcggctgg gacagggtga agcccgcgct 18540

atctacgtca cggcctttaa ggaaatgcgt aagcagaccg taccgaatgc gaatgatgtc 18600

tccgcctgtt acaggggtac gtaccggcat aacgacgcga tagcgcggcg cttcttcggt 18660

gtgggacgcc gtggtgtaga gcatcattgc gaaacgcgac tcccgcacca tctcgcagtc 18720

agttgcaaac tcttccgtgg ttgcactgtc cacatcggcg taagccaggg acgaagacgt 18780

tacggacgca ttgcagcgat aaaacatacc ttcggcggct tctttacctg tagagcttac 18840

cgtggcggta cacgcggcgg tgatatatcc ggggtctgtc ttggggttcc ggcgcgaacg 18900

tttaagcggc tgcataagtt cgatgaactc atcccaggtg ccggaagtcg tggtgtaaac 18960

gttaatgtca gcgcgttcct cacggcgatt gctacgcgac catgagtatg ctagattacc 19020

tgttgacatg tgcttttcct ttgtagtgtt taaggccctg acgttcgcgc gttggggctt 19080

ttcttttatt caaggtcttc tggtgtcgcc tgatagcctt taacttcagc tactgccgaa 19140

tattttccgg ccttactttt aactgctccg tctcgaacca aaacctccag cgacctctca 19200

atgagagagg aactatagta ctgaaaataa gtccggcgca agtcaaggac actacacgac 19260

ccctttttaa atgtaagagc agctactgcc ctaaataccc ttttctggaa atcagtcact 19320

ttacattctc cctaatccac gcttccactt tctccctgtc gaatgttccc ggcatccggc 19380

ggcccataat ccggacacaa caatccggga acttgccgtc tcttagccag ttattcagcg 19440

tgcggcgggt caccccaata agcttagcta cttcattctg tgtcattatc aagccctcca 19500

tctcagttag taaagcaagt atacccacgg cataatggga aatcaagtat aaacgcacct 19560

attgacattc ttattttttt cgtgtaagtt gtctcttgta acttcaagtt acttaagttt 19620

ctgtttcact ttgtgagagc ttagcggttc cgccctaagc ggaaacgcgt taaggcttac 19680

tgaaacctgc aaacttaaaa ctcagtaact tccttcctta gtatctgaac tgccttgggc 19740

gagctatatt ctcatcggca gcatatctct taagtatctg atttccttga tgtgccgtag 19800

attcgcaacc gtaaaatgta acgattagag ggtaggctta aggcctgccc tttagttacc 19860

aggtaataag aataaaggca caaccctggt tgaagaacca caacggcaac gtaactaaaa 19920

tgccgaaaga ttcttagtct actactcagg gattatcgca cttgccttac ggcaagcgct 19980

tacctgacga gggacgggag acaagaatag aaaggcactt gcagaaccta atataataga 20040

gtactactat tacaggttaa cacgacaaga ggatgaacca tgtttaagaa gggtaggtta 20100

gtccgtagca ttgtcgccag gaggtgttac gaagtgttgc accggggtga tgtgagttta 20160

tcggtgaaag atgttagccc caatacgaac ggcaaaagat tctaaagatt ctaaagattc 20220

taaagagtct gagtcgtcgg gtcgtgagtc ttaaaggggg ttgccgttta ttttaattgt 20280

cggtgcgtgc gttgtggtgg ttggtatgag agaagcattc gaacgttggg ccgtagtcga 20340

gggcctgccg gttaacaagg gtctgaagaa agattacccg gtgtatgaga cgttaaataa 20400

cgcagttcag cacgattatt acatagttcc tgatgacggg agaaagcctt tctggaatca 20460

ctacgataag tacgtgactc actggatgcc gttaccagag ccgccggaag tgtaacaact 20520

atttaggatt ttaccgagtg acttacgctg ctgagtagca gcgtaccatt tttgtcaata 20580

tttttctatt tcactagacg caacaatcga agcgactgtt gcaggagatg caacaatgaa 20640

accgaacgac cttgtaacct gggccgggcg taacggagag acccgacacg gcaaaataac 20700

gtcgcttcac ggcatctatg ctcacgtcga gtggtggagg gcctgtgcga agaagcctcg 20760

ttacattctg atgcgccagg gtaaattaat cgtaaaatag tattgacgta accaaatata 20820

ataatgtact attacttcat cgaaacgaga ggggttaaga gatgggcagc aaagacgaat 20880

tattcgagta cctgattgac caactacggc agcaggtaaa cagcaacccg tataagcaac 20940

agtgcgaaga cctggcccat gaggtgctgt cacttaagaa tcagttacgc gatgcgtcgg 21000

cacagattaa ggagttgcat ctggcgttgg tcaaagctac aggtgacgtt gaggcataca 21060

aacttatccg caaaggtggg tacgtgccag agcaatccgg ttgccgtaag gaagacgtgc 21120

agggatgtga acatgagtgg gtagagtttc atggtttgtt tatcgctgag ggggacccgc 21180

gaaatgaacc agggtatacc ttctgccgta aatgtggact taaaggatac tcccgcaaat 21240

gtgagcatga gtggagttac gtcatgggcg acgggcctgg cttttatgaa tgtgacgttt 21300

gcggtgcgcg gagcgaaacg ttaccttatg atttacggtg gttagcataa tggcagactg 21360

tttttgcaca cggtgcgcct tccagcacaa tgcccgtacg gaaggattcg atgcggctta 21420

cgccaaactt cgcaagagaa tgggtaatga gtgtgagcat gagtggagat gtaagacggt 21480

ttttggcaat agacctggtg tcccgtattg ccgtaaatgc ggtgtggagg cgaagcaatg 21540

accagcatct tatttatctg ggtactgtcc gcaggccaga tgcaactcac ggcatcggag 21600

acgttcaaca cattagaagc ttgtcagtca gcggcacggg cggttgagaa cgctcacttt 21660

ctgttccagg gcgacaggcc aattgattca ggagtacgcg ctatctgctc gcctaagcga 21720

cttggtaaac aggagaagtg attatgagtc tcgccgctga tatactgaaa cacgccggca 21780

tcaacctggc acctccttca tcggcggtaa caaagaaggt tacgcgtgtg aaggagaaga 21840

ttaaacgtaa accaaagccg ctggccaaac cgcataacga gatgccggat gtgtatccgc 21900

ggatacctgg cgtgcaccaa ccgaagtatt gcgtaggtaa aggtctatgg cgggcgcact 21960

cctacgacgg taagaaagtg gtaaaccttg gggagttcag cagtcaggcg cgggcgcata 22020

tggcggttaa actgtataag ctgtggcgca agcgtgggta ttccgatacc cctcacaagc 22080

catcaatcag gctttatacg ttcagataaa tccgaattat ctgttactaa aattgtcgta 22140

taaatgttaa tctccgaagg taaactaact ttcggagatt ttttattgtg gacgataaat 22200

acctttggct tagtgtggcg ggcctcgccg ggggcgccgt atcccagatt aagaagcgtg 22260

aggctatttc gccttggttg cgattgtgcc acctggttgc ctcagcatgt tgtgcggtgt 22320

acgcatcccc cataattata agttactatg aattatcaca gtctgagggt cagtacctgg 22380

ttcctttcgg cgtaggtatg ttctggctta agttattcga agctgctgac tcatccctca 22440

gcaactttaa gttaccgtgg gggaaataac atgctcaact caccaatcgg catcatttgc 22500

ctggttgtaa tcatcatcac ctcgttaatt aatatctacg ccgactggat tgaagacggt 22560

cttttcggcc gcctgctgta catggcgtcg gtggttaccg ccgccggagg tctcgcccat 22620

ctctttaccg gtggcacccc gccgttcata attacgactc tcgtcgccat attcgcgctc 22680

aagtcaattc gacacatttg cgtgaaaggc gcccgctaac gcggtgtttc gtcttcatca 22740

tcttccaggt cgtcgacgct ggcagaaacc gcagaaccac cgaatgcctt tccttcgcca 22800

gcaaatttaa tgccgtttag ttttgcacct aaaaccttat atttctcggc gaaccatatt 22860

tcgattgaaa tattagctac gcagccgcta tatacctgct cgccttcaat ctgttcacca 22920

ttaatgttga agtctggttc tacctgcttt tcacccttaa ccgaagtcag tattaatggt 22980

tgttgcttgt tcttggcctg gaagtaaaaa ccttccggga agtcttcgaa cgggttatct 23040

cgttcggcga tatcgcgaac ggcacacttg tccatatgct tcccttcacc gtagttttgc 23100

ttcatccatt tctcggcggc ggcctctccc aatgcttccg acactacggc gaatacggta 23160

tcgcggagtt cttcaatttg cgggtgttcc ggaggtagaa taaaagtacc gttatactgg 23220

cctttggtaa tggaaccgtc atcattttca cggtctttag cacgctcgaa tacgttcagc 23280

catgcctaca aataccggag gatgtatgtc aaaccgaaac atcagtgata acggattaca 23340

cttcaccgcc gcgttcgagt ggttccgggg ataacgttaa aggtgggcgg gaaagtaggc 23400

aatacgccat tcccaacacg cgaggagctg ttaaaacgta acagtttccc cgggccggac 23460

cagaacaagt atctaaatcg catgtggggt aagaagaaat gactaacaac gaatatgaaa 23520

agatgatggt agagtccgcc aacggccgtt ttaagattga agctgtagat gcgcaaatta 23580

aggcgcacca ggccgcgtta gactacctta atgagcatcg gcgggaactg attaccagta 23640

tccccaagct aatcgagacc cacggcacat tagcggatac gtgccgtgag acgggactta 23700

atgagatgac attatccaag taccgccatg acacaaaatg cgagcaacat gttatctata 23760

acaaccgact gatgacccat acgaaaacgt caccggtcat ctacacgaaa cgcggagtat 23820

ctcgcaacga ccgcatgaag ctggaggggt gatggatagt tttctcatcc tcatgggtat 23880

aggattcaca acattactgc ttgccgttgt tatatacctg atgtggattc tgtttctttg 23940

gccatttgtt gaggcggtaa gtcttacaag gatgatgaga gagtattggc gcattaatgg 24000

gtgcaagcga ggtgcctggc gtacgatacg tttattcttt acgtggtatg ccgaaacagt 24060

atttggtcgt aggttctacg ctatccatag tcgcgggtgg cgttgggagg ctgttggtaa 24120

gtggtgggtt tataaagacg agcatgatta aagaggagag tggagaatga ttgagatttt 24180

gaaggctatc gatgccgctg gcgacctggt gttttggttg gcctttctcg gtgtgctgat 24240

aaaacaaccg aatgtgttta acagtcttcc tcgatatctg tggtggccta tgtttttagg 24300

catgggtgta tcgggggcgg cgggcatcct gctgtggttt ttcaaatgag attactaatc 24360

acccctaaca catgggctat cgctgtaagt aactgccatt acggcggtga tggcaaaaga 24420

gcaccacgac acggcctata taactaataa aagccctcta cggagggctt ttctgtacac 24480

aggccttcat tcccctgtac aatccttaat agacgctcag ggggtgtctg atgcgctatg 24540

atggtcatac gcatttaaga gaggattgca tgaaactcaa attaaaacag cctgacgccg 24600

aagtagtggc tgccgcgcac gaagaagcgg ttaacgcaaa ccgtcgccgt aaacgtccgc 24660

gtggtaaaca aagcctgtat cagtcatcca gacattccgc cgaactatgg gacccggact 24720

attgcgacga attaatccgg ttcttcgacc gcacgtcatg ggaactcgta cccacgtcta 24780

aaggcgacga acgcccgctt atccaggaca aaccgccgtc actggcccgc ttcgcattac 24840

acatcggtgt aaccatcccg attatcaagc tgtggctccg agaggttccc gcattcgccg 24900

aagcatatga gacggcacaa gccctggaag aggcgtattt cacggagacc ggtgccgccg 24960

ggatatctgc tacgttcgcc gcggcgaaac tggggcttaa taaaactgtt gtggaagaag 25020

cgcgcgacga accaattagc gaagtaacta ttaaggtggt gtccggtgaa cgttgatatc 25080

acagctacgg aaccacaggg cgcgttcctt aatctgcatt gtaagttccc ggccttcgtt 25140

gcaggcttcg gcacaggtaa atcagaggtc atgtgcaatt ccgccctact cgacagcatg 25200

gaaggtggta gtgattcact tatcgccatg tatgaaccga catacgacct ggtgcggctg 25260

gtggatatgt tcgacacccc ggatgtggtc agggagatta ccgaacgatg gaagcgtaac 25320

gggcatcaca ttgttatgta tccggacgcc agtggtaaga accgtaaatc gactgatgca 25380

agtacctcgg atattgccca attacagaac gcgggtttcg agatacgtgc gaaatcagtt 25440

aaccctgcgg ttaaagaccg tgtagcatcg gtgaataaag cgctggagtc tggtagatta 25500

atggtcaacg agcaggcttg cccagttaca gcacgttgcc tggagcaaca ggcttacgat 25560

aaaaacggta taccggataa gacaagcggc aacgaccacc agaacgacgc gacaggatac 25620

cctatcacct acgaaatgcc gttggttaaa cctgtttccc atatcccggt tacttttgca 25680

ctttaagagg attattaaat gttaacagca aacggccagg gttctggcgt aaaaaccaag 25740

caccgcgaat ggctgcacta cgcaccgaaa tggcagaagg tgcgccatgc gcttgcgggg 25800

gacctggttg gctacttacg caacgtcggc attaatgaac cagataaagc atacggcgaa 25860

gcgcgtcagg cagaatacga ggcgggcggc atcgtataca acttcacccg gcgaacgctg 25920

tcgggaatgg tcggtagcgt catgcgcaaa gaaccagaaa tcaatattcc gaaggagctg 25980

gaatacctgc ttaaaaatgc agatgggtct ggtgtaggcc taatacagca cgcgcaagat 26040

acactcatgg agattgactc ggtaggccga ggcggccttc tcgttgatgc tccggaaaca 26100

gccgcggcca ccgctgccga acaaaatgcg ggcttgctta accctacaat cgctttctac 26160

actaccgaga atatcattaa ctggcgactc acgcgcgtag gttctgtaaa ccgggtaact 26220

atggttgtgc tgcgcgagac atgggagtac aacgaaccgg gaaacgagtt cgaaactaaa 26280

tacggcgagc agtaccgcgt gctggacatt gacactgatg gtaattatcg tcagcgactg 26340

ttccgtttcg atgcggaagg cggagcgcag gaagaggttg tggagattta cccagattta 26400

ggggattggc cgagcttaat atcgggcact accggaacag tgctgataac gaggaatcaa 26460

gttttgtagt tggtcaacct acactgttta tctaccccgg cgaaaactta accccgcaat 26520

ccttcaagga agccaacccc aatggcatca aatttggcag tcggtgtggg cataaccttg 26580

gttatggcgg tagcgctcaa cttattcagg caggcgaaaa caacctggcc cgccagaata 26640

tgctggacaa agaacagcag gctatccaga ttggtgccca acttattaca ccatctcagc 26700

aaattaccgc agaatccgcg cgcatccaac gcggcgccga tacatccgtt atggccacaa 26760

tcgctcgtaa cgtaagtcag gcgtataccg atgctttacg atgggttgct atgatgttgg 26820

gtaagccaga agatactgaa gtcgagttcc agcttaacat ggatttcttc ctgcaaccta 26880

tgaccgcaca ggacagggca gcatgggtgg cagacattaa tgccggatta ctgcctgcca 26940

ctgcttatta cgctgcgttg cgtaaggcgg gggtgactga ctggacagac gctgatatta 27000

aggatgcggt agccgaccag ccattaccgg tcgctactga agttcagggg gaaatccctc 27060

aatcggcgca acaacaagag cagtaaacag aaaggccccg ttatggggcc ttagttttaa 27120

cgtatattag ccgcgcattt acgcacaacc cgaggtatcg tctctttcag ttcaccccat 27180

gtattaatgg cctccatgtc aagaagcgtc accagggctt tctggatgtt tttaacggaa 27240

caatacctgt ctcctcctca cacacacgca taaagttttt gcagtcatcg gtttttagta 27300

tctgcgcaag atttcttggt tggagtccgt tatcgaacct ccatctgttc atctggatgg 27360

ttaatgttga ggcatctata tactcatcac gtgtaaattc cacctcgcca tacggaaatt 27420

ctaaagtctg cacattcatc tgtttttctc ctcttaattg tatcgtttaa attatattcc 27480

taatttttgt ggttatgcaa cctttaacta ccaaaatgaa agcgaatagg attatttctc 27540

gcagactaaa tggccttgat tatgcaccct gatgagttga gtgtataatc accacaccaa 27600

aggcgggagg ggtatatgag tcttttagaa tcgattatca gccatcaaat atggctgcaa 27660

cgcaacgcca gtagcgaagt aaaagacctg gcaccattta tctcgcaaat gcgtgatgag 27720

gttaagcgac aggtgctgct gtttggtgat gacagcagaa cggcagcgag actcaccatc 27780

atgctgcggg aacttgaaca agcactgaac gggattacgt cagaatggta cgagaaatta 27840

ctggctgacg ctcgtgagct ttctgactat gaaattaact ggaacgtaaa aaccctgtcg 27900

accaacgtta acgctaattt cgtaacccca gccgccgagc aagtgtgggc cgccgctact 27960

ttcgcgcctc ttgaactaag cgaaaagcca gttgattttg tttctctaat gtcgggctgg 28020

ggtgaaacgg aagtcgcgcg cctggtaact ggcgttaaga tgggatttgt gcaaggccag 28080

acaacacggc agatagttaa gaacgttgtc gggcccggtg gactggcgga tatttccgaa 28140

cgtaatgcgg cgacggttat ccgtacagcc ctggcgcacg tatctaatga agcacggcaa 28200

caggtttacg cgcagaatga cgatatcatc acgaaatacg aatgggtgtc caccctcgac 28260

tctcgtactt ctgccgtttg caggtcccga gacggaatga cgtgggaaat tgataaaggg 28320

ccgatgcccc ctgcccattt tggctgtcgg tcgaccacgg caccagtaat cagtccagaa 28380

ttcgacttcc ttgataaggg cgcgaaacgg gcggcccggg gtgcagatgg gggtcagcag 28440

gtaagcgcag acaccaccta ttacgagttt cttaaacaac agccggcctg gttccaggac 28500

gaagcgcttg gccctgtaag aggtaagatt tttcgtaata gtgggataac cccggaagaa 28560

tttcgtgtaa tatcggtaga cggattcggg cgtccgttaa ctcttaaaga gatggcggag 28620

ctcgataaac gtgttgccaa atacctgaaa gaggaataaa gatgggattt ttcaaagtta 28680

aagatgtgcc gtcacgccgc gtagttcagt actcacgtgt gtctggtgct ggcgaaggtg 28740

ttgtttatat taaagatgaa tctgttcttg gtgagtcggt agatgaaatg ccgttcgccg 28800

ataagaccgg actggtagca atcaccgacg gtatcctgta cgaagtgtcg tatctggatg 28860

gagcaggtga tgtgtacttc gatacgcagc cagcagatgt agaattgaaa gacggttccg 28920

caaaattaac cgttgtcgtg aaaggtgtgg gtagaaggcc gagagaatct tgtttcacag 28980

caacaaaaga aggcacaagc tgagctgacg aagaaaacac aacggcagca gcagaatgat 29040

actaaggccg cctccgccga atcggaaggc aaagaaaatt cggaggctat cacccatgaa 29100

gtcatcaaat atgttacccg tcctggccgt actgtctgtg agtttcctcc tgaacgggtg 29160

tcaatcaaac gacgcgccac tgagaatgct aattccatcc ccggatatga cactaatgca 29220

accgccgtgc aagcttattc ccccgagcag tgacgctgat gaagacctgg ccatcgacgt 29280

acgtaacgcg gagtgcacac ggcaacttcg cttgaaagta ttccggttgc aggagtacat 29340

aaggaatatt ctggaatagt tgccttagta agtggaataa tttattcttg ttacagaaac 29400

accgggtggc ccggtgtcct aaagtccagg ggacatattg actatgaatc gttttttaca 29460

ctatccactc caggaagaag ctggagtaga agataaatcg ggtgcaggtg atgcaccaaa 29520

aatgtacacc gccgaagaag tgcaggcctt gattgagaaa gaagtggccg ggctcaaggc 29580

aaatcaggaa gcgctcctta gcgagaaaaa ggaagctgct cgccgggcta aagaagccga 29640

agaagaacgg cagcgcgccc accaggaggc cctgaaggct gctggtaaaa tggacgaatt 29700

tgaaaagacg attcgtagtc agtatgaacc tgtactgaaa gagaaagaag aacgctacgc 29760

ttctttagcc gcgcgaattc ttggaagtga acgtaaggct gttttgggtt ctttcgccgg 29820

tgatttcatc accccggaag cggtagaaat cctggcgccg ttcgttaaga ccgaattcga 29880

gggggaagac atagttacta aattcatggg tgcggacggc aatgttgtta ctaccgaccc 29940

tgagcagttc cgcaaatacc tgcgcgaaca taaagctttt tcgcatttga ttaaagcaaa 30000

tgcagcttcc ggtggcgggg cttccgggaa taaaggcggc ggggccgcac cagcgtttaa 30060

agatatgagt gaaagtgagc gattggctct ctataaatca aaccctgccg aatttgaacg 30120

gcaacttaaa gccctgagga aataaataat ggcaatcacc actattggta acatcgtaac 30180

tggcaatatc cctgtactgg cgtcctatat gacggaagac ccggtagaga aaaccgcgtt 30240

tttcaactcc ggaattttga ccccaactcc gtatgctgcc gagattgccc gcggaccgtc 30300

taacatcgct aacctgccgt tctggaaagc tatcgatact tctatcgagc ctaactactc 30360

gaatgacgta taccaggaca tcgcgacccc gcgcgctatt cagaccggtg aaatgatggc 30420

ccgcgttgca tatctgaacg aaggtttcgg tcaggcagac ctgactgtag aacttaccag 30480

ccagaatccg ttgcagtccg tcgcatcccg tctggataac ttctggcagc gtcaggcgca 30540

gcgtcgtctg attgctaccg ccctcggtct gtacaacgac aacgtatccg ctactgatgc 30600

gtatcacgag cagaacgaca tggttgttga cgtttccgct accatgggct ttgacgctgg 30660

cgctttcatt gatgctactc agactatggg tgatgcattg atgggcaacg gcggtgaggt 30720

tctcggtgct atcgcgatgc acagcttcgt atacgcacag gcccgtaaag ctcagcttat 30780

cgacttcatc cgtgacgctg agaacaacac catgttcgcc acctaccagg gctaccgtgt 30840

tatcgtcgac gacagcatga ccgtagtagg ccagggtgcg cagcgcaagt tcatctccgt 30900

catcttcggc cagggcgcta tcggttatgg tgaaggcaat cctgagatgc cgctggaata 30960

cgaacgtgaa gcatctcgtg ctaatggtgg tggtgttgaa accctgtgga cgcgtaaaac 31020

gtggttgctg catccgtttg gttacagctt caccagcgct gtaatcactg gcaacggcag 31080

accctaaaac ggttcaatgg tataaggata ataacgcaat ctccggtgca acagacctta 31140

cctacacgaa ggctaactcc gtagccgcgg actcaggtac ttacaaagtc gtcgcacaag 31200

acgggtacgg caacattatc tcggatagca cagtagtaac cgtaggttaa atatacgcgg 31260

ccttcgggcc gctttaagga ttagacatgg cagataatta tgtagtacgg ggacaataca 31320

agggtgtggt cgaggttgac gggcagttag tcccgatgcg cgaagaagcg aacccggaag 31380

cattaatcga aactcagcca gcggccgaag aaccacatta caacggaggt ggggaaccta 31440

agcagcgtcg ccgccgtaaa agtgtagagg aatagcatat gccgcttatc gtagaaacag 31500

gacaaggctt acctaacgcc gattcttacg tcagcctgga agatggccgc gcaacggcgg 31560

ctaagtacgg cctcgaactt ccagaagacg atactaaggc tgaggctgcg ctgagaaatg 31620

gcgctgtata tgttgggctt ttcgaatcgc agatgtgcgg tcgccgtgta tcggcgaacc 31680

aggcgctggc gttcccgaga acaggtatta ccctgcacgg attcccacag ccgtctaacg 31740

ctataccgtc gctagttatc caagctcagg taattgccgc agttgagtat ggcactggta 31800

cggacgttcg ggggtctaca gacgggcgag aggttcagac cgaacgggtt gagggtgcgg 31860

taaccgtgtc ctactttaag aacggctact caggcagtac agtaagcatc acggcagccg 31920

atgattcgct acgtcctctt ttatgcggta gcaataacgc ctactccttt aatgttttca 31980

ggggttaatc atggttaaga ctaaatcgga aatattcgcc ttaattgggg caaacttccc 32040

tgataaccag tccgggctaa ttacaccgga aaagttgcgc gaagttacca cgcaaatcgc 32100

ggattcaatg ctttacggcg ttaaggaagt agaggtactt cgcgcgtcgt ctacggatat 32160

tcaggcgtca actaccacag gtacagcgtt aaccgtatcc tttggtgggg cgcagaaaac 32220

aagcgctgac cctgtgatga ttaatgcatc cggggtggtt acgttcaacg ccgctggtaa 32280

ctacgctatc cgtgttaaat tgcaggctgg gcgcaccggg gcgagcggaa catccatcct 32340

tctgtcgcgt gttctgctcg gcggcgccca attcggttca ccagccgcta ctaaaccggc 32400

aagcgcagac gccacgaatc ctattgaatc gcgtgtggta gttaacgccg tcgctgggca 32460

aaacttcgcg gtagagatta tgcgagacgc ggccgggtct aactttggtg gattgtaccc 32520

tcaaacagca acggttactt cctggggcgt agccccatcc gcattgctgg tcatctcaag 32580

actggaggcc acataatgag caccgctttc agtaaaagga tgcaaggtgt aggaacttta 32640

ctctaactaa caatttgccg tatgcgcagc gccttgagta tggttggtcg caacaggcgc 32700

ctcaaggctt cgttagggtt aatgtcagca ggttccagca attattaaat gaagaagcct 32760

ctaaggtgaa ataatggcaa catattttga ggacttaacg aaagcattcg acacagcact 32820

ggtggcattt ggcaccgaca acgacatcat ggttgcgtta gagaacatag acgcaccgac 32880

gtctacggat gtaccatatt tggcgagtta tatgttgttg gccgatacgg aacaagctga 32940

cctgttctgg accgagcgac gggcaggtat ttatcaggtg gacattaacg ttgggtctac 33000

cttgggttgc gcccctctca accggttagc tgataagcta aacgccacct tcgccgccgg 33060

taactgtttt agtcgtaacg aaatctgtgc tgaggtacaa tcagtaagcc tcggtcctct 33120

tattgttgag aatggatggg cgaagaggcc tctctcaatt aatttcatag cctttacagc 33180

gaggattaga taatggcgtt acaaccatac aaaggcgcga tgaccgcgca attttacgtt 33240

cttgaggcga cgccgggggt gacgccagcg aacccggtat ggcagccgct ccgcaacact 33300

gggggtattc ccgccgtaac gcgcgacgcc ctcatttcca acgagctcga cggcagtcgt 33360

gaaacctcat ccatccgcac cggtaaccgc caggtaacag gtgaatacgc tattgaactg 33420

agcgcgacca gtcaggacga gctgttggcc ggtgcgatga ctagttcatg ggtggccggg 33480

tcaactctat ccggcattgg tatcaccgta gacccagcag cgaaaacttt tacacgcgct 33540

gccggtagct ttgtgacaga cggcgttgaa gtaggcgacc tggtgcaatt cgatggtttg 33600

tcgggtaata acgataaagc tttcctcgtt accgcagtaa ccgctacagt cgtaaccggt 33660

gcgggtatcc agcataccct taccgccgaa tccgacgtcc gggccgattt gcgtatcgca 33720

gataaacttg aaactggtaa cttgtgtaaa acctattcaa tcttgacatg gttgaaaggt 33780

aaatgcggaa acccggattc atacatcata acccgcggag tcgagtttac cgggttcact 33840

atcgaacagg cagttaacgc gatggtgacc ggctctttcc cgttcatcgg cttgaatcag 33900

gaaatcctac aagcaccgcc gagtggttca gatttcacga ccaattttag cgcccgtccg 33960

tttgcatcgg ttgacgtatc cgcctatgac ggcgccgctc cgcttaaact tatagacacg 34020

ttcaccatta ctaacgacaa cagcgcgtcc gcacagttcg agttaggaaa taacagcgtg 34080

gcatttgtcg aacgtggccg cgcagctaac accttctcgc tggcgggtaa gctgtacgac 34140

atgacgttat tgaataaatt cctgaacgaa acgcaaatgg aggtatcttc tgttctgaac 34200

ggcccagacg gtgccatgag tttcacctta aaacgcgctt cgttgacgtc agcaactccg 34260

gaaatcggcg gccccgaatc tgtcaccctt tctcttgagg gccaggcaac cggcaaccag 34320

ttccagtctt caattgttat ccagcgcatt aagtacgcct aagaaaaagg ccccgaaagg 34380

ggccttagtt ttaaatcaaa ccttcttctg ttaacttacc aataatccac atctcacctt 34440

ctgcaaagaa catggcctgt gaatgcccgg tttctgtttc acgcataatg ccgtaaccct 34500

tatcgatgaa ccattgctgg aagacacgac cgcgttttac agagcgattg tatacgccaa 34560

attgttcaag ttgtttattc atccacacgg cagattgacc gaacttctgg gcaacctgcg 34620

tcgcgttata tagattattg cggtcaacta tacggtcgta gacatcggcc tttggtgcca 34680

tttccttgtt ctcaagcgca aggcgttctt tctcctccca caagtccgca gccaacctta 34740

aagcctcccc cattgtcttg ggcaccggta aggatggttt gctcgcttgt gactccagtt 34800

cctgccaacg gcggatgatg gcagcgcgca ttttgatgct gtaccctgaa atcaggatta 34860

atgtttcctc tttatccaaa agaagcattt tatacgtgcg ccccttgctg tctttgtaat 34920

cggctgactt ttcagccgat tgaatctcag acaacatttt gcggatatca gccagaacat 34980

ttttatgctc tttccccgtc aattccgcaa tctcacggct ggacatggtt tgtgcttcac 35040

taacattcat taatccgttc atggtttcat ctcctcttgt tggtgtggtt atatttaacc 35100

ccgaacacgc ccaccgccca aaccagaccc cccaccgcga cgacaaaaaa aagccccgaa 35160

aggggccccc cccccaccca ccaacaccac cacggcaccc ccgcaacccc cccagacccc 35220

caaccgcccc cgccaagccg gcccccgcgc ccccgcgc 35258

Claims (8)

1. An acid-tolerant salmonella bacteriophage, comprising: the Salmonella bacteriophage is Salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) with the preservation number of CCTCC NO: M2018765.

2. The acid-tolerant Salmonella bacteriophage of claim 1, wherein: the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorumphase SG4P1) has acid resistance under the condition of pH value of 1-4.

3. The acid-tolerant Salmonella bacteriophage of claim 1, wherein: culturing for 8-10 h under the condition of MOI =0.001, wherein the titer is 3 multiplied by 10 ¹⁰ PFU/mL。

4. A composition comprising the acid-tolerant salmonella bacteriophage of any one of claims 1 to 3, wherein: the composition at least contains one strain of Salmonella pullorum phage SG4P1(Salmonella pullorum phage SG4P1）。

5. The acid-tolerant Salmonella phage composition of claim 4, wherein: the composition also comprises myotail phage BP-66(Myoviridae sp. BP-66) with the preservation number of CCTCC NO: M2015146; myotail phage BP-63(Myoviridae sp. BP-63) with the preservation number of CCTCC NO: M2015145; the long-tail bacteriophage BP-12(Chilikevirus sp. BP-12) has a preservation number of one or more of CCTCC NO: M2015141.

6. The acid-tolerant Salmonella phage composition of claim 4, wherein: the composition also included chlorine dioxide at a final concentration of 0.0002%.

7. A reagent or kit comprising the acid-tolerant salmonella bacteriophage composition of claim 4, wherein: the kit contains the salmonella pullorum bacteriophage SG4P1(Salmonella pullorum phase SG4P1) or Salmonella pullorum phage SG4P1( Salmonella pullorumphase SG4P 1).

8. The use of the acid-tolerant Salmonella bacteriophage of claim 1, wherein: the Salmonella pullorum bacteriophage SG4P1(Salmonella pullorumphase SG4P1) for preparing feed additives.

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