CN112680423B - Wide-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria and composition, kit and application thereof - Google Patents
Wide-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria and composition, kit and application thereof Download PDFInfo
- Publication number
- CN112680423B CN112680423B CN202011598316.7A CN202011598316A CN112680423B CN 112680423 B CN112680423 B CN 112680423B CN 202011598316 A CN202011598316 A CN 202011598316A CN 112680423 B CN112680423 B CN 112680423B
- Authority
- CN
- China
- Prior art keywords
- ec35p1
- escherichia coli
- phage
- bacteriophage
- coliphage
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of coliphage, and particularly relates to a broad-spectrum coliphage isolate capable of cracking four bacteria and application thereof in sterilization and bacteria prevention. The invention mainly discloses a coliphage EC35P1(Escherichia coli phase EC35P1) with the preservation number of CCTCC M2020438. The phage is a virulent phage separated from the nature, and experiments prove that the phage has no toxic action on normal microbial flora, and the DNA of the phage cannot encode virulence genes, so that the stability is high; the phage has a wide host range, can crack Shigella, salmonella and enterobacter cloacae besides Escherichia coli, and can realize large-scale industrial production. The bacteriophage provides excellent strain resources for developing novel antibacterial preparations, and has good application and development prospects.
Description
Technical Field
The invention belongs to the technical field of research and development of coliphage, and particularly relates to a broad-spectrum coliphage capable of simultaneously cracking four bacteria, a composition and application thereof.
Background
Escherichia coli belongs to gram-negative bacteria, is a resident bacterium in the intestinal tracts of humans and animals, and can be classified into pathogenic and nonpathogenic groups. The antigen components of the escherichia coli are complex and can be divided into a thalli antigen (O), a flagellum antigen (H) and a surface antigen (K), the escherichia coli can be divided into more than two hundred types according to the difference of the thalli antigens, some special serotype escherichia coli have pathogenicity to human beings and animals, and especially cause severe diarrhea and septicemia to infants and young livestock. The Escherichia coli has stronger heat resistance than other intestinal bacilli, and partial bacteria still survive after heating at 55 deg.C for 60min or 60 deg.C for 15 min. Coli can survive in natural water for weeks to months and survive longer in feces at lower temperatures.
Avian colibacillosis is a conditioned disease, and is easily caused under the conditions of poor sanitary conditions and poor feeding management. Avian colibacillosis is a general term for various diseases caused by Escherichia coli, including colibacillosis, peritonitis, salpingitis, umbilicitis, synovitis, air sacculitis, ophthalmia, pericarditis, perihepatitis, vitelline peritonitis and other diseases, and has serious harm to poultry breeding industry.
Salmonella Enterobacteriaceae, gram-negative bacilli, are predominantly flagellated and motile. The confirmed salmonella genus has more than 2000 serotypes according to the classification of kaufman-white, and can be classified into A, B, C, D, E … … and other 34 groups according to the difference of the antigenic structure of salmonella. Most of salmonella causing human diseases belong to groups A to E, and the salmonella mainly comprises more than 10 serotypes including salmonella choleraesuis, salmonella typhimurium, salmonella enterica, salmonella gallinarum, salmonella ducks and the like. In nature, salmonella has a wide range of animal hosts: poultry such as chicken, duck, goose, etc., livestock such as pig, cattle, sheep, horse, etc., and various animals, fishes, rats, etc., can carry bacteria, and even salmonella can be separated from cicada and some insects. Because salmonella is present in the intestinal lumen of these animal hosts, the meat of poultry and livestock can be infected with salmonella during slaughtering and processing; after slaughtering, the sick animals are sold directly for easier transmission of salmonella, and the eggs or egg products can be polluted by salmonella.
Salmonellosis is a generic term for acute or chronic diseases of birds caused by a salmonella of the salmonella genus. Disease caused by salmonella pullorum is called pullorum disease; the disease caused by salmonella gallinarum is called fowl typhoid; avian diseases caused by other flagellated motile salmonella are collectively referred to as avian paratyphi. Salmonellosis is ubiquitous throughout the world and seriously compromises the development of the poultry farming industry. Some salmonella have been reported to carry genetic plasmids with drug-resistant factors that can deliver resistance to multiple antibiotics, which can be disseminated particularly in susceptible bacteria, and which pose difficulties in treating salmonella infections.
Enterobacter cloacae, a bacterium of the genus Enterobacteriaceae of the order Enterobacteriaceae, is widely found in nature, can be detected in human and animal feces water, soil, and plants, and is one of the normal intestinal bacterial species. Enterobacter cloacae has become an increasingly important pathogen for nosocomial infections, and bacterial infectious diseases caused by the enterobacter cloacae often affect multiple organ systems, including skin soft tissue infections, urinary tract infections, respiratory infections, septicemia and the like. Enterobacter cloacae can produce pathogenic bacterial endotoxin, and the bacterium has strong resistance to disinfectant and antibiotic. Enterobacter cloacae is reported to cause morbidity and death in broiler chicks.
Shigella, also known as Shigella dysenteriae, is a gram-negative bacterium. The bacterium is cold-resistant, can grow on a common agar culture medium for 24 hours, and forms a semitransparent smooth bacterial colony with the diameter of 2 mm; the shigella forms colorless colonies on the selective intestinal bacillus culture medium, and the size of the colonies is 0.5-0.7 multiplied by 2-3 mu m. Shigella is sporulate-free, capsulously, flagellate-free, mostly fimbriae. Shigella is an enteric pathogen in humans and primates and can cause bacillary dysentery. In addition, researchers find that Shigella can infect chicks, so that the chicks are attacked to cause a large number of diseases and deaths, the morbidity can reach 100%, the mortality after administration still reaches 31.84% -33.13%, and great economic losses are caused to the chicken industry.
The bacteriophage is a virus specially used for cracking bacteria, mainly comprises chemical components of protein and nucleic acid, is widely present in soil, air, water and organisms, and has strong specificity. Because of its strong bactericidal power, bacteriophage can be used as an anti-bacterial infection agent, and has been paid attention by many scholars at home and abroad since the beginning of the 20 th century.
Sunli works et al (the Chinese animal infectious disease journal, 2018,12,10) isolated virulent enterohemorrhagic escherichia coli phage with broad phage spectrum from a sewage sample collected from a farm, and conducted biological characteristics such as plaque, phage spectrum, morphology (transmission electron microscopy), genetic material, acid-base and thermal stability, one-step growth curve and the like, and studies on controlling enterohemorrhagic escherichia coli contamination in a breeding environment.
Chinese patent CN106591241A discloses a novel enterohemorrhagic Escherichia coli O157, which is used for preventing and treating enterohemorrhagic Escherichia coli O157 pollution caused by sewage discharge or reuse.
Chinese patent CN110129283A discloses a short-tail highly lytic coliphage named PD38, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation date of 2018, 9 and 26 days and the preservation number of CGMCC No. 16391. The bacteriophage has good lytic effect on Escherichia coli, has good killing effect on Escherichia coli in food and culture environment, and can be used for preventing and treating diseases caused by Escherichia coli in chicken.
Chinese patent CN109251899A discloses a drug-resistant coliphage and application thereof. Classified and named as Escherichia phase vB _ EcoP-EG1 with the accession number: CCTCC M2018562, and the preservation date is 8 months and 21 days in 2018.
Chinese patent CN106754751A is enterohemorrhagic Escherichia coli bacteriophage and application thereof, the preservation number of the bacteriophage strain is CCTCC NO: m2016539, deposited at the China center for type culture Collection, university of Wuhan, China, at 29.9.2016, and classified and named as enterohemorrhagic Escherichia coli phage vB _ ECM _ MIE, Entero-hemorrhagic Escherichia coli O157, H7 phase vB _ ECM _ MIE; has high-efficiency sterilization capability on EHEC.
Korean patent KR102003786B1 discloses a novel Escherichia coli-specific bacteriophage EcoH7 and a method for preparing the same. The bacteriophage EcoH7 has very high specificity to escherichia coli, can solve the problems of drug resistance of escherichia coli to antibiotics and escherichia coli residue in food, has a wide host range, and can be widely used in the fields of antibiotics, compositions, feed, disinfectants or cleaning agents.
U.S. Pat. No. 4, 10265353, 2 discloses a myoviridae phage ESC-CHP-1 isolated from nature and capable of specifically killing enterohemorrhagic Escherichia coli strains, deposited under the number: KCTC12660bp, the infection of entero-hemorrhagic escherichia coli can be prevented and treated using a composition comprising the bacteriophage as an active ingredient.
U.S. Pat. No. 4, 7635584, 2 discloses an isolated bacteriophage having strong lytic activity against the Escherichia coli O157: H7 strain, and a method of controlling the growth of Escherichia coli O157: H7 in various environments using the bacteriophage and/or progeny or derivatives derived from the bacteriophage.
The above technique has the following drawbacks: the research on the Escherichia coli phage mainly focuses on the lytic aspect of the enterohemorrhagic phage, and the research on the aspect that the Escherichia coli phage can also lyse bacteria of other genera and the application aspect is rarely reported. Therefore, it is urgently needed to find a novel coliphage isolate which can kill various bacteria simultaneously and has high stability, good safety and high potency.
Disclosure of Invention
One of the purposes of the invention is to provide a broad-spectrum coliphage capable of simultaneously cracking four bacteria, a composition and application thereof, and the composition has the characteristics of high stability, good safety and high titer.
The invention adopts a wide-spectrum Escherichia coli bacteriophage capable of simultaneously cracking four bacteria, wherein the Escherichia coli bacteriophage is Escherichia coli EC35P1(Escherichia coli EC35P1), and the preservation number is CCTCC M2020438; the Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) has a nucleotide sequence shown in SEQ ID No. 1.
Further, the Escherichia coli phage EC35P1(Escherichia coli EC35P1) has good tolerance under the conditions of pH 4-11 and temperature 4-70 ℃, the MOI is 0.1 for 8 hours, and the titer is as high as 1 × 1011PFU/mL。
By the technical scheme, the Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) has a high cracking rate on Escherichia coli, and also has a good cracking effect on salmonella, enterobacter cloacae and shigella.
The invention also aims to provide a composition of broad-spectrum coliphage capable of simultaneously cracking four kinds of bacteria, and the coliphage EC35P1(Escherichia coli phase EC35P1) has a wider host range and strong tolerance, and can be mixed with other substances to meet specific application requirements.
The second aim of the invention is realized by the following technical scheme: a composition containing the broad-spectrum Escherichia coli bacteriophage capable of simultaneously lysing four bacteria at least contains one Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P 1).
By the technical scheme, the Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) and other phages are combined for obtaining a wider cracking spectrum range. By way of illustration, the proportional relationship between the E.coli bacteriophage EC35P1(Escherichia coli phase EC35P1) and other bacteriophages can be determined by those skilled in the art in connection with the present invention and the actual field of application and general knowledge in the art.
The present invention in a preferred example may be further configured to: the composition also includes a chemical disinfectant.
The present invention in a preferred example may be further configured to: the adopted chemical disinfectant is one of 0.05 percent of didecyl dimethyl ammonium chloride or 0.01 percent of benzalkonium bromide.
The present invention in a preferred example may be further configured to: the composition also comprises Staphylococcus aureus phage JIP2(Staphylococcus aureus phage JIP 2).
The Escherichia coli bacteriophage EC35P1(Escherichia coli phase EC35P1) and other antibacterial agents are mixed for use, so that the antibacterial broad spectrum is obtained, and the specific killing effect on Escherichia coli, salmonella, enterobacter cloacae and shigella is achieved. 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. As an exemplary illustration, the ratio of the E.coli bacteriophage EC35P1 to the other antibacterial agents can be determined by one skilled in the art in connection with the present invention and the actual field of application and general knowledge in the art. The E.coli phage EC35P1 of this protocol can also be used in combination with other phage-forming compositions.
The invention also aims to provide a reagent or a kit containing the composition of the broad-spectrum coliphage capable of simultaneously cracking salmonella coli, enterobacter cloacae and shigella coli.
The fourth purpose of the invention is to provide the application of the broad-spectrum coliphage capable of simultaneously cracking four bacteria.
The fourth object of the invention is realized by the following technical scheme: escherichia coli phage EC35P1(Escherichia coli EC35P1) was used as a biological bactericide or disinfectant.
By adopting the technical scheme, the Escherichia coli phage EC35P1(Escherichia coli EC35P1) is used as a novel safe environmental biological bactericide, can specifically kill Escherichia coli, salmonella, enterobacter cloacae and shigella in the environment, and improves the distribution of microorganisms in the environment.
The coliphage EC35P1(Escherichia coli EC35P1) is used as a biological bactericide for the culture, transportation and preservation of livestock and poultry products, and can prevent and treat the pollution of pathogenic Escherichia coli, salmonella, enterobacter cloacae and shigella during the culture, transportation and preservation of livestock and poultry.
Spraying 10 Escherichia coli EC35P1(Escherichia coli EC35P1) alone or in combination with other antibacterial substances onto the surface of food contaminated by Escherichia coli, Salmonella, Enterobacter cloacae and Shigella5～106The phage of PFU/mL can completely inactivate the bacteria within 12h, and can be applied to preventing and treating the bacterial contamination on the surface of food.
Escherichia coli phage EC35P1(Escherichia coli EC35P1) is used as a bactericide and sprayed in a food production workshop, so that Escherichia coli, salmonella, enterobacter cloacae and shigella in the environment can be specifically and continuously prevented and controlled, and meanwhile, the pollution of the bacteria in the food processing process can be prevented and controlled.
Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) is used as a novel safe environment disinfectant, and can effectively kill Escherichia coli, salmonella, enterobacter cloacae and shigella in the environment.
The fifth purpose of the invention is to provide the application of the broad-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria.
The fifth object of the present invention is achieved by the following technical solutions: escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) is used as feed additive, health product, preventive medicine, therapeutic medicine or medical apparatus.
By adopting the technical scheme, the Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) is used as a potential drug or health product for treating and preventing bacterial infection caused by Escherichia coli, salmonella, enterobacter cloacae and shigella. The Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) can be used alone or in combination as a potential feed additive, and can be added into feed to specifically and continuously prevent and treat the pollution of Escherichia coli, salmonella, enterobacter cloacae and shigella in feed production, storage and animal culture. In addition, the four bacteria in the intestinal tract of the animal can be inhibited from infecting, and the resistance of the animal can be enhanced.
The Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) can be used as a medical instrument for treating and preventing bacterial infection caused by Escherichia coli, salmonella, Enterobacter cloacae and Shigella.
The invention also aims to provide a composition of the broad-spectrum coliphage capable of simultaneously cracking the four bacteria, or a reagent or a kit of the broad-spectrum coliphage capable of simultaneously cracking the four bacteria.
The sixth object of the present invention is achieved by the following technical solutions: a composition of broad-spectrum coliphage capable of cracking four kinds of bacteria simultaneously, or a reagent or a kit of broad-spectrum coliphage capable of cracking four kinds of bacteria simultaneously, is applied to preventing and treating diseases caused by colibacillus, salmonella, enterobacter cloacae and shigella.
The application has the following beneficial effects:
(1) the Escherichia coli phage EC35P1(Escherichia coli EC35P1) is a strict virulent phage and has high toxicity to host bacteria; the composition also has high toxicity to salmonella, enterobacter cloacae and shigella; has a wide host range.
(2) The Escherichia coli phage EC35P1(Escherichia coli EC35P1) is a virulent phage separated from nature, and is safe and free of any side effect as proved by a toxicological experiment; the test phage DNA is unable to encode proteins that may pose potential health risks and does not contain virulence genes or undesirable genes; the present invention does not involve any genetic modification of the test phage.
(3) The Escherichia coli phage EC35P1(Escherichia coli EC35P1) can be well proliferated on a non-pathogenic bacterial host; culturing for 8h under the condition that MOI is 0.1, and the titer is as high as 1 × 1011PFU/mL, can carry on the large-scale fermentation culture; the invention provides a high-quality phage strain source for the industrial production of phage bactericides.
(4) The Escherichia coli phage EC35P1(Escherichia coli EC35P1) has good stability: the culture solution can survive for 41 weeks at room temperature; and can be stored for 24 months at 4 ℃; the Escherichia coli phage EC35P1(Escherichia coli EC35P1) was bathed for 24h at 50 ℃ and the titer was reduced by only one order of magnitude; the water bath lasts for 10 hours at 60 DEG C8Presence of phage in PFU/mL; at the same time, the survival rate of the product is 80 percent at 70 ℃ in water bath for 2 h. The coliphage EC35P1 has good high temperature resistance.
(5) The coliphage EC35P1(Escherichia coli EC35P1) has good application and development prospects, and can be prepared into a medicament or a reagent for treating or preventing avian chicken omphalitis, adult chicken pericarditis, perihepatitis, air sacculitis, peritonitis syndrome, pullorum disease, avian typhoid and avian paratyphoid caused by Escherichia coli, salmonella, enterobacter cloacae, shigella and the like by technical personnel in the field according to the record of the application and common knowledge in the field; the agent or reagent can be prepared by those skilled in the art according to the description of the present application and the common general knowledge in the art, and can be used for treating or preventing other diseases caused by Escherichia coli, Salmonella, Enterobacter cloacae, Shigella, etc.
(6) The coliphage EC35P1(Escherichia coli EC35P1) can be used as an active ingredient to be applied to preparation of a composition or a rapid detection reagent or a kit so as to detect various diseases caused by Escherichia coli, salmonella, enterobacter cloacae and shigella, and the coliphage EC35P1 can be used for detecting the Escherichia coli, salmonella, enterobacter cloacae and shigella carried in a target sample in the forms of test paper, kits and the like or screening target pathogenic bacteria in a clinical sample, and can effectively ensure the detection sensitivity because the stability of the phage is high.
(7) The Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) of the present invention can be used alone or in combination for killing Escherichia coli, Salmonella, Enterobacter cloacae, Shigella at a concentration of 103PFU/mL in E.coli Medium 109The killing rate of the phage of PFU/mL to the colibacillus of the concentration reaches more than 99 percent. The Escherichia coli EC35P1(Escherichia coli EC35P1) can be used as an effective component of various products for environmental disinfection, for example, the Escherichia coli EC35P1 can be used for disinfecting and decontaminating water distribution systems, medical facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the forms of liquid soaking, spraying, combined use with an aqueous carrier and the like, 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 solution, TSB medium, LB medium, chloride free water and the like.
(8) The coliphage EC35P1(Escherichia coli EC35P1) can effectively kill Escherichia coli, salmonella, enterobacter cloacae and shigella on the surface of chicken, prevent and control the pollution of the Escherichia coli, the salmonella, the enterobacter cloacae and the shigella in the storage process of meat such as chicken and the like, 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 infection with escherichia coli, salmonella, enterobacter cloacae, shigella, in the form of liquid immersion, spraying, use in combination with synthetic ingredients, and the like, particularly for cooked or non-sterilizable 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.
(9) The coliphage EC35P1(Escherichia coli EC35P1) can be used singly or in a mixture, 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 Escherichia coli, salmonella, enterobacter cloacae and shigella. Hosts infected with E.coli, Salmonella, Enterobacter cloacae, Shigella include humans, livestock (pigs, cattle, sheep, etc.), poultry (chickens, ducks, geese, etc.), and various beasts, fishes, rats, etc.
(10) The Escherichia coli phage EC35P1(Escherichia coli EC35P1) 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 infection of Escherichia coli, salmonella, enterobacter cloacae and shigella. The product form can include but is not limited to the forms of carrier carrying, concentrated injection or medicament soaking, etc. applied to the controlled host body surface, oral, rectal, intrapleural, etc.; 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 injection, pleural cavity injection, transvascular injection, and the like; 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 E.coli bacteriophage EC35P 1.
FIG. 2 is a diagram of the morphological structure of the E.coli bacteriophage EC35P1 under a transmission electron microscope.
Detailed Description
The following examples serve to further illustrate the present application, but do not limit the effective scope of the present application in any way.
In the following examples, the reference numbers of the strains are the same as the reference numbers of the same company.
The Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) has a preservation number of CCTCC M2020438, the preservation unit is China center for type culture Collection, the preservation time is 2020 years, 8 months and 20 days, and the preservation address is Wuhan university in Wuhan, China;
the Escherichia coli bacteriophage CL9(Escherichia coli CL9) has the preservation number of CCTCC M2018937, the preservation unit is China center for type culture Collection, the preservation time is 2018,12 months and 28 days, and the preservation address is Wuhan university in Wuhan, China;
the Salmonella phage SG8P3(Salmonella pullorumphage SG8P3) has a preservation number of CCTCC M2020205, the preservation unit is China center for type culture Collection, the preservation time is 2020, 6, 12 days, and the preservation address is Wuhan university, Wuhan, China;
the Staphylococcus aureus phage JIP2(Staphylococcus aureus JIP2) has the preservation number of CCTCC M2016285, the preservation unit is China center for type culture Collection, the preservation time is 2016, 5 and 26 days, and the preservation address is Wuhan university, Wuhan, China;
in the example which follows, it is shown,
the formula of the TSB liquid culture medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride and 1000mL of distilled water;
the formula of the TSA solid culture medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 15g of agar and 1000mL of distilled water;
the formula of the TSB semisolid agar culture medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 7g of agar and 1000mL of distilled water;
the SM liquid formula is as follows: 5.8g of sodium chloride, 2g of magnesium sulfate, 50mL of 1mol/L Tris-HCl, 0.25g of gelatin and 1000mL of distilled water.
Example 1 isolation preparation and purification culture of E.coli phage EC35P1
In the application, a source sample of Escherichia coli bacteriophage EC35P1(Escherichia coli EC35P1) is collected from domestic sewage in Jiangning district of Nanjing city, Jiangsu province, filtered by double-layer filter paper, centrifuged at low speed and normal temperature, and the supernatant is filtered by a filter membrane of 0.22 mu m.
Separation of phage: and taking 10mL of filtered supernatant, adding the filtered supernatant into 10mL of 2-time TSB culture medium, simultaneously adding 1mL of phage host bacterium EC35 logarithmic phase bacterial liquid, placing the mixture at 37 ℃ for culturing for 8h, taking the culture, centrifuging the culture for 10min at 8000rpm, and filtering the supernatant by using a 0.22-micrometer filter membrane for later use. And (3) adding 0.5mL of phage host bacteria log-phase bacterial liquid into 5mL of semisolid TSB culture medium at 48 ℃, uniformly mixing, pouring onto a TSA plate, and preparing into a double-layer plate containing host bacteria. Dripping 10 μ l of the filtered supernatant onto the coagulated double-layer plate, air-drying under aseptic condition, and culturing at 37 deg.C for 8 hr to form phage spot.
And (3) purifying the phage: picking bacteriophage spot to 1mL SM buffer solution, shaking for 1min, performing 10-fold gradient dilution, and taking 102、104And 106And respectively adding 0.5mL of log phase host bacteria into the diluent, uniformly mixing, standing for 15min, adding 5mL of semi-solid TSB culture medium at 48 ℃, immediately pouring the mixture onto a TSA plate, uniformly shaking, flatly standing for 5min, solidifying the mixture, culturing in an incubator at 37 ℃ for 8h, and observing to obtain a double-layer plate containing a single plaque. Picking up single plaques into 1mL SM buffer solution, purifying for at least 3 times according to the method, finally picking up single plaques with consistent shape and size on plaque forming plates, referring to FIG. 1, placing in 50mL TSB culture medium containing 1mL log phase host bacteria liquid, and shaking at 180rpm at 37 ℃ for 8 h. The culture was centrifuged at 8000rpm for 10min, and the supernatant was filtered through a 0.22 μm filter to obtain the purified E.coli phage EC35P1(Escherichia coli phage EC35P 1).
Example 2 Electron microscopy of E.coli phage EC35P1
The phage EC35P1 culture supernatant prepared in example 1 was used for electron microscopy: dropping 20 μ L sample on copper mesh, allowing it to settle naturally for 15min, absorbing the excessive liquid from the side with filter paper, adding 1 drop of 2% phosphotungstic acid (PTA) on the copper mesh, dyeing for 10min, absorbing the dye solution from the side with filter paper, drying, and observing with electron microscope. As shown in FIG. 2, the morphology of the Escherichia coli phage EC35P1 observed under an electron microscope is that the phage is tailed phage, is not folded, and has a polyhedral symmetrical head and a telescopic tail, wherein the head is about 90-105 nm long, and the transverse diameter of the head is about 50-60 nm; the tail length is about 95-110 nm, and the tail width is about 15-25 nm. Based on their unique size and morphology, the coliphage EC35P1 was systematically classified as Myoviridae (Myoviridae) according to the international committee for virus classification (ICTV) definition.
The preservation number of the Escherichia coli bacteriophage EC35P1(Escherichia coli phase EC35P1) is CCTCC M2020438.
Example 3 E.coli phage EC35P1 particle preparation and genome extraction and sequencing
100mL of the phage EC35P1 prepared in example 1 was added with DNaseI and RNaseA at a final concentration of 1. mu.g/mL, incubated at 37 ℃ for 60min, dissolved with 5.84g of NaCl (final concentration of 1mol/L), and placed in an ice bath for 1 h. The mixture was centrifuged at 11000rpm for 10min at 4 ℃ and the supernatant was transferred to a new centrifuge tube. Solid polyethylene glycol (PEG8000) was added to a final concentration of 10% (w/v), and after complete dissolution, the ice bath was allowed to stand for at least 1 h. Centrifuging at 11000rpm for 20min at 4 deg.C, resuspending with small amount of SM solution to obtain phage particle concentrated solution, and storing at 4 deg.C.
And extracting phage nucleic acid by using a lambda phage genome DNA kit and sequencing. Through nucleotide sequencing, the Escherichia coli phage EC35P1(Escherichia coli EC35P1) has a nucleotide sequence shown in SEQ ID No. 1.
Example 4 determination of E.coli phage EC35P1 Titers
Using SM as a diluent, the stock E.coli phage EC35P1 (prepared in example 1) was diluted stepwise in 10-fold gradient to l08And (4) doubling. Respectively taking l05、l06、l07And l08And uniformly mixing L000 mu L of diluted phage culture solution with 300 mu L of host bacterium liquid, and standing for 15min to ensure that the diluted phage culture solution is fully combined with receptors on the surfaces of bacteria. Adding the above mixed solution into 4mL semi-solid agar medium cooled to 50 deg.C, mixing, spreading on solidified solid agar plate, solidifying, and spreading onAnd (4) carrying out inverted culture at 37 ℃ for 6-8 h. Three replicates of each dilution were taken and counted as the average of the three replicates of that dilution. Wherein, the titer of the phage (PFU/mL) is the average plaque number multiplied by the dilution multiple
As can be seen from Table 1, the Escherichia coli phage EC35P1 had 10 h after 12h of culture10Titers above PFU/mL.
TABLE 1 titer of E.coli phage EC35P1 after 12h culture
| Incubation time | 4h | 8h | 12h |
| Phage EC35P1 potency (PFU/mL) | 5.5x109 | 1.0x1010 | 1.5x1010 |
Example 5 E.coli phage EC35P1 virulence gene or undesirable gene deletion detection assay
The present invention selects 103 virulence genes identified as originating from lysogenic phages within pathogenic bacteria (table 2), and determines whether they contain the above virulence genes by determining the whole genome of the E.coli phage EC35P1 and performing bioinformatics analysis on it. The results show that the test phage of the present invention did not contain the following virulence genes.
TABLE 2 major known virulence genes of lysogenic phages in pathogenic bacteria
Example 6 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 of Escherichia coli phage EC35P1, control group given an equal amount of physiological saline, continuous administration for 15d, the test mice were killed by neck-cutting, and visceral status was examined.
The experimental results show that the coliphage EC35P1 at this dose has 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 7 determination of the optimum multiplicity of infection (MOI) of E.coli by the E.coli phage EC35P1
And (3) selecting a single colony of escherichia coli EC35, inoculating the single colony into a test tube containing 3mL of TSB culture solution, and performing shaking culture in a shaker at 37 ℃ at 160rpm for 8h to obtain a host bacterial suspension. The bacterial suspension was transferred to 10mL of TSB culture medium at a ratio of 1:100, and cultured at 37 ℃ with shaking at 160rpm until the logarithmic phase. Pure culture solution of phage EC35P1 (prepared from example 1) and host bacteria (MOI ═ phage count/bacteria count) were added at a ratio of multiplicity of infection of 1000, 100, 10, 1, 0.1, 0.01 and 0.0001, respectively, and TSB liquid medium was added to make the total volume of each tube the same. The culture was carried out at 37 ℃ for 8 hours with shaking at 160 rpm. After the culture is finished, 10000g of the culture medium is centrifuged for 10min, supernatant is collected, and the titer of the phage is measured by a double-layer plate method. 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. The results are shown in Table 3.
The result shows that the Escherichia coli phage EC35P1 titer reaches the highest (1X 10) under the condition of 8h culture11PFU/mL), its MOI is 0.1.
TABLE 3 titer of E.coli phage EC35P1 at different multiplicity of infection
Example 8 thermostability assay of E.coli phage EC35P1
1mL of phage EC35P1 pure culture medium (prepared from example 7) was loaded into a sterile EP tube and exposed in a water bath at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C for 2h, 12h, 24h and 48h, respectively. After the action time is over, the sample tube is taken out and immediately placed in an ice bath for cooling, and the phage titer is measured by adopting a double-layer plate method after the sample tube is properly diluted. The experiment was repeated 3 times.
As shown in Table 4, the E.coli phage EC35P1 showed only an order of magnitude reduction in potency in a 50 ℃ water bath for 24h compared to the control, and 10 in a 60 ℃ water bath for 48h8The phage of PFU/mL survived while still having 80% survival rate in water bath at 70 ℃ for 2h, thus demonstrating the good high temperature resistance of the E.coli phage EC35P 1.
TABLE 4 titer of E.coli phage EC35P1 at different temperatures
Example 9 test of pH stability of Escherichia coli phage EC35P1
Adding 900 μ L of TSB medium with different pH (1-14) into sterile EP tube, placing the EP tube in thermostatic water bath at 25 deg.C, allowing temperature to equilibrate, adding 00 μ L of pure culture solution of Escherichia coli bacteriophage EC35P1 (prepared from example 7) to give initial titer of 1X1010PFU/mL, standing at room temperature for 1 h. After the reaction time is over, the sample is diluted properly and then the titer of the phage is measured by adopting a double-layer plate method. After thatThe above process was repeated after 4h, 8h, 24h and 96h, respectively, and the experiment was repeated 3 times.
The results are shown in table 5, the escherichia coli phage EC35P1 has no significant change in potency between pH4 and pH11, which indicates that the escherichia coli phage EC35P1 has good stability under neutral, slightly acidic and alkaline conditions.
Under acidic conditions at pH1 and alkaline conditions at pH 13, the titer of phage EC35P1 decreased to 0 after 4 hours of treatment, indicating that phage EC35P1 can tolerate a pH of 1 to 13.
TABLE 5 pH stability of E.coli phage EC35P1 (initial titer 1X 10)10 PFU/mL)
Example 10 survival stability test of E.coli phage EC35P1
5mL of E.coli phage EC35P1 pure culture medium (prepared in example 1) were dispensed into sterile test tubes, and placed at 4 deg.C, 25 deg.C, and 30 deg.C, and the titer of phage was determined by double-layer plate method after regular dilution.
The results are shown in Table 6, in 4 ℃ conditions of Escherichia coli phage EC35P1 storage for 50 weeks of titer only reduced about half, still have 6X109PFU/mL; the titer order of magnitude is not reduced by more than one order of magnitude within 12 weeks of the Escherichia coli bacteriophage EC35P1 storage at 25 ℃, and the titer can still be detected by 41 weeks; the E.coli phage EC35P1 can maintain the titer on the same order of magnitude in 8 weeks at 30 ℃, and the titer can still be detected by 32 weeks; the bacteriophage EC35P1 has strong lysis capacity on host bacteria at each preservation temperature. The E.coli phage EC35P1 is suitably stored at 4 ℃.
TABLE 6 survival stability of E.coli phage EC35P1 at different storage temperatures
Example 11 E.coli phage EC35P1 testing for the lytic Range of E.coli and Salmonella
Respectively taking the titer of 1x108Stock solutions of PFU/mL E.coli phage EC35P1 (prepared from example 1), Salmonella phage SG8P3, E.coli phage CL9 were spotted to determine the lysis profile of the phage.
Single colonies of 100 strains of animal-derived Escherichia coli were picked, inoculated into a test tube containing 3mL of TSB, and cultured at 37 ℃ and 160rpm for 8 hours to obtain bacterial solutions of the respective strains. And mixing 300. mu.L of the bacterial suspension with the semisolid culture medium respectively, spreading the mixture on a common agar plate, and dripping 5. mu.L of bacteriophage EC35P1 stock solution on the plate. And after natural air drying, culturing at 37 ℃ for 6-8h, and observing the result.
Single colonies of 100 animal-derived salmonella are respectively picked up and inoculated into a test tube containing 3mL of TSB, and the test tube is cultured for 8 hours at 37 ℃ and 160rpm to prepare bacterial liquid of each strain. And mixing 300 mu L of bacterial suspension with the semisolid culture medium respectively, spreading the mixture on a common agar plate, and dripping 5 mu L of stock solutions of the phage EC35P1, the salmonella phage SG8P3 and the Escherichia coli phage CL9 on different positions of the plate respectively. The phage culture solution can not be contacted when the sample is added, so that the test result is not influenced. And after natural air drying, culturing at 37 ℃ for 6-8h, and observing the result.
As a result, as shown in Table 7, the Escherichia coli strain was cleaved with the Escherichia coli phage EC35P1 at a rate of 88%. The lysis rate of the Escherichia coli bacteriophage EC35P1 on salmonella is 60%; in contrast, the Salmonella lysis ratio of Escherichia coli CL9 was 2% and that of Salmonella phage SG8P3 was 86%.
The example shows that the Escherichia coli phage EC35P1 has high lysis rate on Escherichia coli, has good lysis effect on Salmonella, has a small difference in Salmonella lysis capacity from that of the Salmonella phage SG8P3, and has a better Salmonella lysis capacity than that of the Escherichia coli phage CL 9.
TABLE 7 lysis profile determination of Escherichia coli and Salmonella by Escherichia coli phage EC35P1
Note: "+ + + +" is completely clear, "+" is moderately clear, "+" is slightly clear, and "no cleavage is" - ".
Example 12 E.coli phage EC35P1 to Enterobacter cloacae and Shigella lysis range test with titer of 1x108The lysis profiles of the phages were determined by the dot-drop method using PFU/mL E.coli phage EC35P1 (prepared from example 1) and E.coli phage CL9 stock solutions.
Single colonies of 100 animal-derived Enterobacter cloacae were picked, inoculated into a test tube containing 3mL of TSB, and cultured at 30 ℃ and 160rpm for 8 hours to obtain bacterial solutions of the respective strains. And mixing 300. mu.L of the bacterial suspension with the semisolid culture medium respectively, spreading the mixture on a common agar plate, and dripping 5. mu.L of stock solutions of the Escherichia coli phage EC35P1 and the Escherichia coli phage CL9 on different positions of the plate respectively. The phage culture solution can not be contacted when the sample is added, so that the test result is not influenced. And after natural air drying, culturing for 6-8h at 30 ℃, and observing the result.
And respectively picking 100 single colonies of the shigella of animal origin, inoculating the single colonies into a test tube containing 3mL of TSB, and culturing at 160rpm for 8h to obtain bacterial liquid of each strain. And mixing 300. mu.L of the bacterial suspension with the semisolid culture medium respectively, spreading the mixture on a common agar plate, and dripping 5. mu.L of stock solutions of the Escherichia coli phage EC35P1 and the Escherichia coli phage CL9 on different positions of the plate respectively. The phage culture solution can not be contacted when the sample is added, so that the test result is not influenced. And after natural air drying, culturing for 6-8h at 37 ℃, and observing the result.
The results are shown in table 8, the lysis rates of the coliphage EC35P1 on enterobacter cloacae and shigella were 56% and 70%, respectively; the lysis rates of the coliphage CL9 on enterobacter cloacae and shigella are 6% and 30% respectively. The Escherichia coli phage EC35P1 has better cracking effect on enterobacter cloacae and Shigella.
TABLE 8 results of lysis-spectrum determination of Enterobacter cloacae and Shigella by Escherichia coli phage EC35P1
Note: "+ + + +" is completely clear, "+" is moderately clear, "+" is slightly clear, and non-lysing is "-".
Example 13 application of Escherichia coli phage EC35P1(Escherichia coli EC35P1) to field experiment of pullorum disease in chicken
360 chicks were randomly divided into 4 groups (Escherichia coli challenge group, Salmonella challenge group, Enterobacter cloacae challenge group, Shigella challenge group) of 90 chicks each. Adding 1x10 final concentration into breeding drinking water tank of 4 groups of chickens5cfu/mL E.coli, final concentration 6X105Salmonella cfu/mL, final concentration 7X105cfu/mL Enterobacter cloacae, final concentration 9X105cfu/mL Shigella were used to challenge the chicks. Randomly selecting 30 out of 90 chicks in each group, and adding 5x10 to the drinking water to obtain final concentration5Preparation of PFU/mL E.coli phage EC35P1(Escherichia coli EC35P1) (prepared according to the method of example 7). The chicks were observed daily for 2 weeks for morbidity and mortality (see table 9). And positive control groups were also set for each group (4 groups each at a final concentration of 1 × 10)5cfu/mL E.coli, final concentration 6X105Salmonella cfu/mL at a final concentration of 7X105cfu/mL Enterobacter cloacae, final concentration 9X105cfu/mL shigella) and a negative control group (4 groups were both non-challenge treated and given equal amounts of saline). Mortality rate (number of chicks died/total number of chicks) × 100%.
The results are shown in table 9, and the mortality of the chicks in the 4 challenge groups was low in all of the Escherichia coli phage EC35P1(Escherichia coli EC35P1) treated groups, indicating that Escherichia coli phage EC35P1(Escherichia coli EC35P1) had therapeutic effects on all of the four bacteria-challenged chicks.
TABLE 9 Effect of Enterobacter phage EC35P1(Escherichia coli EC35P1) on mortality of chicks
Example 14 experiment of the Bactericidal Effect of the composition of Escherichia coli phage EC35P1(Escherichia coli phage EC35P1) in liquids
The titer was 5x109PFU/mL coliphage EC35P1(Escherichia coli phase EC35P1) stock solution, didecyl dimethyl ammonium chloride with a final concentration of 0.05% and benzalkonium bromide with a final concentration of 0.01% were mixed homogeneously in equal volumes to make 1:1 composition 1.
The titer was 5x109PFU/mL E.coli phage EC35P1(Escherichia coli phase EC35P1) stock solution, titer 5X109pFU/mL Staphylococcus aureus phage JIP2(Staphylococcus aureus)Aureusphage JIP2) stock solutions were mixed in equal volumes to make 1:1 composition 2.
Culturing Escherichia coli to logarithmic phase, subpackaging into 4 test tubes, and diluting with TSB liquid culture medium to obtain Escherichia coli with final concentration of 1 × 103cfu/mL, where 2 tubes were inoculated with composition 1 and composition 2, respectively. Control and blank (CK) groups were set, and the control group was given a final concentration of 1X103cfu/mL of E.coli; the blank group was given an equal amount of physiological saline. After 4h the residual amount of E.coli was determined as described in example 6. See table 10 for results.
TABLE 10 Bactericidal Effect of compositions of Escherichia coli bacteriophage EC35P1 in liquids
Table 10 test results show that: the composition 1 and the composition 2 with the Escherichia coli phage EC35P1(Escherichia coli phage EC35P1) not only have good bactericidal effect, but also have no antagonistic effect on other composition components. Compositions 1 and 2 of the Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) have potential for application as a biocide.
Example 15 prevention and treatment of Escherichia coli contamination during preservation of beef samples with Escherichia coli phage EC35P1(Escherichia coli phage EC35P1) and compositions thereof
1. Method for preparing compositions 1 to 2: the same procedure as described in example 14.
2. Autoclaved beef was cut into 75 cubes of 1cm square, divided into 5 groups (single phage EC35P1 group, composition 1, composition 2, control group and blank group), and each group was set into 15 pieces, and placed in a sterile plate. Experimental groups were inoculated 1X10 per block3cfu/mL E.coli and dose 108PFU/kg of test phage; control group was inoculated with 1X10 per block3cfu/mL Escherichia coli and an equal amount of sterile water; the blank group was given equal amount of sterile physiological saline. Each treatment was incubated in a 37 ℃ incubator. Placing a piece of beef in 10mL sterile water every 2h, shaking sufficiently, and determining the size of the liquidEnterobacteria content, experiment was repeated 3 times. See table 11 for results.
TABLE 11 results of prevention and treatment of Escherichia coli contamination during preservation of beef samples by Escherichia coli bacteriophage EC35P1 and its composition
Table 11 the results show: after 8h, a large amount of escherichia coli grows on the beef surface of the control group; in each experimental group, Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) and the addition of composition 1 and composition 2 thereof always control Escherichia coli on the surface of beef at an extremely low level. Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) and composition 1 and composition 2 thereof can be used as biological bactericide, effectively kill Escherichia coli on beef surface and prevent and treat pollution of Escherichia coli in the preservation process of beef. Of course, the coliphage EC35P1 can also be applied to health products or medicaments.
Example 17 prevention and treatment of Enterobacter cloacae contamination during preservation of beef samples with Escherichia coli phage EC35P1(Escherichia coli phage EC35P1) and compositions thereof
1. Method for preparing compositions 1 to 2: the same procedure as described in example 14.
2. The autoclaved beef was cut into 1cm square cubes 75, divided into 5 groups (single phage EC35P1 group, composition 1, composition 2, control group and blank group), and each group was divided into 15 blocks, and placed in a sterile plate. Experimental groups were inoculated 2x10 per block3cfu/mL Enterobacter cloacae and a dose of 108PFU/kg of test phage; control group was inoculated with 2X10 per block3cfu/mL Enterobacter cloacae and an equal amount of sterile water; the blank group was given equal amount of sterile physiological saline. Each treatment was incubated in a 37 ℃ incubator. A piece of beef is taken every 2h and placed in 10mL of sterile water, the beef is fully shaken, the enterobacter cloacae content of the liquid is determined, and the experiment is repeated for 3 times. See table 12 for results.
TABLE 12 results of prevention and treatment of enterobacter cloacae contamination during preservation of beef samples by coliphage EC35P1 and compositions thereof
Table 12 the results show: after 8h, a large amount of enterobacter cloacae grows on the beef surface of the control group; in each experimental group, due to the addition of the Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) and the composition 1 and the composition 2 thereof, enterobacter cloacae on the surface of the beef is always controlled at an extremely low level. Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) and composition 1 and composition 2 thereof can be used as biological bactericide, effectively kill enterobacter cloacae on the surface of beef and prevent and treat enterobacter cloacae pollution during the preservation process of beef. Of course, the coliphage EC35P1 can also be applied to health products or medicaments.
Example 18 control of Salmonella contamination during preservation of beef samples by Escherichia coli phage EC35P1(Escherichia coli phage EC35P1) and compositions thereof
1. Method for preparing compositions 1 to 2: the same procedure as described in example 14.
2. The autoclaved beef was cut into 1cm square cubes 75, divided into 5 groups (single phage EC35P1 group, composition 1, composition 2, control group and blank group), and each group was divided into 15 blocks, and placed in a sterile plate. Experimental groups were inoculated 3x10 per block3cfu/mL Salmonella and a dose of 108PFU/kg of test phage; control group was inoculated with 3X10 per block3cfu/mL salmonella and an equal amount of sterile water; the blank group was given equal amount of sterile physiological saline. Each treatment was incubated in a 37 ℃ incubator. And (3) putting a piece of beef in 10mL of sterile water every 2h, fully shaking, determining the content of salmonella in the liquid, and repeating the experiment for 3 times. See table 13 for results.
TABLE 13 results of prevention and treatment of Salmonella contamination during preservation of beef samples with coliphage EC35P1 and compositions thereof
Table 13 the results show: after 8h, a large amount of salmonella grows on the surface of the beef of the control group; in each experimental group, due to the addition of the Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) and the composition 1 and the composition 2 thereof, the salmonella on the surface of the beef is always controlled at an extremely low level. The Escherichia coli bacteriophage EC35P1(Escherichia coli phase EC35P1) and the compositions 1 and 2 thereof can be used as biological bactericides, so that salmonella on the surface of beef can be effectively killed, and the contamination of the salmonella in the preservation process of the beef can be prevented. Of course, the coliphage EC35P1 can also be applied to health products or medicaments.
Example 19 prevention and treatment of Shigella contamination during beef sample preservation by coliphage EC35P1(Escherichia coli phage EC35P1) and compositions thereof
1. Method for preparing compositions 1 to 2: the same procedure as described in example 14.
2. The autoclaved beef was cut into 1cm square cubes 75, divided into 5 groups (single phage EC35P1 group, composition 1, composition 2, control group and blank group), and each group was divided into 15 blocks, and placed in a sterile plate. Experimental groups were inoculated 4X10 per block3Shigella cfu/mL and a dose of 108PFU/kg of test phage; control group was inoculated with 4X10 per block3cfu/mL Shigella and an equal amount of sterile water; the blank group was given equal amount of sterile physiological saline. Each treatment was incubated in a 37 ℃ incubator. Placing a piece of beef in 10mL of sterile water every 2h, fully shaking, determining the shigella content of the liquid, and repeating the experiment for 3 times. See table 14 for results.
TABLE 14 prevention and treatment results of shigella contamination of coliphage EC35P1 and composition thereof in beef sample preservation process
Table 14 the results show that: after 8h, a large amount of shigella grows on the beef surface of the control group; in each experimental group, due to the addition of the Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) and the composition 1 and the composition 2 thereof, the shigella on the surface of the beef is always controlled at an extremely low level. The Escherichia coli bacteriophage EC35P1(Escherichia coli phase EC35P1) and the composition 1 and the composition 2 thereof can be used as biological bactericides, effectively kill shigella on the surface of beef and prevent salmonella pollution in the preservation process of the beef. Of course, the coliphage EC35P1 can also be applied to health products or medicaments.
Example 20 prevention and treatment of Staphylococcus aureus contamination in storage of beef samples with Escherichia coli phage EC35P1(Escherichia coli phage EC35P1) and compositions thereof
1. Method for preparing compositions 1 to 2: the same procedure as described in example 14.
2. The autoclaved beef was cut into 1cm square cubes 75, divided into 5 groups (single phage EC35P1 group, composition 1, composition 2, control group and blank group), and each group was divided into 15 blocks, and placed in a sterile plate. Experimental groups were inoculated with 5X10 per block3Staphylococcus aureus cfu/mL and dose of 108PFU/kg of test phage; control group was inoculated with 5X10 per block3cfu/mL Staphylococcus aureus and an equal amount of sterile water; the blank group was given equal amount of sterile physiological saline. Each treatment was incubated in a 37 ℃ incubator. And putting a piece of beef in 10mL of sterile water every 2h, fully shaking, measuring the content of staphylococcus aureus in the liquid, and repeating the experiment for 3 times. See table 15 for results.
TABLE 15 results of prevention and treatment of Staphylococcus aureus contamination in beef sample preservation process by coliphage EC35P1 and its composition
Table 15 the results show: after 8h, a large amount of staphylococcus aureus grows on the beef surface of the control group; in each experimental group, due to the addition of Escherichia coli phage EC35P1(Escherichia coli phage EC35P1) and composition 1 and composition 2 thereof, staphylococcus aureus on the surface of beef is always controlled at an extremely low level. Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) and composition 1 and composition 2 thereof can be used as biological bactericide, effectively kill Staphylococcus aureus on beef surface and prevent contamination of Staphylococcus aureus in beef preservation process. Of course, the coliphage EC35P1 can also be applied to health products or medicaments.
Example 21 preparation and use of a kit for Escherichia coli phage EC35P1(Escherichia coli phage EC35P1) and compositions thereof
The kit contains 5-10 mL of titer 5x109PFU/mL of a combination of Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) or Escherichia coli phage EC35P1(Escherichia coli phase EC35P1), 1L of TSB semisolid agar medium, 1LTSA solid medium.
The using method of the kit comprises the following steps: taking out the titer of 1 × 107PFU/mL of a liquid of Escherichia coli EC35P1(Escherichia coli EC35P1) or a liquid of a composition of Escherichia coli EC35P1(Escherichia coli EC35P1) which can be formulated with reference to composition 1 or composition 2 of example 14, the lysis profile of the test phages was determined by the two-layer plate-drop method. 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. mu.L of the suspension of the strain to be detected was mixed with 1mL of TSB semi-solid agar culture and plated on a plate, and 10. mu.L of a liquid of Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) or a composition of Escherichia coli phage EC35P1(Escherichia coli phase EC35P1) was dropped 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 present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by the application law within the scope of the claims of the present application.
Sequence listing
<110> Philippinecaceae (Nanjing) Biotech Ltd
<120> a broad-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria, a composition and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 44460
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggtaagtaca ctactatttt acactaacag cttgacacga tagccgcgac aaaccgaggt 60
tggctataac accatcgagc aatactcctg catgttcttc attgtatgct tctacccaca 120
aagggatggt gacgcccttc aggtggtcgc tgtacacgtt gtacctatat acctgtttgt 180
gtgctttgtc aacacgtaca acagtaaggt taccatcctc gtaaggagga agctcaacgc 240
ctttcttgct ccagaagtta cggaactcag cactattaac aatgccgtgt acaacacgct 300
cagtaagttc tgttttgagg tcagcacgtg cctttgtact gataaagaaa cgacaagaga 360
agaaagcatt gacgtttatc acatcaaccg catacacaac gttatgctct tcagtagttg 420
gtgctttata aacaatgtat ttagttttga acaggtccat agtactctcc agggaggaag 480
actggcgggc ttgcacccgc ctaagttatt acagcagttc tgctttcaat gcagcgatac 540
catcgagcaa tgctttgatg tccgggttag atgccgccag gtcagcaaca actgactgac 600
gacgacgttc cagctctttc tcaacttcca ggtcgaggcg tgctgacgct tcacgcagta 660
agtcttcaac ctgctgacga cgctgatact tgctaaagtc aggcacgtca accagccatt 720
tgagagttgc agtgttgtaa ccagaagtat tggcggcact tacagacacg acatcaacga 780
gcttggtacc gttcagctct ggatagttaa cattacgaac gtcaacaacc gctttatcac 840
ctacacttac tttgccgatg tacttgtaag tgtacacacg accattcggt tcaaacttaa 900
caccaacaaa cttcagtttg tcgtctgcaa taacctcttc ctcaactttg aagttgattg 960
cagtgttctt gacaggtgca gccgctttga gcttagcgac catgtactgt tctgaatgag 1020
acggacggca cagacggaaa acgtcttcgt tataggtcag ccagttaccg gtctcatcca 1080
cgcatgcaat ctgattagtg ccatccagtg aacgaacacc cgggcgattc ttctcaacac 1140
gatacagctt accttctttc aggttagcgt aagaacggcg ggcgataccc cagttgtgtt 1200
tttcagattc gtacttagcg aaacggtcaa cgctgaagtt ctgcttcaga ccacggtcgt 1260
cgattaccca taagttatcg taagccccgc caaaagaacc taggttggca tacactttgc 1320
cctgcgtcaa cagggaagtg tttccctcgt caacacagat aacaaaaccc agagcttcgt 1380
gtgctttacc gctgcggtca aatactgtag tcatttcttg tttccttgta taccagttta 1440
attgtatgcc agagttattc cctgcgcatg tgctttattg cttaaccgtt aaagacatta 1500
tatcattagt gagtggtgac gtacacattt attttaaggc agaagtgcaa atagcatcac 1560
acaaacaata atgagcacgg tcgtagtctt caaacaagaa cacttcgcca ttggcgacaa 1620
cttcattact gtttacctgc agggtaatac ctgaatgcag aagcacatcc atagcctggt 1680
ccttgttctt gtacgggtcc caaataagct ggaagcctga cccacaaagg agacatccct 1740
tgagctccgg aagagttgac tccttcacag tcacattaca aatcttagcc agtttagcac 1800
gagaatcgag agtactcatt tacgtttacc tttacgtttg tgtagcggag tatatgtttt 1860
ctttggtacg ccaaacatgg ccgcatatgc accgacttct ttctttgttg gcttatccac 1920
aatttcgtca atcacgatta agtcatggtc gatattgcga aagacttcag ccgctctttt 1980
ctcaacagga gtaaagtcaa taaccagctc tttgcctatc aggctttctt tgagcttgtc 2040
aaatagccat ttgtcacgcg gaccaggatt gcccataaac ataacacgca tctgcttatt 2100
gtccatggat gatatacccc gttatttgtg ttgccaggta caaaccgtat accacacacc 2160
caactagtat ggcatcaaga atcagatgag ctatcttcat gtttgacctc cggaaggaat 2220
gagttatcaa ccttgatcag tacgttacgg ctgttaacca cttccaggct ggtgccatcc 2280
tttttgtggg ttacacgaat aaatgcaaca ccctcagcct gtgcctgctc aatagcttta 2340
ttcagcacaa cattatcctg taccatatga cgtacgcgag gaaggatatt gcaaagaggt 2400
tccatctcat ccattcctgc agtacgcatg acggacttga gaatgccaag atgatgcttg 2460
taacggtctt caagattata cttcttgctg ttatctgatg ccatgtaagt ctccaaaagt 2520
tagggggctt gtgctaagcc ccgtttaatt actcggcttc ttccgtagct gccggggtga 2580
atgttacagt cggaatacca ggcgaggtct tcacaacagg gtcgacgata gccagttgtt 2640
ctggtgtcag gttacggtat gcagtaactg acatttcata cttggtttta aacagcttgg 2700
caacaacagt cttcggtaac ttcttcatta cgtcaggcac aagcttctgg tctagtgagc 2760
ggttaacctt ctgctcaacc ttgagtttac caggtacgcc atgcatgctg aactcaaccg 2820
tttgtgtacc ttctttacca cgattgataa tcatgtcgcg ttcaagacgt gctttaatgg 2880
tccgacgcaa cgtctcctgg tcagctttcg cttgtgccat ccagcggttc agattcgcca 2940
gcttgtcgta cagctgttcc gtgtccatcg attccagttc ttgtgggttc aatgccatca 3000
gttgtttcct caggttgcat gtgcatagtt gcaccaatta gtagacaata gttattgaac 3060
attgtacgta gtgcttggcc cgcaacaata tccgttatgt ctattatacc acttttagtg 3120
gtgacgcgta aagctttttc tttctttgtt ttctccacaa atgcaatgct gatgatttct 3180
tccgcatgaa ttacgtggat ggtacctgca aactcaccag tcttagctgg tgcctggtac 3240
actaacgaca tagacggttg gttactcact taattgctcc tatttatata agtccatcca 3300
gttacgatac tcgtcataaa accggtcaaa ctcgtttttg gtgagtgcaa tagcaacacc 3360
atttaccaga tgcatggttc gtgttttgat gttgacatac atgacgttgc tgtacgggaa 3420
caagctaccg tcattgtcag tgaagtaagt cggtgtaaca tgaagcacat cgcgcaatgc 3480
aaccagcgct tcaacagctt ctgtatggcc ttcgcggcgg aacgtcatgc aaggcggtgc 3540
accgtcttcg tcctgagtac cgcgcaaagt aaggtacaca gtttcagcaa catcattgtg 3600
tacgactacg ctgatgtcaa cgatgtcgct ggtacggaaa gtaacaaagt gctgtacaat 3660
tacctgttcg tcggtattag gactgacgcc ctgaaaggtc ttgagtgtgt gaaacattgg 3720
ttgtatctcc ttaacagaat gtaagtctat tatgtatata aaggagggga aagtaaatag 3780
atataaatgt ataaaataca taaaagtcta tttactctgg tttaataacg tgtatataat 3840
gagagcctca caaacaagag gagagactca atgataaagc caaaactgtt gcggcatatt 3900
cgcaacggta ttaagctact cgccgaaggc aatgaatatg cctgcaagca acgcggtatt 3960
acaaaagtcg gcctgttcaa taaagagtta ccgactcgct actttaccaa atcaccatta 4020
acactcgtac ctgcagcgga gtattataaa gaccccgaag cagctcgggc taaaatcaag 4080
gaaggtcttg accgacttgt agaggaaggc tatttgacag tacgtcaaca cactgaatca 4140
tcttattact acaagccaac ttctaaggta accagcaatg acaatgacga gtcagccgag 4200
tgatattctc ggtctgaaat actttaagcc agaagggatt ccccaagtct tcaaagatat 4260
gaagcgctgg gccatctttg agctgacgta cgaagacgag accctcgaag ttcaatccaa 4320
acctaagaaa cctgcaagag cgccatcagg ccgttccttc agcacaactg tgcctgaaga 4380
gtggatgact ttcgacgacg ccctgacacg ttgtattcgc aatcgtgaaa tgaagattaa 4440
agctgtacag ggtaatatca cctttgtgcc ggctttgatt gttcctcgcc agtggtactt 4500
tgttgacctg gacaaccatg atgataatcc agacattgaa gcaacccaca aagcgataat 4560
tgaggggacc cgtggtgctt atgctgagac gtcaatctct ggcaaagggc aacacattgc 4620
aatcccttta ccctggactg cagcaacatc taaagagaaa gacgaacagc tggacatcaa 4680
aatccaaaag gctgagatgt tcctggtaat gacaggcaat gtattgagcg agttcaatgc 4740
taatcctgtt tcagcccgtg aatggcatgc ggcaattgag aagttctttc ttgaagcaag 4800
cgcgcctgat attgatgttg agtttgagca agacgaagac cgtggcgaag agtatgacaa 4860
agagctgtac gaacagttag ccgataatac tcgctgggct ttagagcact atctgaatga 4920
gcatgcacca gacggtgttg gtgttagtaa tgacggttct gaacgtttga gccgcatact 4980
caaagacttg cttcgtgtaa cccgtaacta tgaagttaca caacgcatct ttatggccag 5040
caaagctgca gcatatgaag gtcgcagacc aagccggcat aacttgtctg ttgataagta 5100
ccatcaatgg ttcagtcgtg ttggtaagac tgtgctgaaa gaaatgcaac gtgacggctt 5160
attcgttaag accaagtttg ctcttgacat caacaaagag ttagccaaga attcaggtgt 5220
ctccctggct gagaacgttg agttcaaagt caacgatgat attctgccat ctgatgtatt 5280
cgtcaggacg gcaccgtccg ggtttaaacg gcttattgct gagatacagg ataacatatc 5340
tcctgctaac agggttaacg actatgccat cggcacagct cttaacatcc tgagcaactg 5400
cgccggtcgc aagtatgttt gtccagtagg cggtcatgct aacttcctgg taaccaacat 5460
cattcttgtg ggtggttcat caatcggtaa atcgctgtat accgagttgt tcccacaaat 5520
ccaggcaagt gtaccagaca catcgcctat cagccttaac cgtatcccaa gggaacagac 5580
ttttgcaacc aggacgtttg ctgaactgat gtccaacccg tcataccatt cagtccagtt 5640
gttctaccct gaatttggtc tggctttagg ttctggctta cgcatgaatc ctaataaccc 5700
ggacaacttt cagaaagccc tgatggacgc ttcaaccaag cgtaaggttg gtggtattct 5760
gacaggtatc aaacgtgcta atgctgacaa tgatgtcaag acagtaagcg aaccgtgcta 5820
ctctatcctg ggtgactcaa ctcaagagct tatccttgac aacacccgga agcaagactt 5880
ctcaagtggc tttttgcccc gcttcctgtt catccctaac tatgaacgag cggagtttgc 5940
caaacctgag aaggtaggac gtcgccagca gattcgccgt actcaattct ccaaagagct 6000
gattgagaag ctggaagcaa tcgcttatgt gaatgctatc ccgcctaacg gtaaattaag 6060
gcacgacccg attcctattt acgacgagtc ggacgatgac gacttcctgt acgagtacca 6120
gtgcaatatt aatgacatgc gccagtatta cagggataac gaagtagcat ctgcctttgt 6180
gggccgaatg ggtgagtatg tattcaatat agcagcactt attggattac tcgacaactg 6240
ggatgctcct gtaatgacca gggacaacat tgaatgggct tacaaatatg ttcttcgttg 6300
tataaccgcc tgggtcaata acacaagcag gatagtcgca ccaccaacta ccaacgccga 6360
agtagtggac ggcttcctaa gagtctacag ggacattttg gccctttaca ccaaaagagg 6420
atggtctggc attgtcaagc attatcccga aggtgttgta agaagcttac gggaagacca 6480
tctgaaaatg aacgggctga gtatatatgc tatccggcaa tcattaagtt ggttcaagtc 6540
caattatggc tttaacataa ccgcaaataa caaggttatt gacggtatgc tacaggacat 6600
gattgaccag gactatttag cagtccaaat atacaaaccc acaagaggga aatcggcaaa 6660
tatctactgt ttaaccgaac ggggatacga cgaagccaag aaacttaagt aaaatcaagg 6720
gcttaggtag attatatact taagccctta ttttatatac tctttttggc tttttaaatt 6780
agctataatt aagtatagag caaaaatatg gtcctaagct cttaatttat atactcattt 6840
tgattttata atatatggcc tcgataaata aaaataaagg gggtaataaa ggggatacaa 6900
gtaccggata aaagggggta tatatttatt ttattattat tattattatt atatattatt 6960
aattatatta gaagtaataa aaacagctac ttagaggcgt ctatatgtat gatccggttt 7020
atctctttat taatatcaga accggatgaa aaaaatttac gctggcgaaa aaaataaaaa 7080
atccggattg ataaataaat ctgtagacgt ttacactata aaaaagatgt gttacaatat 7140
ttatcaatgt tgctgattac caaatctttg gattatatac gaggctgaat aaatggttaa 7200
tattagacag aaaggacaga actttgagcg caaaatggct aagaagttca atagctcgtt 7260
cggattaact gttgagcgca atcttcaaca gtctattaat ggcggggatg acttactggg 7320
tgtaccgttt ttcagcgttg aactgaagaa gcacaatacc aagtctatcg ggacatggtg 7380
gagacagtgt gcggcatcgg caaaggctca gaacaagatg ccggtactga tatacgaact 7440
accaagaaaa cagcctaccg tggtgatgaa taaggctgat gtgattgact gttttccaag 7500
tggcacgact tttaacgagg catcgaaccg cctctttgtg ggtctagtag aattaacatg 7560
ggaacaattt tgccgggtct acaactatta cgttgcgccc ggcactcaat atcacgtcaa 7620
ttaatcttgt ttcagctcgt cacaaatgac acgctgattt ttggcaggga taataccaag 7680
atttagcagg gtgtatggac tatcagcgac acattgttca acggtagtcc attcaggttt 7740
ggattcaaca attttgtaac tacaatcatg agtagccaca catgttgcca ggattaagcc 7800
aatcacaagt aatcaccttc aaccaggaac tcaaacaatt ggttgtaaac gttgcaacct 7860
ttagcgaaag agctttcagc aggtttaccg tcgcggacga tcatcatgta ctcaccgcca 7920
tgaatatcca cccagtattg catgccacct ttgacagggt tacgttgtaa gaatttaaac 7980
attatttgct ccttattcca agtttattca ataaccaaat tgtcatgtca acccaggtta 8040
acggtacagt tttcgggggt tgataattcc agcagcttgc agttctttgt atttctgctt 8100
ggcgccgctc cgcttcgcgt aataccgcat cgtcagaagt gccggcagaa aaaagatcgg 8160
taacaccaga taaatcgcgt tgattaacac gtcgagcaat gtcatataaa tcatccttag 8220
aaaacataag ccaataccat aagaattatc ataaagaccc caaccagctt cctcatcgcc 8280
cgaccttcag catatgcagg attcacaaca gaatactcaa caacatgttt acagcggacc 8340
ataaggcctc cacttgtggg ttaattcaag catgaggact ttcatcaaat cctcaggatt 8400
gaattaatta ccagcttcta atcaagccct gtcagcgcca ggacttgact aatatcactc 8460
tatcattacc tgaactacta ttatacactc tttattgaag cgagtaaaca cttttccacc 8520
attagtgcgt ttagccccag attttagagc tgactggcac cagatatgcc actcctcaac 8580
tgtcaagaat ttttgttcag ctttgggtgg caatccattg gcaaggtcta ccgtagtaaa 8640
tttgactagt ggcatcactt gacctcaaat tttgactcgt cgtattccgg atcagtcacg 8700
tcccgggtcg cacaattttt gattgcttcc cagtagcgtt gggcatcatc accatcgacg 8760
taaaccgtcc agtggccgta accaccttca aatctgtccc aggcttgttc ccagactttg 8820
ccatcttcca gcttgtacca agttggacga ctgtcaaact cacgaattgg atggtacccc 8880
atcaagggat tcatcaggtc gataatgtct tcccgactaa tctcctggtt ctcccacaac 8940
accgtcatga attgctcaaa cttgaaggcg tttggcagaa ctttgagtac tttaccgtca 9000
caatatacgg tatagatacc atcttcgcat tttgcactgt acatgtttct ctctcctgtt 9060
gattgtgaat aatttcaaaa atgaactatc acaactttgg acagttcatt gttgaaacta 9120
tgctaacttt gttgcgatct gaattgtcag ccggaaatct tccatcaagt tatcaaactc 9180
tttggaacca ccaacagcat cagatatttg atctttcaca atttgggcat ttgattccag 9240
taattcaatc aggtgcgcca attcgtcttc agtcaatttc agatctttca taatgcatcc 9300
acccaagcca taacttttgc cagtcttaca acgtcttcat ggtcccgctc gccatcagaa 9360
aattcgctgg cgatgatacc cataattgac gccagattca tgttgatgat gtaggcgtgt 9420
tggcgcactt caccaattcc tgaatctatc accagttccg caaattcttg tgaaccttca 9480
tcccattcag cccaatactg ctcagcataa aagtccagtt cttttttcat ctcaatgtca 9540
gtcattttga ttactcctca gaacctgaaa atttgtcgtc aatggcttca acaatataat 9600
caagagttga agtgtgagaa ctatcagaaa aagtgcccat cttctctaac tcaatcatca 9660
tatcacgcaa ggccatcgcc tggtctttgg tcagtttgat tttaagcatg tttctctcct 9720
gttgattgtg aataactttc attgatggca ctacttgagc gccatctcaa aaattactct 9780
tcagtttctt gtgcttcagt ttcaggtttt acttcagtat caggtttttt gccagttact 9840
gatttgcctt cggtcacttt accagatttt acatcattac ggtagtatga catatatgac 9900
tggataacac gttttgctcg aatatcatcc gcttccattc cgcaagcttt catcgcatca 9960
gcgagcgcca atttggaatc ttccttatcc atcaggttgt aatctttgaa caccagatag 10020
caggtatctt tagttgacgg accagatgac gatttgcgag agccaccagc ggatgctcgt 10080
gttgtcgcaa ctttgacttc ttcaacttcc agttggttca gctcgtcaaa caaagccttc 10140
accagcactt ctttggagtc tttgacgctt acagtacttt catcattaag cgacttaatc 10200
atcttaatga tctggttgtt ggcaaagtac tgaagactat ccatctgcac tacaatagtg 10260
ccaagacttt cagtagtcag accagatttc acagactcga cagaaaaagc ggaagtcagg 10320
taaacgtagt tgatagttgt catttgatgt atcctcgttg atagaatttt tgatatgtgc 10380
caatccttgg caagtaagtc caaacatgac aacttttgtc aagttgtcat gattgaaatc 10440
acttcgcgct tgcgttttca acggcttgta caactttcaa caaatgcctg aagtcatcat 10500
gatagatttc attaaagcca tctgccgcaa agttttcgcc aattaagatc agtctttcgc 10560
ggaccaagtc aatcaatagt ttattgtcag ttatttccgc acctgtaaca agttcaacaa 10620
ataattgctg tacgttatca taacggatgt tgctttcaat acacacttgg cgcatgtctt 10680
caactattga catgattgtg acttgcattt ctacgtcaga agtgatatta tttagtttca 10740
taatagttac ctttgtacta gtttccaggt gtgactactg ttaccaatag tcactagttg 10800
aaactactac ttgtgttact atcaacaaga aacatgttac caaattgtta aagagcaaaa 10860
ggtaccattg acatctacca gatgtacctg ttgactggtt aagtactacc tacctaatct 10920
aaaccatacc aatatccttt agaccagtta gactggtcag ttagtcatca cattcaataa 10980
atatataata gaccaatcgc tattgattgt acataaaaga attgcaagtt tatgcaagtt 11040
tttagaaatg ataatcactt gcgattgata gtggttctca tatgcaattg atatagtttc 11100
acatgtaatt attcaatcca aaatggtcgg caggggttat aataacatag gggtgccact 11160
taaaggagcc ggcgaaatta tttctaaaaa tataatttca atctgaatta tagacgcttt 11220
gtactatacc ggccgaagta gttcacacgg taattgtacc aatcataata gttaccagag 11280
taatcatttc aattgaaata ggtgcggcgg ggggtaattt cgcaggggtg ctacccgcca 11340
ggagccagcg gaattttttt attttttggt atttttgcct tatcaggacc aatcatcatc 11400
agagtgatac aacaggagct aaccttttgc ctaacacaat aataagtgaa gcccctctaa 11460
aattttttta tatttttcgg aaattgtgta gacactcact aaaaagtggt gtactttctc 11520
tcctccacgc gctatcatta atgtgaatca tcaaacggcc tcacggtctc ctacttgtgg 11580
gtaacattat gaaaattgtt caattagagc catatccgcc tcaattggct atcgtttgta 11640
gcgaaaaaga gcatgaagag ctccaaaaac tgatggattt gcctgatttt ggcaactgga 11700
atgaccgaga aacacgtaat gcatctacct ttacattctc tcgcgttaac tacccgtacc 11760
tgttaatcgt cgtcgagctg taccacccgg acgacctgaa gtacaacacc atttcccacg 11820
aaggcattca cgtaatgtca tcgctgatga actatgtagg ccttaaatac gacccagaaa 11880
acgatgaatg gtatgcatat cagcatgatt tcatcgtaaa tgccatttgt aaggcccatg 11940
acgagtatac agagcgccaa aaagcgccaa aaactaagcc aaaaatcgaa acaggtaacc 12000
atcctgcaat actgccacaa gacgttatga actcgctttt gactgacact actggggact 12060
ttttaggtga ttaatataat ctttgcaatg ggccaaaatg gggaattcgg gttatctccg 12120
gagtttatga agcatcaggt tgactcgtgc aagaccatgg aagaggttcg tgcacttcct 12180
aaatcgggtc tgccctggaa atgtccgtca gacatgctct ttttcaggac tatgactaat 12240
tcaattgcga cagaaccagg ctataatgca tatcagcaac tacgtggaca attcccaaat 12300
gaccagttgc gaaatatgat agcagatgta acggtaaaaa tggtcgggca tagcgtattg 12360
ttagccggtc gtaatacgta tatgactatg agtctgccta tgtcagccca cagaatactg 12420
ctgccggtat ctcgccagat tatggcttca gaaggctaca ataatctggg tgagatggca 12480
gcagaccttg aatccagagg tactgacgtc tggattgtag gtggtgccga gttgatactt 12540
tcggcccttg aagagcatgc aaaagactta ctccgtatcg acaacatgtt tatcagtaca 12600
atcaagcaat caggtccaag tgatatttta atggatcgta gcaaattaat gatgtacatt 12660
gatagcgact tcacttataa tgatgaatat gttgataaca aacaagtgct gatacaacgc 12720
tacaggagtt tacacaaatg aacatcataa tcgaggggcc cgacaatgct ggtaaaagta 12780
cacttattga gttccttaaa tctcgtactg gccgcgatgt tattcataat actgttgata 12840
aagaccccga atcggtcatg gctaaacaga ccaaagaaaa cgcgttggaa ggcaattgca 12900
tttatgaccg tagcgctgtt atatctgagt atatctattg cctggttctt caacgtacgc 12960
ctgtcatcaa tattaatgta tcacatgtag cagatatgtg tgacaacgcc atcgttatct 13020
tttgtatgcc gcctgttgaa aaggtactgg caactactaa agacgaaatg cctggtgttg 13080
tggaaaatct tgaggcgtta tacaatcagt atgacaacct cattgacgag ctggttatga 13140
tgagtaagca gttcttcatt tacgactgga ctgttgaaga agacggtcct gaagcagtta 13200
tcgaatacat caacgaacgt attaataagg agtggaacaa atgagcccag tatacaatac 13260
ctcaatcaca ctgtcagcaa ccgtagggga cgtcaacaag accgttactg tgggttcaac 13320
taacccgaac ctgtcaggcg aagacctgct gcgcctggcc aaacgtgctc ttgaagataa 13380
cgaagtgact ggtactgaag gcgtcagcat cgaaattaac gtcgaaggat gtccacacag 13440
catcagtatt gcgacttctg acggtgaaat tgaaaagttc aacgatgagc tggaatatcg 13500
tactggtaag atgttcagca gtgcgttaaa ctggtacttg tgcaaggcct cgggttatac 13560
tggtgctcct atgtcagacc gtatgttagc tgatatgtca gctcgcgaaa ttgctaaagg 13620
ctgggcacat gtacagatgc tgcaggagca tatctgcaaa ctggaaagct gcaataaaga 13680
tgcgtctgag cttatcactg cctataaacg gcagattacc gacctgcagc agttgatggg 13740
taaacgtgct gaagagtatg aggctgagat tcgtagcctt aacggtacag ctgttgctaa 13800
taaccgtgct ttcagcgaag agcgtaaagc gcttattgcc cagcgggacg ctgaaaaacg 13860
tcgtgcagac gacctggcag accgtctcaa ctttatccgt aagcatatgg atgctatgga 13920
aggggtaatc tgatggacct gttcgatatg gtcgaacatc ctgaagtaaa cggcttggcg 13980
gaagctatgc cgtttagctg ggatgaattc gtagcaggca aacctgctgt aacccacaac 14040
gggcagattg catattacgg aggccatagc gaacatccga tgtgcactca cctgtacaac 14100
gctatcacaa ccatatccga cgaaaacgtt attaagtacg gaaaagaggt aatgcctttt 14160
cttgtagatg aacgcggtta cttcgaaggt aaatggttcc cgtttctggt agaaatggcc 14220
taaaaaccgg ataaatataa aggcgtctaa gctagttctt catctatata agctacttat 14280
tcgcctttct actgtagact ttctcatcat ctctctagac acttcatttt tatattctgt 14340
ttacttactt cgcgtaactc gttataatcc cgactatccg gattaacgag gacgataccc 14400
atggcaaaac cgaaaaaaac ccgtgaggca acagaagagc tgttggccac gatgcgacgt 14460
atccaactgg acccgttgga agttatgcaa cgggctatca tacttgctga acgtgaagac 14520
gactacaaag cgatgatgga tggtgcactg ggcgttatgc cgtacatgta tcctaaactg 14580
aaagaatccg ttgtcaaagc agacatcgac cagaacctgt ctggtaatgg tgtcaacctg 14640
aacattacaa taggcgacca gaaagtggta gatactactg gggaagagta ataaatgaat 14700
cttgactttc acctgcatga cagacaaggt gccgctctgc agtctcaggc aactgagatt 14760
ctatacggtg gtgctgctgg tggtggtaag tctcacctta tgcgtgtaat gtcaattatc 14820
atgtgctgca tgatacctga cttgcagatt taccttttcc gccgaaacta taacgacctt 14880
acggctaacc acatggttgg tcccacaagt tatcctgcat tgctggacgg tatgtgcaaa 14940
gctggcaagt gcaagattaa ctactcgagc aacacgatta aattctggaa cggtgcgaac 15000
atcaagctct gccacctcca gtacgaaaaa gatatttata cctaccaggg tgcggagatt 15060
cacgttctgc tgatggatga gcttactcac ttcacagaaa cgatgtaccg cttcttgcgt 15120
aaccgtgtcc gactcggtgc tctcaaagtc cctgagaact ttttccggcc actgccactc 15180
atcttttgtg ggtcaaaccc cggtggtgtt ggtcacaact gggttaagcg cacttttgta 15240
actgctgctc ctccgatgaa aatcgttaag caggaaaaac gcgaaggtgg tatggcgcgt 15300
cagtacattc cggctaaaat gactgataac cctacactga tggaaaacga ccctgactac 15360
ggcgaccgtc ttgaaggtct tggtgacccg gctcttgttg ctgcgatgaa agaaggtgat 15420
tgggatattg ttgctggtgg tgcgcttgac gacgtatggt caaccaagtg tattctgccg 15480
cgtatgcgta ttcctaaagg ctggactatc gaacgttctc ttgactgggg ttctactcac 15540
ccgttctctg ttggtatctg ggctatcgca aatggtgcct cgcttacaga cgacgaaggc 15600
aatgagtact ttattccgcg tggttcaatt atacgtatag ctgaatggta tgggtgctct 15660
gaaaaagaag ctaacagggg cattaagtta tctgctgccg aagtcgctca aggtattatt 15720
cgcctacagg aaaatctgca ggctggtgga tggattcaaa gcaacgttaa tcctggtccg 15780
gcagatagtc agatttatgc gtgcaaggaa aaagacgttg agactatcgc gaagaaaatg 15840
gaagacgaag gcgtaacctg ggtcaaggcc gacaagtcac aaggttcgag aataaatggc 15900
ttagagctat tccgtcagct tctgaaaaat gttaagatgg acgaaacgga aaagccgggc 15960
atttacttca tggaaaactg tccagcagcc ataaacacgt tgccggttct gccgcgtgac 16020
aagaacaaca cggaagatgt cgacacgaac tctgaggacc acgtatacga tgattgtcgt 16080
tacatggtgt tgcgtggcat caaccgttat actaccaagc tcaaaataaa tatgggacgg 16140
taaaatgtcg ataaccccaa gcagcacgcc taacgtcgga taccagcgtc ctgagctgac 16200
taagaacgct ggtaagtacc gaatgattaa ggactgcatc gagggtcagg aagctgttaa 16260
agcacgcggt gatttttatc tgcctcgtcc tgaccctacg aactgcacag aagaaaacct 16320
gatgcggtac aattcgtacc tgcgtcgcgc tgtgttctat aacgtactac agcgaactct 16380
ttcgggtatg actggcctgg tattccagca gcagcctgag atgttgatgc ctgacgatat 16440
ggaagtgctg aaagtaaata tcgacggtgc aggcattggt gctattcagc aaatgcgtaa 16500
gtgcttcaag tctgttctgg cttttggtcg agcgggtctg ttgactgact tccctaaagc 16560
acgtatcggt gagtttggcg agtccattgg ctttacccgt aaagagctga tggatggaga 16620
tgtacagccg acagtaacgc tgtatatgcc gtggcagatt ataaactggc gagaagagta 16680
tcgcaacggt aaagtgatgc tcactcaggt tgtcatttgc gaacagatac cgggggttac 16740
tgatggcttt cagcaagact acacacccgt gtggcgtcac ctttacctgg atgaagaagg 16800
caattgcaac cagtctttgt gggtcaataa gggcgacaag aatgcgcaga acgtctccgg 16860
tggtgcgttg tatgtcacgc aagaaaatgt tgtcttacgc gactccaacg gcttaccgct 16920
gaagtttatt ccgttccagt tcattggttc tgaaacaaac agcgctgaga ttgaccctcc 16980
gccgatgtac gacttagctg tgcttaacat tgcacactac cgtaactctg ctgactacga 17040
agaatcatgc ttcgtaacag gtcaacccac agtatgggca tctggactta ctgaggcatg 17100
ggttgaagat gttctgaagg gtgaactgcg actcggttcg tttggtggta ttccgctgcc 17160
ggctaacgcc gctgtaggta ttatccagac caaccctaat acccagccgt ttgaagcgat 17220
gaagcaaaaa gaagcacaga tgattgcaat cggtgctcga ctgattcgtg agaatggtaa 17280
agtagaacga cgcgaagttg agattaagaa tgaagcggcg tctgaagctt cgcttatcgt 17340
aactgttgcg cagaacgttg aagctgctta tcgtaaggca atcgaatggt gtggcctgtt 17400
ctacggacac cctaaagaag aaattacgat taacctcagc tacaacttca tctatagcca 17460
gttgacttat caagaacgtc agcagctggt taacgaatgg attgccggtg ctatcacgtt 17520
tagtgaaatg cgtagcaatc ttaaacaagc tggcattgct aagctcaccg acgaagaagc 17580
caagaagcta attgatgaag agctggctga acgcgatgcg cgtgaggtag caaaagaagc 17640
agaaaagaac gctcaagcag ctaagaacag cgatgggcag gaaaataatc cagaaaaaac 17700
tgaataaagc tatttacttt cggtttacct ggctatataa tccttcatgt caaccgcgca 17760
acagccggcc acgagagtct gcagggctca gtggatttcg gatggctggc tgcgcggttt 17820
gacccaaacc tgttcggagc ccacttgagt ggattgagcc cctctcatat aggattgtaa 17880
aattatggca cttaaaacag ttgtaactga ccttaccgaa gttgatgaag gcttccgtgg 17940
cctgtatatc aaagacgaag catctggcgc attccgtttg gacgttgaac cagacgaaaa 18000
aacgctgaaa gaattagaag acctgcgcaa agaaaaacag cgtatggaac agcacaccaa 18060
aaagcttctc gaagaaaaga aaaaagaagc tgaacgtgct cgcctggctg aagaagaacg 18120
cgcacgtcgt gagcgtgatg ttgaatctct tgagcgctct atcaaagaga agcatcaggc 18180
tgaactggaa cagtatacta atcgtcaggc gcagctgcaa gctcagctgg aagcgcaaat 18240
ggttgataac ctggcgctga acctggctaa ccaactctct gacaccccgg cacttattat 18300
gccacacatc aaggcccgac tgcgcgctca ggaaattgat ggtaagtggc gtactactgt 18360
cgtggatgtt atgggtaacc cgactgctac aacgccagat gagctagtgg agcatatgcg 18420
cgctgataag cagtttgcac ctcttgtgcg tggaaccaag gctggcggtg gcggtgccaa 18480
cggcggcgga gctaatcctg gtgggaacgg cggtggcgaa cagcacactt tcgggggcgg 18540
ggatgaccgc atagcacgtg ctcgtgctaa aatccaaaac acacaatggg gcaaataagt 18600
tatgtcactg tatattttta acgaacaagt acgttctacc gccaccgaac tggtcgacca 18660
ggacgtacag aagttcaacg aagcttctgc aggtaccctt atcctgggta accagtacgt 18720
aattggtgat tacgttgagc aggcaatgtg gcagctggtt gacggcatcg ctacccgccg 18780
taatgcttac gctggcacca acaaagttga tgcgcagact ctgggccaga ttctggaccg 18840
ctctgttaaa atcgacggcc gtgttggtcc tctgaacatt accggtacta tgatgcgtcg 18900
catcggtgca ggtgacactg aaccagcagc tgttgttgct gcacaggctt ctgctgcaat 18960
gttccaggat tacctgaata ccactgctgc atgtctggta gctgctatcg gcgcaaacgc 19020
tgatgttgtt ctggacgtaa cttcaggctc cggtgctgct gcaaacatca ccatgcagaa 19080
cctgaacaaa gtcaacgcga agatgggtga ccgttctcag cagattcgtg cgtacatcat 19140
gcactctact gcgttccact ccctcatcga tcaggctatc aacaacacgg aacacctgtt 19200
ccagattggt gacctgaacg tgtacaaaga cttcctgggt cgtcgttatg tcgtttctga 19260
catcccggct ctggttgatg gcgccggtgc ttccgctaag taccgcactc tgggtctgac 19320
cccgtctgct gcaatcgttc aggtcggcgg cctgtacgac atggttacct ccgagcaaac 19380
cggtggtgaa aacatcctgc gtcagatgca gggtgaatac gacttcaacg tgtcgctgaa 19440
aggctacagc tgggtcggtc acaaagctgg tcagtctccg actgatgaag acctggctgc 19500
tcaggcttcc tggtctaaag ttgcgacttc caacaaagac accgctggtg ttatgttgct 19560
ggctaagtct gcaatctaac ccacaaacgg gactaacaaa aggcgagctg acgagctcgc 19620
cttttttatt tacacgtata aagttctggt atataatcgg actaaacttg ctggaggctt 19680
tctatggcat acacacttgt tgtggaagac ggttcaattg ttccgggtgc taactcgtat 19740
gttagcgcag cagaagttga tgaatttatc actaacgaag gtattctcat tcaaccagga 19800
actgacccgt actaccaggc tgccaaagca gctatccgac ttgataacca gtttaactgg 19860
ctaggtcgta aggtcagcaa aacacagagc atggcttggc cccgtactgg tctcggtgca 19920
tgcaatgttg acatcgcgga gtctgtaatt ccgcgagaag ttaagctggc acagctttat 19980
atgatggcca gtgtattgag ctacgacggt gaagaaggtg tgcgtagcga ccaggctttg 20040
cgccgtaaga aagttggtaa cctggagatt gaatacttcg actccaaatc aggcgttacc 20100
ggtgcaactg acctttggac gtcagacgtc aaggtcatgc tcaagcacct tgtgggtgca 20160
tcactgagaa cgcggagagt ataatgattc gtatacgcct tgaacgtcgc gacaaacaaa 20220
agttcgagtt cacctaccgt gcctttaagt cgtttgaagg tcaaggcgta tcagtaggtg 20280
ttcatccaga gcacaacaga cgtataaatc ccagggaccg tataacaaac gccgagctag 20340
caatggtgca tgagtacggt cttaacggtc tgccagaacg aagctttctt cgtagtgctg 20400
ttggtggcaa gggtaaaggc cgacaggcaa taaacaaagc tttccgcgat aatgtacctg 20460
ctgttctcag aggaagcatg actgcgcatg agcttaacga caaaataggt cgtatgcttg 20520
ttgacgctgt tcacgacaga atggacagtg acgtgccacc tcctaacacc gagctgacag 20580
aagaacgcaa acgcgggcca ggtacactta gagaatctat gcagttgtat gattcgatag 20640
gttacaaagt tggcagaaac tggaggctat aatgctcaag ataacttcgc taatgaagga 20700
taaaaccttt gtagcattaa cagatgttct tctccgaaat gattcacgct taccaaacgg 20760
cgacttcgaa gacgaatata cagctgcaaa gtatctgtgt aacattcaac ctggtgcagc 20820
tactaaccag cctaacctgt tgactaaaga cgatggcgag catgagttcc cacgtattgc 20880
gatatactgt ggacaagagc tgaagctcgg agacttcgtt atctacgaag accagccata 20940
ccgtgtattc cgtatcgacc catggagtcg tcatggccac tactacgtta cagcaattct 21000
ccataaagga cctcagggag ctcgttcaga agcttttaac attacctgat ggttcatgca 21060
ttattggtga agagtctggt acagttgatg gtacaccgta tgtttatctg cgtcctggtg 21120
atgcttatga cttcggacca cctagaacca gacaaggctc tgacggcttt gagtacgtca 21180
gcaagccaat gatgacggct gtacgtatta cagctgtcgg taagaatgct gctgagctaa 21240
tgcgtaagct acacatagcg ctgggtagct caccagctaa gcagtttaac cgtttacgac 21300
attatgcaat aaccaaaata tcgaagattg ataatgtcgg tggtgctatt ggtgcaggct 21360
atgcgcagaa gtcaatgatg actgttgaca ttagctatgt gcataaagtt aaaattgagc 21420
aaccgtacat ctacaaggtt gacatcactt gtatggatga caacggtaac aaagcgactg 21480
gtacagtaga ggaaccgtaa tggcaactgg agtagacccg gaactgcaag cgcagctgga 21540
taagctaaca actgctgtcc agctcattca tgacttcggt atgtcagaca acccaaacat 21600
ccctaacccg gaaggcggtg tcatacgcac actcgaaggc attaacgctg caatcaacga 21660
agctatacca aactttgaaa acgctaacga agctgctatc caggcgcgtg ctgctcgtga 21720
cgcggctatt gcagccaaag aagctgcaga aacggcagct gaacaggcca cagtagcttc 21780
gacgtcagca atctatgagt acggcaacgt tgcaagtgca gctacagtga ctataccagc 21840
tgcatctaag acaattgtca gccttactct taaccagcca aacaccgtaa ttaatatcgg 21900
tggtgtatct gacccgtcta ctgttgcgcg tcagattact attgctatta agcaaggcac 21960
aggttctaac aaggtgactt cttgggaccc cagaataaaa tgggcaaaca acagaaagcc 22020
cgttctgtca ttccttgtgg gttacacaga tgttgttacg ttgcttacct ttgattcagg 22080
tgcaacttgg atgggtttct ttaacggtgg atggattaaa taatgcacac gatgttgctt 22140
aaagacggat cagaccatcg agctaatgtc gaaagcctta tcgaaggtca ccaccatttt 22200
cttgagcgca atactggcag aactgacgac gctacgcgtc agcactatat cttcaaccca 22260
gaaggagtag tgtcgaacaa ccgccatttt attgcacaca ctatgatgga gtatcaacct 22320
aacggtgatg ctccgtctga aagtcagtcg ctgctaatac tcggcgaaat tcacgcgtac 22380
cttgctacta aggaacagcg gtacctggac aaagcaattg agtacttcaa tgcttacgtc 22440
aagtactatt acgaggggga cccaatccct gatactcctc gccgctggat cgctaactgg 22500
ctttgtaatg gcaaagagcc tgtgttagcc agcttcccaa tcaaccctga ttcaccaaca 22560
caaggcggct ataaaaatgt gccagttaaa tttgttaacg gcaaagcaca aatccctcat 22620
ggttctcctt tctggggcga gtacctggat gttgctactt gggcccacag gggacacatg 22680
gcttggcctg ctattaatgg cggtgtccgt gttattaaaa ataaagttga ctgggacgat 22740
atttacaaca attatcgcat cactaccatg ccagctgacc cgtggaatca gttagcctgg 22800
attgactggc cgaggtatct gggtgaacca agctacactg tggactggtc agctgctgaa 22860
cctcctgtac atctcatcga gtatattgtt gcatggacca ataacaagat tggtgttctg 22920
cctggcaaaa acgatgagct atggggaggc gagattcttg agactggact gcctgactct 22980
cgcagaggtg agattcaact ccgtgaccac actgttaacg gtgtgtatct gcttaactat 23040
gccgttaagc ttcctgttga gcacggtggt tatttgctta agcgaaatga agtatggcac 23100
aacagaccgg taaacgttcc actgtacggc gagcttcaac gcggtaatgc tgcggatgct 23160
gagttatggt tctgcgactg ctgctatatg ctgtggcgaa ttactaaaga gcaaaagtat 23220
tacaacgctt ggcgagctgt cctgtttacg ctggaggagt atattgatat tgataaacaa 23280
gaccagttct tccgtcaaga ctctcacgcg tcatctccgt ttactgatgg tatctcttat 23340
gattggtctt atcctgaact ggcacaagct atatattcac gtgatgaaaa tggttatata 23400
gttctacgcc agaaccaaaa agctcaatca tctctggagc aacaggctgt atggtttaag 23460
tgtaagccaa cagctaagat acgtacagag gttggcggca aagatgatgc tggtcagaat 23520
atcagtgtca aggtggaaat gtacatgaac cgtgaaaagg ttgacgttga gcaaaacatc 23580
tatcgttttg tcacacagct acctgctctt ggtgcttcac ctgctaacta cgacattccg 23640
ttaagcgatt ttgtcgcagg tactaagcca gatggttctg agtatatctt agctgatatt 23700
cgttcatcca ctgattatgg caatgccact attgcagtgt cttaccgtac tgacatactt 23760
gacaacagac gtggcaacac tgtccgtatg acaataccgg atagttcgtc aggtgccatt 23820
ataggctttt ggctactgga atcttcccag gcacctttaa actcgctgac gtatacttct 23880
acaggtccgt tgtgcataaa agtaactgac gcgcaaggct ggagttgggt acgtgattta 23940
cctgccgcgc caggatgggt aactcagcag attcgcacct gggagttcag gctggcgtca 24000
taccaggaaa acacaggtac accacctacc agcccacaat tcggtgagat taagcagttc 24060
gcgtttgtac tgcctgacgg tgttaatacg tcgacaagta ttagctggta ctgtgtgaat 24120
gatgttccgc ctcgctataa ctggccaatc ggctatacga ttaagtatcg tcttactttc 24180
tcggctgata acccgtacac agctaagcta ggcgactgta ctgtgctgga ctatgagaat 24240
gataacctcg cttacacgcc aggtgttatt ccgttctcca acatttataa ccctgattct 24300
aagcagttcg atggttggca cggtatacct taccctggct atcagtatcc gtttgtcttt 24360
acgcatgacg atactgcagc aggtacaatc aggctgaaca acatggttaa ctttttgtac 24420
gattctcagc aatggtacta taagaagttt ggtcagctag gtcctggtgc atctgctttt 24480
atctggaacc gttgggataa ctttaaatat ggtgtacctg atacgtttac catgtatcac 24540
tggggtaatg gtagtgcatg gtcaggttac cagcctcgtg cttatttctc cgcagcacgt 24600
ggatggtatg agctagtaaa ccgtggtaag ccagttccga gtaagcttat tgcttatgtg 24660
gataactggt caaaatggtt gcttgacttc atggaaaggt caggaggtgt gtcgccttct 24720
aacttcccgc aagaaggcgt tgctctgcca gtcccttcag acttcacagg tcacatgtgc 24780
ggactatggc ttgctggtgc agctatgtca gccctttgtg ggtcaacggt gtcaggactt 24840
gataagctga tggatatgct tgtcaatgag ctggaagacc actatgacat aactggtatt 24900
ccggggcacg taatggatgg tggttggtcg ccagcgttac gccttgaaac tggtacagga 24960
cctgaaagca acgccatgta ctatggtttt tactctggcg aaatattgcg agggttgggc 25020
ttatacctgc tgtacaaaaa caaaggggct aaagcagata tttatgattt aggcccgtaa 25080
ccactattta cttaaccgag gcgccagcct ataataagct ggcgcaccca ataagaggat 25140
tctccagatg gcaaatcgta ctacagccaa ccgtcctgtt aaagacgcag acctgcgtca 25200
agagtcggtt actcagttcc cgttatccaa gccaaccaag cttgacgaga acggaaaatt 25260
cattccaacc ccagttaccg gtgtaactgc tacgcctaag actaagtcaa ttgctgttgg 25320
tgagtttaca actctgtcag cagcagtagc accggcagac gcagatgacg tcaccgtgtt 25380
gtgggaaagc aatgcgcctg acaaagccac tgttgatggt tcaggtcgtg ttcagggtat 25440
caaagttggt tcagcaacta ttactgctaa atccaagctt gacccttcca aaacagatac 25500
tgtagcaatt acggtaactg ctccgccggt agccgttact ggtgttacgt tagcgcctaa 25560
gtcgccaagc gttgtagtag gctccactgt acagctgact gcaactgtac tacctgctgg 25620
cgctactaac aaagctgtta cctacgaatc aaacgaccag gctaaagcta ccgttagtgc 25680
aactggtctg gtgaccggta aagcagctgg tgtcgtcacc atcactgtca aaacagctga 25740
tggcagcttc actgacaccg cgtcagtaac tgttactgct gagtaaggag ctgattgatg 25800
tcgcttccac ttaatgaggt tgtcgacgtc cagttcagta agagcaacgg gggtaaaggt 25860
tttaccccca atgttccgct ggaaggtata atgctttcca actacgagta ctcaatcgta 25920
gcgggtgatg accctgttca gctagttgtg cgttacattc caaccaacgc tactgagcgc 25980
ggtatggcat ggaaatctag cgacccggac gttatcactg ttgatgagct cggatttgtg 26040
aagccactgg cggaaggtgt tgcatacgtg actggtacgt ctgttgacgg cggttataca 26100
gtacgggcaa agtttaacat tgctcgggca cctatccttg ttgaatctgt tacagtgtct 26160
cccacaagcg tgacgcttga agaaggcaag actcaacgcc tgacatacac ctatttacct 26220
gctaatgcag acaaacctga tttctactgg atttcagatg atgagtctgt tgctatcgtt 26280
aacgacagcg ggcttgtcac tgccattgga gatggcgaaa caactgtaaa cgtgcagtac 26340
ggtgttgaca gctctatatt cgatagctgt actgtaactg ttattccgcc tgttgtacac 26400
gttacaggag ttgagtttct tactcagtca cctcttaacg ttaatatcgg aagcacagct 26460
gggactagta ttcgtatatc gccagaagat gccactgaca aaacagtgac atacgaaagc 26520
agcgcgccta ctgtagcaac tgtggatact gacggaaatg ttactggtat tatagctggt 26580
aacgccactg ttactgttaa gacgaatgac ggtgataaga ctgacactct tgacattata 26640
gtcagggtgc ctgctgtaaa tgttaccggt gtaagtattg acgaaggcga tacagcttcc 26700
atggaagtcg gagacactct tcagttgcat gccactgtga ctcctgaaaa tgctactgac 26760
ccgtctgttg tatggagtgc tcctgtatca gaagttgcgt ctgtgactga agctggtttg 26820
gtaaccgcta acgctgcagg cttaattgac gttactgtta tcactaatga cgggaacttt 26880
actgacagaa tcgaggttaa agttacagac cccagaatcg cagttactga tgttgcatgg 26940
gctcctgact ctccgactaa agtaagcgct ggtcatcagg tgcagactaa aattgttata 27000
acacctgaca acgcaaccaa caaaaaagtt agctacaata gttcaaaccc agacgctgtt 27060
actgttgaca ctaacggagt tatatacggt caatcacctg gaacttcgac tattacagtt 27120
acaacagacg acggtggact tgttgcttca tttgtagcga cttgcacaga ggtggctgtt 27180
attacaccag atggtcttgg tggtattcca gtcggtggta ctcagcagtt cacttacact 27240
gttagcccgt ctgatgctga cctgactaat attcgttatg agtctgctaa cccagatatt 27300
gcaactgttg attcgtcagg cgttgtaact ggtgtagcta gtggcggatg taacatctgg 27360
atttacgcaa ctagtaatgg cgctgatatt agtgcagcta cttgggcaca aatttctgag 27420
aaggtacagg tataccctca gtatgccgaa tggcttgtgg gtaacaccaa acagcttgat 27480
gttgagattt atccgtctga ctctaagagc gttaccgtta cttatgaatc gtcaaatgac 27540
gctatcatta gtatagatgc aaatggcttg atgactgcta agtctgaagg ttctgcaact 27600
attacggcta acgttgtgat gaacggtgtt gctgcttctg gtacaacatc tccttacgtg 27660
tctgagcttt ctgttactac tgattcgctg cctgcactta ctgttggcaa cactcagcag 27720
ctggttgtaa ctgttagacc tgaatacgct aagacagacc caggctataa cattcagtat 27780
acaactacag acgatactgt cgctactgta agtaataccg gtgttattac tggcgttggt 27840
aacggtggat gtcgtattgg tgccacagta actgtgagaa atgctacagc gtcggacagc 27900
tcttatcagt ccgtcgatgc agcataaaaa ttaattaact ttgcactggc catttacttg 27960
gttaaactta ctgagtataa tggccggaaa gtccacccaa gaggtaaaca aaaatgccgt 28020
tgaatgtaaa ccagattgta aacgttcaac tgaacaacac tccaactggt atcagcaaag 28080
gtgacttcgg taagctggct cttttaactc aggaccctgg cactgctttt cctgacagcg 28140
acaacggaca ctatgttgaa atcggcagct actctgaagc cgagtcttat tggggtactg 28200
gttcagttgt acttggcgcg ctgaagattt tctttgcaca atctccgcgt cctcgttcag 28260
tagttatcgc ctcatggagc aacacctcta ctctggatgc ggctattaaa gcattcaacg 28320
atgtgtatcc tgactggtat gttctgcgtc cgcttccgcc tactggtgag ctgtctgacg 28380
atgacatcag tttgctggcg tcaactgttg ctgcttacga taaaaaacgt tgcagctaca 28440
ctaccactaa agcaactcat attgaaaaca gcgactccaa cccgattaaa gttctggctg 28500
gtaaaggcta caacaatatg tggcttcagt acgataagaa caaccaacac tatgcagaca 28560
tttctgctat ggcgcgtgct ctgtctgtta acttctccgg taaccgcact acgattacca 28620
tgaagttcaa acaagagcct ggtattacac cgaacaacac tttaacgctc actgaagcta 28680
acaagtgtaa agcgctgggt attaactggt acacctacta cagcaccttt gctatgcttg 28740
cagaaggtac tgtcctcggt acgtccaagc cgccgcgctt ctgggatgaa gtacatggtc 28800
tcgactggtt ctgtaatgct gtccagactt acgtcttcaa ctcaatcgct acgtcaccca 28860
cgaaggtgcc gcagacgaac aaaggtcacg agcgccttac cggtgctgca gctgttgctt 28920
gtcagcaggc tgtcgacaac ggcttggttg cacctggtat ctggtacggt gatgagtttg 28980
gtacactgtc ttatggtgac cgactcgaaa caggtttcta catctacatt cctgacgtag 29040
atgaccagcc tctggatgag cgtgaagctc gtcaggccgt tgtaatgcaa atcgcgatta 29100
agctggcggg tgcaatccac tccagcgaca tcctgattaa cttcactcgc taaggagcag 29160
atcaatgatc atcgattctt ctgaatgcgt gttgagcctg aatggtgttc agctgtccga 29220
ctgggcagac ggtgccgatg tcattgacgt gcaatgggtg gacccagctt ccacctatct 29280
tgcaggtgca gatggcaatg gcattcagat tatgaacccg tccaaagcca ttaagctaac 29340
tatcaaggtt aagcaaaatg gtccggatgc caaatggctt aacgaccagt acaacctgca 29400
gcgtaacaac attcgtggat ttgtcccgtt tgtgctgcaa gccaaagacc tgctaaacca 29460
ggatttggca tggggcaaaa aaggttactt taccgatggt tccccgtgga ctcgcggtaa 29520
cggccataac ccgactacct tcgctattgt gttccaccaa ggtggtatgc agttaactga 29580
aggctttgac cagtaacaat agaggaaagc acaaatgact gagcccaaat catttgacat 29640
cgacgacgta acgtacaaca tgacactcgc aaacgcgctg caagcctgga cggtcctgaa 29700
aaaagcaggt aagctgttca aaggcattgg caacgcggac attgtgggta aagacggcaa 29760
agtggacggt aagaaagcaa tgctggtcct gttggacgct gttctggaga acgcaggttc 29820
tgacgagatg acagctattg aggaactaat cttccgcaat actgttgttg tagttgatgg 29880
caaaccgcgt aagctgtctg actgtaaaga ccaacacttc aacgcacatc gtagccatat 29940
cttcggtgtg ctcaaggagg gattggtata tcagttcgcg gattttttca aagggaatgg 30000
cctgtcaggt ctagcgggtc tagccacgcc cttgacgaaa taaaacgcaa gcaacagtca 30060
gggacgcaag aaactgactg gttcgtttgg cttcctattc tcaagggtca tgtgaccctt 30120
gcagaattaa agtcgtctta cagcttgtgt gatttgtgcg acctgcatat tgctattctg 30180
gactatgccg agttagagga aatccatggc gaccataatc gatagctttc tggtaacgct 30240
tgagagtaaa gccgatagtg ctggctttga cctcttcgac cagctgtccc gccgttttgt 30300
gattaccctc ggcgatgtga tcaatcttgc tgagcgcgcc gctggggcgc tcggtaggat 30360
gtttgcgcct gcgttagagg ctgacgtact cgccgccaag attaaaggct tcggctacga 30420
cgtcgaatcg acgtggaaaa cgctgatgga tatgtctctg caaatgggta caccattcca 30480
ggaggcacta gacggcttta ctaaacttaa agcgtatggc gttgacccgt taaacggttc 30540
gctacaaatg ctcaaaacaa tcactgctgc aacaggcggt gatttggcac gtaccattgt 30600
tgcgtatggc caggcgactg cgatgggttc attgcagggc caggaaaaga accagttcat 30660
taacgccggt gttgacatct ggggcgcgtt gtcccgctac actggtaaga agcttggcga 30720
gcttcaggat atgatgcggc agaagcaaat tactgccgac atactgacta gcgcgctcaa 30780
acaagaagcc gaacgtttaa aaccaattgc ccacgaagtg tcacagacac tcaaagcgca 30840
gttaactaac ctgagcacaa tttggtatgc tgctcgttct aagattaccc agaccagagt 30900
atgggatgag gtaactaaag gcgcgataag tatggttaag tcactcacag cagtgcttaa 30960
cgataccaca cttactgcgg catttaaaaa gttcgacgaa gttgtattca atacaattga 31020
aaacgtaaaa cgacttatct acgttatcgg ggataacttc ccgttaatgg ttgcaataac 31080
ctcatacgca ctgctggcgc ttattccagt gctagctaag gtcggcaacg cctttgtggg 31140
tttagcagcc aaaatactta tgaatccgct cacgtggatt gtactggcta tcggcacagt 31200
tattgctgta ctcgctgacc tcgatgatgc tctggcagga cgtgagacag ttgttaagtg 31260
gtcaaaagat gttgtagccg catggggaat tgtcaagcag tatatcgtag ttattgcaga 31320
ctttattctg tcaatatttg acggtcgctt gtggaacaaa ctcggtaact ggtggcaagg 31380
tttcctcggc gaagttaagc aaggcataga gcaaataaag ctgtggttgt ctgaagcgct 31440
ttatcagcta cctggcggta agttcctgat ggcgttgttt ggtaagctcg attctacgct 31500
tggctttgct ggcaacaaac ttgtgacagg tgctgaggct actgttaatg ctgtgtcgcc 31560
taccactgag cagggcggaa gcttctggag tagagcactc aactggtcta tgcgtaactt 31620
caaccctatc acaggggaga tgacagctgg cggtcaggct atgcagtcag ccataaatac 31680
tgctaagcaa agccagcagc agcagaacgc aactgttatc caccaggata atagcgttaa 31740
agttgatgct actattaccg gtgtacagaa cccacaagaa gtaggccgtg aagtcaatga 31800
ccaggtcaaa caggcgcaga aagagaacgc tactgtaagg gctaacaaca aaacggcggt 31860
aacacaatga gtgacatctt tgctggaatt actgatgctg ccaacaactt gtaccaggac 31920
gcccgtgaca gactcatggg cgatggtaca accaccttac gcttccgcaa ctgcggaggt 31980
attaagatgg atgctgtcac caatgaggag catcagtctg accttgacat cgctgacaat 32040
gaacttgagt ccggggctaa ggcttcggac catgctgcag tacaaccaaa agttattact 32100
gtaactggcg ttgtagttgg ctatatcgac gagtcgattc aagagtcaac agttaacgac 32160
gtaaccggct tacggtctat ggatttcttg gatgatattc agcttccgca ggtagtgtct 32220
actgtcctcg aaggaacaag ggactttgct gttaacaagc tagtctcatt catagactgg 32280
ggcggtttag acgccactat ttcacgctct ttagttccat ggcttcctga ctttagtata 32340
gctgaacaac ttaagtcttc ggacaacatg aggattgaac aggtataccg tacactgctg 32400
gatttacaga aaaacgttat ttactgtgac gttgatactg gtatcttcaa gtatacgaat 32460
atgcttttga agtctgttcg tgttgtgcag gagaaagatg gttcagctac gtttaccctt 32520
acgttccgtg aagtcattga agtaccgatt gtagttacaa acgctgtagc cgctaagtcg 32580
acagggcgcc ctgctaataa cggcactaag aaaagcggac gtgcacaagg tcagggtgat 32640
gccaccagta agaaggacat aaacacaact acaccgatta gcgagaaacg tgtaaacgat 32700
aaccgcggtt ttggtgtaac actcggtgac atgtttggct ttaacttacg aggcatattc 32760
tgatgatatt taccacaact gctttccttg acattatagg taatgatggt tctggtattt 32820
atgacttgga acctacctcc gagggtaagc aatcaatatc aatttctatc gctggatata 32880
cgtataacat tgagttgtac tataacacgg taggtcagct ctggcaggct acagtgactg 32940
acatatcaag taatgctgta ctttgttcca gttatgcttt acaatatggg cgtattgctt 33000
ttggtaactg ggacacgcgt atagcgttgc tggtaagtga caaatcctcc agccgcatcg 33060
gtcctatgac tacagatgac atgggaggac gttgcagtgt ctacatcgtc gacaaaacag 33120
cctacccgtc agtttggccg cgcgttaaag cttgaggtca gcaacggtaa agatgaaggc 33180
ataatcatct caaatcttcg tgttaccttt gacattcaaa agggtaacac gaagcatcca 33240
aataaagcga caatcacagt gtttaacttg actgaatcca accgttataa gttggcgtca 33300
ggtacctggg attacgtccg tctctctgtg ggttacggtc ttgacaacat agtttatcgt 33360
ttgatattcg aaggcgatat tactcgtgct aacgaccaac gaaacggcct tgacattcag 33420
actgttctcg aatgtggcga tgcccgtaaa tcttacaacg atggctttgt taatacgacg 33480
ctggctgctg gttctacgta tgacagtgtt gttgacgttt gcctggctag catgccaaac 33540
gttgaagaag ggtacgtctg ccttgacagc aacaaaccag ctttcctgag agcacgtact 33600
ctgttcggac aaacgcacag catactcgac caggttgctg ctgccaacaa ggctacctgg 33660
tctatacaag acaataaact caatatcatt cctaacgact ctgttactga ctacgacgtt 33720
gttgtgctgt cagaatcgtc tggtatgatt ggtgctcctc gcggcacaga caaaggtctt 33780
gaggttactt gcaacctgaa tccttctctg gccatcggtg gtcttgtaca agtaaattct 33840
atgtttacac agtacaacgg acagtataaa atcgacacta taagatttcg gggtgacaat 33900
aaaccgaaag gaccgtggcg tgctgacttg acggttctcg gtggacagtt cgcgaagaaa 33960
gttcgtcgac tcaagaagcc acgcaaactg tcgaagattg agtacgacta cgttactgaa 34020
cccggtgttg accatacagt aggcggaggt agctgatgtc agctattgac tataatacag 34080
caacacttga cgaggccatt gaggctgcac tgtttgctgc aatgattggt aagcgccact 34140
gtttacctgg ccgcatagta tcgtttaaca gcaacctaca gactgttgtt gttgaaccta 34200
tggtcagtgc tgaaaacgaa gatggttcaa tgagaccgct accaccagca gctgatgttc 34260
ctttgttcca gctaggcggt ggtgattttg ttataacgct tgaaccaaag aaaggcgacc 34320
cgtgcttact tcttgttgcc gacagatgta ttgacgtatg gtatgagctg gctgaaaacg 34380
ctataccagg tgacttccgc cagaatgacc tgtctgactg cttctgtctt gtgggcttca 34440
gacctaagcc tctgaagata acaaactgga tggaaggtgt aactatacgt aaggttgacg 34500
gcagtcacta tatcaatatt aataatgctg gcaaggtatc gataaaggct acagcagtgg 34560
acttgcttga tacaccaaca cttaatgcac caaacacagc ggccaagttt aaagaagttg 34620
acattggcgg cgttaagatg tccaaacaca gacaccaaga aaacggtgat ggtggtggta 34680
ttactaatgg cccgcaaaac taggtaaccc acaatgcgaa ctagacgact cgattcaaac 34740
tgggactgga attttgggcg tggccgactt gactacgccg atacgtctga gtccattgaa 34800
caacgcatcg tgcagatact ccgctgctat actatggact gggtacatga cctggactat 34860
ggcattgact ggttcggact aatggagaag cctgttcgat gggctgacct tgaggctgca 34920
atcaagacag caatacttgc ggagtatgga gttagttcta tcgagagctt taccctaagc 34980
ttcgaccctg actcacgtca cgctgtttat tcggttgagg ttaatgatat ttaccacaac 35040
acaacctcta ttaacagaag tattaattta accggagaaa actgatgggc tctgtaacta 35100
gcacaggcta tgttcccgtc aagctcgacg actggatcag cagattcgaa acgctcttcc 35160
gtcgtgtgta tggcgcggat attgatttgt cagctaagtc tgctgacgga caaatggttg 35220
gtattctggc gcagatgttt gcagaccagg acgagcaagt tgcaattctg ttttctatgc 35280
ttgaccctaa ccaggcatct ggtgtatacg tagaccagtt ccttgcctac gtagcgcttc 35340
agcgtaaagg cgagatgtca acgctgttga ccagggttaa gattgaaggt gaccgtaaca 35400
ctattatacg tactccttac tacgtaactg acagctcgaa taacgtgtac cagcttaact 35460
ccactgttgc gctggatgat aacggtgtag tcgaaacaag cttcagttct gttaagactg 35520
gacgttttgt cgtacttgca aacgatgagc ttaaaccaca gaccatcgtt tctggtgtgc 35580
gtaaagttac caacatgcaa gtgtcgtctg gcggtgcgtc cactgagcta gactctgagg 35640
caatagaccg tgcgtataac tcttacggca aacctggcac gaacagctta gacggtattg 35700
ttgctgagat tcgtcagatg actgatgtta tcgcgtgtaa cggttacgag aacgaagaag 35760
atactcggga cccgttaact ggcttacctc ctcacagtat gcagattgtt gtggatggtg 35820
gttcgcctgc tgctattgct gactgtatct tccgccgtaa gactaatggc tgtaagcaga 35880
ttggcactgt gtcttatcag attacggata agtcaggtac acagcgcaca atgaaatgga 35940
gtcagcctac ttatgttgag gcttatgctt cactcgttgt agcccgtaag gaactgttta 36000
ccgacgtttc cgaggaactt attaagaatg ctattctgtc caaggtatac aatatcggtg 36060
aagaggtatc aacctacgag ctgggtcagc taattcaaga agcgaatggc gtaagcccta 36120
acttctacat caagactttc tttgtgggta aatcagctaa tccgtcgaca gatacgattc 36180
tgcgtatcac tgctcttgag cgtgcaatat tcgctgcaga ccgtatcact attgcagtgg 36240
tgaatgctga tggctgaata tcataaacta cttattcaac agtacgctga taagccaaaa 36300
gctgttgcaa ctattaaggg cttcgatgat gcaattcaag gtatctggga cggcgcccaa 36360
caagttgttg atagccttaa tatcgataaa gcttttggta aaggtcttga taatgttgca 36420
gcccgcgtcg gcgcatctcg tgttctcaag aaaggcgtag accgtaagtt cttcggcttt 36480
actaacaaag taccagacgt gcttggcttc ggtggttcgt ctggtaataa aggtggcccg 36540
ttctatcgct acggcgcatc agtatgggac ccggtaattc ttgacgacat tgacctgcgt 36600
acttacatca agatgaaaat ctggaagaac atgcaagacg gtactatgcc gtatctaatt 36660
gacttcctga ttgaattctt cggaatgaat aacttcgtta taactgacaa cgaagatatg 36720
acaatgactc tggagatata cagtactctg acaccactta agcaagtgct gattcagaac 36780
tacgatatac ttccgcgccc cacaggggtg aagattgaac ttaacacgag cgttgacgtc 36840
acggatagtc tgtaccgtta cgctactgta aacttaccag cacaggtgaa tgtatatggc 36900
tgatgtacaa acaagacccg ataacgacat cttcgccagc tctgcgggta ctggtgaact 36960
gctgccgttc ggtgaccttg ctcgtggctg gggtgctacc cttggctact cagaaggtat 37020
tcctccgatg gagtggttta actttatcgg taaccgtact gacaaaggta tccactacat 37080
ccttcagcag ggtgttgata tatgggtaga aactgagacc tacccggttg gtgcactggt 37140
taaagttaac gccgacaata tgctgtaccg ttgtctggtg cagaacatta atatgccacc 37200
gtctgctaac cctacacagt ggcgccctgt acttgacctg actgctgcaa ctgctgcagc 37260
acgacaggtt ggtaaagcct ctggtaacgt aatccaggtc ggtgcatttg gcttaggtgg 37320
caacgctgag gtagttacta atgctaacag cattgtaaac gacggtatct accagcttcc 37380
agcgtcaggt acgggcggac ctactaacgg tgttgagact gagattctgc atatcaactg 37440
ggatgagaaa aacgcttcac agtggggacg caccaagggt aagcgcgtaa tagccatgcg 37500
cgagaaggtt aataatacct ggggcggatg gtcatactct tacgggcctg acaataaacc 37560
tactgcctct gaactgggta ttaatcagga cttcatccct atggatggtt ctgactcaat 37620
ccgcggcaac ttaggtactg tcggttggct acatattgca cgtactactt ccgacttgct 37680
acgtgctgct aactccgccg gcgtaggtac tgttgagtat tctggtaaca tcgcaggtgg 37740
tactgctgtt caggagctgc tgacttcagt ttctgtcgcg gctaacagta atgttgtcac 37800
tgctgctcgc tggggatctg tacccatgtc gcacatccag cacatccttg tggctggcgg 37860
tgctacacag attaacatgg cggtatgtcg ccctggtggt attgccgacc gccgtacagt 37920
agctttgcag gtggatggtc ttaacagccg agtaactacc acaaatagct ttattttatg 37980
cgagaatggt gttagagtat attcacctaa caacccgcag cctatcgata cttcagccgg 38040
taacgttcgg ggtattcgac tcggcgctcg tattgctgct ggtgtatccg agtcaacgtt 38100
ctaccctggg catgtaatga ctggctggcg cttcggcaat aagaaagagc tgcgcggtgg 38160
tacatactgg tcagcgccgt tacagtatta catcaacggc acttggatta acgtaagcaa 38220
cgtatcataa aggtttatta tgactaagca taaggtgttc cgtaacttta ccatgtacca 38280
tcccgaggta acagaagaga acgagaacat tgtaaatgct ggtgcaatct tcctcaagga 38340
tgaggaaggt cgcgactggt acaatctcgc aaaagagttc gctatcaaat atgaaggtgc 38400
aacgttcttg caggtagata agaacggagt tgtaattgcg gcacataccg acccgactat 38460
gttccatcct gtgggctgcg atatggtagt aacatacgac ccgcctttca acatcttcga 38520
gactgttggc gaatggctgt tcgacggtaa agagtttacg cttaacgcac caatgatgaa 38580
ggagattgct cgtgagcgta aagaacaagc tctgtccatc gcttcgacca aggtcagtgt 38640
tttacgtgac acagttgata gtggcaatgc taccgatgaa gaaaaagagc tatttgctaa 38700
atggcgtgat tacaggattg ctgtcaatcg tcttgacatt aaccttggtt ccgctatggt 38760
tgttccacct atgccggagt aaaccatgca actgtcattt ttcaacttta cagaatacaa 38820
cccacaaagg acggcggaga cacttccgct gattaagcaa ggcgttgtat tcatcaagga 38880
taacaatggt aatgactggc gcaagctggc taaagagctg gctaagaata aagagctggt 38940
tcattacatt gttgtggaca agtacaatgc tgttgccgcg tatgctaacg atcctacgct 39000
actgtttcct gttaattgcg atttgcatat tgttgagtct gtccctgttg ctaagtttgc 39060
ggattgctat tacgaatggt cgtacgttaa tggcaagttt gttaagcact ctgaactgca 39120
gaaggtgatt gcagaagaac gcctgcagga agaacaggct tgggttgaag gacaactcga 39180
cactgaagaa gatgaggaac gtattatcgc tctccataaa ttccgtgccg atttgttgct 39240
gcttgttatg tcagatgcgc cgcatatcga ctggcctgta cgaccgtaac taattgctac 39300
cagcaagctt gtgcagttgc tggtagtgtt tcttttacgc ttgagtcgtg ctataatcca 39360
ctctgggtat acaataaacc ggagatatgg gatgtttgac tacaacgaca agtcttccct 39420
tattggccag tttgtggctt gggtaacaat aacagtaagt actttatcac taagtgattg 39480
gggtactctt atcggtatac tcagttcctt ggtgatgctg attgttgtta cgtataacca 39540
aacacggcga accaaaatta tgtcggatta ttacaatcgt aagaagccga tagaggatga 39600
agtcgcatcg aaagaagagg tgttgcgtga gtctgaaatc agcagcgact aaaggaggtt 39660
gtagcgtgct cgctatcatc gggcttgtat tgtcgatgac aagtacgcat gtacgtacta 39720
ataaagaagg tcttgaaata atcggtaatg cagaaggttg tatgcgcaac ccgtatgtat 39780
gccctactgg ttatttaaca gttggcattg gtagccgcat ctacagcgat gagcccgctg 39840
tacggcgcga aggcctgaca gaccaggaga ttgctgaccg ctgggtcaag aatattcaag 39900
aggccgaaaa atgtgttaat gactggttcc acggtaaaga tatgaacacg aaccagttca 39960
gcgccatgac ctctatggta tttaaccatg gttgcactag actccgtaag aacaaggatg 40020
gttcacctac caaaatatat actgctgctc gtaaacaaaa ctggaccgaa atgtgtaacc 40080
gcataacgga ctgggatatg ggtggtaagt ataggggctt gacaatacgt aagcaaaagg 40140
aaaaagcgct atgtctaaaa ccggtgcaat aattgcagtt attctcggcc tcgccattgt 40200
gttcctgtcc ttgtgggtac gtgagctgaa aggcgatatt accgaccgcg acggtataat 40260
caaacagctt aacaagacta atggcgagtt gtctgctgct aatcaaacgc tgattaattc 40320
tgtaaaggat gaacgtgagg cctcagccaa acgcgccagt aagatagctg aacttgaaca 40380
acaactgcgt gataagaagg gtaaatatga cgaagcgaca aaatatgatt cgtgcgctaa 40440
taccgttgct ccttctgccg ttcttgaact catgcgctga taagcctgaa gttgtttatg 40500
ttgacagggt agtcaaggtt tatccgcctg gcgcatacct ggttaaatgc cataagcctg 40560
aattaacagg taacacctgg aaggcaatag gtcagttagc tattgaacgt ggaacagctc 40620
ttgatgattg tgcagacaag atggacagca taattaactg gagtaacaaa ggggccgagt 40680
gatggcccct ttatttgatt aagcctgcag gaagcctggg aacatatcag cgcgtaaagc 40740
agcgcccttc agtaaccaga tttggtcttt agcctgtgca agcgcattgt cctgagctac 40800
ttgtttgccg atgtctgcat tgaactctgc agggtcaaca caaccagaag tacctacacc 40860
taactggaag ccatccagtg ttgccacaca tacagttaca gttgtattcg gtacctgcca 40920
gaaatgatac tccagtttat caacaagagc gttaacttct ttaaaatcta ctttcattat 40980
cgctttcctt tcttctcgtt atactgcgcg caggatgcac acaatactgt accgggtaag 41040
agctgacgtc gtttctcttc aatgtcttcc ccacactggt agcacgtcag cgggcttacg 41100
cctgtgtatc tgacacggtt cgacagcgct gcgtcccggg caacttgttc agcttcagaa 41160
gccatatcat taatgtccga tgtgcgcgta gtctcaatca ttattttccc tcttcttcac 41220
aatacagggt ttctaattcg tcgcatagct gttgcgtcca gcaatcatcc tcaccacgta 41280
tctggaagca cagcacagga atatcctgtc gtaaaatatc gctcttgtaa atctttttgg 41340
caacagcttc tggttcttcc tggaatatct tcataaacca ctccaggcct gacgtgcagc 41400
ctgaggcgtc tagtatctcc ttcgccctta acttaagcgt ttcatctacc tcatactcta 41460
aacctatctt agacatatta tagaccttta attagtgtgc ctctgcccag ttactttcta 41520
ccttagcatc agtaagaaca ggtacacgaa gcggtagtac agtttccatg acccgtacga 41580
agtcatagaa cccatcttct gcttgcttac cagttggtag agagaagtta agctcatcgt 41640
gtaccgtcag gcgaggtacg ccaatcacgt caaagtaacc gtcttcatac cctttcagca 41700
ttgcagactt catctgatct gctgacgacc cttgcagcac tcggttgatt gctttgtggg 41760
tgtctgccag tttaatgttc gggccatact cctgacgtgc ttcttcaaat ggtaaaggca 41820
tcttaccgaa tccaccacgt tttgggaccc atttgttgaa ccacgtcttc cggcccataa 41880
aggtttctac gtacccctgg ctataacata aatcttcata atgcttcatg gtagcaccaa 41940
tgaatggtgc accctgatgg tacgccttca gaacaggttc tgcttcttca ggcgtcattg 42000
ctaacttacg ggcaagagca ttaagcatca tgccatacaa caagccaaag ttgatggtct 42060
tagccggttt acgctctata acgtgacctg aggcttcctt gataatatcg accataacat 42120
tgtggtaatc tgcttccggg ttagccgcga aaattgctcg taactcatca gcgcccgggc 42180
cgactgcaaa atgcgccatg aatcgatatt caatttgggc gtagtctgct gatccccatc 42240
tgtgtccgac atccggtacg aaacaagcac gaaggagtgg tcccagtatt ttgtgtcgag 42300
acggtatatt ctgcagatta gggtcagatg agctaaacct accactaacg gttccgccgt 42360
catcaccgcg catttgatgg aattgaccgt gaaggaagcc atttgtattc atccccaata 42420
tatagccacg aataaacgtg gactggattt tctcataaga cttaatctca gacaccagtt 42480
ccgcaaacgg atggtgcaag cctgctaaga agtctgccgt aaacgacgga ttgcccgccg 42540
ctgttctgaa gtactccaga cctaaatggt caaatgcaag agctaagtcc ggacctgagt 42600
taacgttaat gttaaagcca atctggtctt taatctgctg gtgcattttc cttatttcag 42660
cgctaagcgt atcatccgcc tctttggcct tgtcaaggtc aacacgcaca cctgcaaaac 42720
gcatcttaag cattaatgga atgagcttgc actccatata gaatacgtct gttaaacacc 42780
agcgacttaa caatggccac tgggctttga taatgtgtat tggtaagtcg gcgtcagatt 42840
cagcatagta acccacaaga cgaggaggcg ttttgtagat gtactttctg gcttcacctg 42900
ttggcttaca gccataagca tctgaaatcc attggtacat ctcggctgag tctttacctt 42960
cgcctacata ctttcgtcca agtccttcaa gtgcaacacg cgcgcctggg tcaagtaatg 43020
cttccgcgaa ttgtacatcc atgcacaatc ctttaacttc cacgccttct gctgcaagcc 43080
agcccaagtc atatgtgatg ttagcaccga ctttaacttg gttaggtcgc gagagttcga 43140
gccgaagcca accaagtact tcctcagctg gaaggttatc ctcttcgccg agctcgtgtc 43200
tgattgggta gtaagctttg aagccgttgt ccgtagcgat actaactcca acgatatgac 43260
cctccctcct ggcccaacca ctaccatttg tatccatccc tgggtcgtat gtttcacagt 43320
caagaccgat aatctttgca gaagacaaat ccgggtaatg cgcgcgcggc cgccatccag 43380
tatccctgac gtatggtata ggacggttga taggtttaac attctttgca ccttcgaagc 43440
tatcgaataa aaagctcatc agtcccctct cacatacgca attacaactg tacgagttga 43500
gttttccatt ttgagcgcct gactaccaag tcctattctt tccatgcctt tagccagttt 43560
actcatctga ctaataacac agttgaacgg tttactaaac ttagtcgact ggaacttaat 43620
gtcgacggac atctttggaa taatcttatc tggtaagtct gcctttgcag ttgactcacc 43680
tgttatctcc aggatggctt gaccttcgtc gtacccagta agctggtcca tgacttccag 43740
gaactcaggg gtgatggcct gcatatccat ttccatattg aagaacttcg atacgtctgg 43800
ccatttggct gctagtaccg gggcatccat ctgtacgcca tccgcaaagc cgaagaacat 43860
ctggccgcgg gcgtatccta cacttgtggg ttccatcttc agcttaacca gggcctcaag 43920
cattggtttg gctatggcac attcaaattc aaaatctacg ttcaacttct cgcgtatcat 43980
atacttaccg ttagtggcgt aagcataacc atcgcgcaga agtaaagatg ttgcccatac 44040
ctgcggtgct tcgttaggta cccatttggc taaggcagtt aataccggga gtaattcgcc 44100
atgcgctgaa taccagttgt taactgtagc aagtggcggt attggttcat taagcgcttg 44160
cattcttgtt ttaagtacac ctgctgtgac tgtgaggttt ccagcggctg taatgttaaa 44220
agtaatctgt tcagttcttg ccgcattaac ggccttacga aatgattcag catccacttg 44280
caggtctgga aatcctttac atggcgctcg tagtaatacg ttcttgtagt atgaatggat 44340
tgttccgtct gcaatccata ttttgcccat aagaccacta tcagtatact tgatgggacg 44400
agcttgttga acggcagtct gtatttgttt ggtgtcatag ttaaacttgc tcatagtata 44460
Claims (9)
1. An application of coliphage EC35P1 with a preservation number of CCTCC M2020438 is characterized in that the coliphage EC35P1 is used for preparing a preparation or a composition for inhibiting or killing enterobacter cloacae or shigella.
2. The use of the bacteriophage EC35P1, having the accession number CCTCC M2020438, according to claim 1, wherein said formulation or composition further comprises a chemical disinfectant.
3. The use of the bacteriophage EC35P1, having the accession number CCTCC M2020438, according to claim 2, wherein the chemical disinfectant is one of didecyl dimethyl ammonium chloride or benzalkonium chloride.
4. The use of the bacteriophage EC35P1, having the accession number CCTCC M2020438, according to claim 3, wherein the final concentration of didecyldimethylammonium chloride is 0.05% or the final concentration of benzalkonium chloride is 0.01%.
5. The use of the escherichia coli bacteriophage EC35P1 with a accession number of CCTCC M2020438 as set forth in claim 1, wherein the preparation or composition further comprises the staphylococcus aureus bacteriophage jis 2 with a accession number of CCTCC M2016285.
6. The use of the coliphage EC35P1 with the collection number of CCTCC M2020438 as claimed in claim 5, wherein the preparation or composition consists of the coliphage EC35P1 and the Staphylococcus aureus phage JIP2 in equal quantitative ratios.
7. The use of the bacteriophage EC35P1 with the collection number CCTCC M2020438 as claimed in any one of claims 2 to 6, wherein the bacteriophage EC35P1 titer in the preparation or composition is 5x109PFU/mL。
8. The use of the bacteriophage EC35P1 with the preservation number CCTCC M2020438, according to any one of claims 5 or 6, wherein the titer of the Staphylococcus aureus bacteriophage JIP2 is 5x109PFU/mL。
9. The use of the coliphage EC35P1 with the preservation number of CCTCC M2020438 as claimed in claim 1, wherein the preparation or the composition is used for preparing a medicament for preventing or treating diseases caused by Enterobacter cloacae or Shigella.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011598316.7A CN112680423B (en) | 2020-12-29 | 2020-12-29 | Wide-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria and composition, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011598316.7A CN112680423B (en) | 2020-12-29 | 2020-12-29 | Wide-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria and composition, kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112680423A CN112680423A (en) | 2021-04-20 |
| CN112680423B true CN112680423B (en) | 2022-07-08 |
Family
ID=75454181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011598316.7A Active CN112680423B (en) | 2020-12-29 | 2020-12-29 | Wide-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria and composition, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112680423B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111481574B (en) * | 2020-04-17 | 2023-03-14 | 吉林省农业科学院 | Combined phage preparation for treating piglet diarrhea |
| CN113444695B (en) * | 2021-06-30 | 2022-05-27 | 武汉格瑞农生物科技有限公司 | Escherichia coli bacteriophage with high fermentation efficiency and good clinical effect and application thereof |
| CN113913391A (en) * | 2021-08-05 | 2022-01-11 | 瑞科盟(青岛)生物工程有限公司 | Cross-species cleavable Escherichia coli bacteriophage RDP-EC-20128 and application thereof |
| CN113755450B (en) * | 2021-09-03 | 2023-06-27 | 广西大学 | Escherichia coli phage GN4-1 and application thereof |
| CN113789305B (en) * | 2021-09-10 | 2023-05-12 | 南京农业大学 | Lytic coliphage preparation and its preparation and application |
| CN113881641B (en) * | 2021-10-19 | 2023-06-23 | 广西大学 | Escherichia coli phage EP01 and application thereof |
| CN114317458B (en) * | 2021-12-31 | 2023-07-14 | 浙江省农业科学院 | Coliphage with bactericidal effect and application thereof, bactericide and medicament |
| CN114908063A (en) * | 2022-06-15 | 2022-08-16 | 湖北大学 | Be applied to phage that live pig bred carbon emission reduction |
| CN117701512B (en) * | 2024-02-06 | 2024-06-11 | 江苏大方生物工程有限公司 | Salmonella phage and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110846283A (en) * | 2019-10-30 | 2020-02-28 | 菲吉乐科(南京)生物科技有限公司 | Escherichia coli bacteriophage with wide lysis spectrum, and composition, kit and application thereof |
| CN110846282A (en) * | 2019-10-30 | 2020-02-28 | 菲吉乐科(南京)生物科技有限公司 | High-temperature-resistant escherichia coli bacteriophage and composition, kit and application thereof |
| CN112029732A (en) * | 2020-09-05 | 2020-12-04 | 菲吉乐科(南京)生物科技有限公司 | High-temperature-resistant salmonella bacteriophage with wide lysis spectrum and composition thereof |
-
2020
- 2020-12-29 CN CN202011598316.7A patent/CN112680423B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110846283A (en) * | 2019-10-30 | 2020-02-28 | 菲吉乐科(南京)生物科技有限公司 | Escherichia coli bacteriophage with wide lysis spectrum, and composition, kit and application thereof |
| CN110846282A (en) * | 2019-10-30 | 2020-02-28 | 菲吉乐科(南京)生物科技有限公司 | High-temperature-resistant escherichia coli bacteriophage and composition, kit and application thereof |
| CN112029732A (en) * | 2020-09-05 | 2020-12-04 | 菲吉乐科(南京)生物科技有限公司 | High-temperature-resistant salmonella bacteriophage with wide lysis spectrum and composition thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112680423A (en) | 2021-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112680423B (en) | Wide-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria and composition, kit and application thereof | |
| CN112029732B (en) | High-temperature-resistant salmonella bacteriophage with wide lysis spectrum and composition thereof | |
| CN110438091B (en) | Novel Klebsiella pneumoniae phage and application thereof | |
| CN110846283B (en) | Escherichia coli bacteriophage with wide lysis spectrum, and composition, kit and application thereof | |
| CN113416712B (en) | Wide lysis spectrum salmonella bacteriophage and application thereof | |
| CN113583972B (en) | Escherichia coli bacteriophage capable of reducing antibiotic resistance and application thereof | |
| CN113583971B (en) | Salmonella bacteriophage capable of simultaneously cracking escherichia coli and application thereof | |
| CN113201502B (en) | Coliphage for piglet diarrhea and application thereof | |
| CN113337480B (en) | Broad-spectrum coliphage and application thereof | |
| CN111254121B (en) | Salmonella bacteriophage and application thereof in medicine for preventing and treating salmonella infection diseases | |
| CN111647567B (en) | Acid-resistant salmonella bacteriophage and composition, kit and application thereof | |
| CN108471756A (en) | Novel shigella dysenteriae bacteriophage and application thereof | |
| CN113755450A (en) | Escherichia coli phage GN4-1 and application thereof | |
| CN115786279A (en) | High-temperature-resistant pigeon-derived salmonella typhimurium bacteriophage, bacteriophage composition and application thereof | |
| CN113583966B (en) | Salmonella furciosus bacteriophage and application thereof | |
| JP6112961B2 (en) | Novel bacteriophage exhibiting lytic properties against Salmonella and compositions containing the same | |
| CN113430176B (en) | Stable and efficient salmonella furnacalis bacteriophage RDP-SA-21004 and application thereof | |
| CN112538463B (en) | Novel aeromonas hydrophila phage, and composition, kit and application thereof | |
| KR20210142679A (en) | High Temperature Tolerant Salmonella Phage with Wide Dissolution Spectrum and Combinations thereof | |
| CN113913391A (en) | Cross-species cleavable Escherichia coli bacteriophage RDP-EC-20128 and application thereof | |
| CN115161288B (en) | Acid-resistant and high-temperature-resistant Proteus mirabilis bacteriophage, composition and kit thereof, and application of acid-resistant and high-temperature-resistant Proteus mirabilis bacteriophage | |
| CN111349618B (en) | Escherichia coli bacteriophage composition and application thereof | |
| CN114574450B (en) | Ultraviolet-resistant Proteus mirabilis bacteriophage with broad cleavage spectrum, composition and kit thereof, and application of phage | |
| CN114480307B (en) | Pasteurella phage vB_Pmu P_PS30, phage composition and application thereof | |
| CN118126959A (en) | Proteus mirabilis bacteriophage, composition and kit thereof and application of phage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |