CN113528462B - Phage composition and application thereof - Google Patents

Phage composition and application thereof Download PDF

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CN113528462B
CN113528462B CN202110884141.4A CN202110884141A CN113528462B CN 113528462 B CN113528462 B CN 113528462B CN 202110884141 A CN202110884141 A CN 202110884141A CN 113528462 B CN113528462 B CN 113528462B
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CN113528462A (en
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潘强
任慧英
孙虎芝
刘广芹
葛超
罗志祥
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Qingdao Phagepharm Bio Tech Co ltd
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Abstract

The invention discloses a phage composition and application thereof, wherein the phage composition comprises Escherichia coli phage PD07 and salmonella phage PYC04; the Escherichia coli phage PD07 with the preservation number of CGMCC No18865 is preserved in the China general microbiological culture collection center in 11-month and 4-month in 2019; the salmonella bacteriophage PYC04 with the preservation number of CGMCC No18867 is preserved in the China general microbiological culture Collection center in 11-4.2019. The phage composition is a component for effectively preventing and treating salpingitis by compounding the Escherichia coli phage PD07 and the salmonella phage PYC04, and compared with single lytic Escherichia coli phage, the phage composition greatly widens the sterilization range, increases the sterilization activity, and can improve the laying rate of laying hens and enhance the physique and disease resistance of the laying hens.

Description

Phage composition and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to a bacteriophage and application thereof.
Background
The common cause of salpingitis in chickens is caused by biological pathogenic bacteria infection, and escherichia coli and salmonella are the most common causes. A white pus-like inflammatory exudate often flows from the fallopian tubes, and the feathers under the anus are often contaminated with inflammatory material. Inflammation of the fallopian tube causes thinning of the eggshell, and the proportion of the eggshell, the deformed egg and the broken egg is increased, so that the egg selling phase is deteriorated. Salpingitis affects the egg quality reduction, and serious inflammation diffusion and systemic infection can occur to cause death of chickens and cause great economic loss.
In order to ensure food safety and avoid drug residues in eggs in China, the laying hen group is forbidden to use antibiotics, and the traditional Chinese medicines and the like are commonly used for conditioning and preventing salpingitis at present. However, the traditional Chinese medicine has the defects of long treatment time, unsatisfactory effect, high medication cost and the like. At present, no safe and effective medicament for preventing and treating the salpingitis of the chicken exists.
Therefore, the prior art is in need of further improvement.
Disclosure of Invention
Aiming at the problems, the invention provides two strains of virulent phages PD07 and PYC04 which can be used for treating the salpingitis of bacterial chickens and a phage composition taking the two phages as main components, can kill pathogenic bacteria escherichia coli and salmonella causing the salpingitis of the chickens comprehensively, quickly and without residues, improve the laying rate of the laying hens, improve the quality of the eggs and enhance the physique and disease resistance of the laying hens.
The technical scheme of the invention is as follows:
the invention provides an escherichia coli bacteriophage PD07 for preventing and treating chicken salpingitis, which has the preservation number of CGMCC No18865 and is preserved in the China general microbiological culture collection center in 11-month and 4-month in 2019. The bacteriophage PD07 has a wider cracking effect on chicken-derived escherichia coli, and can be used for preparing a bactericide or an environmental disinfectant for preventing and treating chicken salpingitis.
The invention observes the form of the phage PD07 under an electron microscope, and the phage PD07 has a polyhedral head structure and a flexible tail, the length of the head is about 86.67nm, the transverse diameter is about 106.67nm, and the tail is about 120nm, and belongs to myophage (figure 1).
The invention also provides another salmonella bacteriophage PYC04 for preventing and treating chicken salpingitis, the preservation number is CGMCC No18867, and the salmonella bacteriophage PYC is preserved in China general microbiological culture collection center in 2019, 11 and 4 months. The phage PYC04 has a wider cracking effect on chicken salmonella, and can be used for preparing a bactericide or an environmental disinfectant for preventing and treating chicken salpingitis.
The form of PYC04, which is a long-tailed phage having a polyhedral head with a transverse diameter of about 60nm, a long diameter of about 66.67nm and a tail length of about 133.33nm, was observed under an electron microscope (FIG. 2).
By phage whole genome analysis, we obtained: the whole genome sizes of the phages PD07 and PYC04 are 78626bp and 41843bp respectively, wherein the content of the phage PD07 genome G + C is 49.89%, the phage PD07 genome contains 271 Open Reading Frames (ORFs), the content of the phage PYC04 genome G + C is 49.50%, and the phage PYC04 genome contains 62 Open Reading Frames (ORFs). The phages PD07 and PYC04 do not contain drug resistance genes and virulence genes and do not contain genes related to lysogenicity.
The invention also provides a phage composition for preventing and treating chicken salpingitis, which comprises the coliphage PD07 and the salmonella phage PYC04.
Alternatively, the ratio of the number of live coliphage to salmonella phage is 1:1; the content of each bacteriophage is not less than 1 × 10 8 pfu/mL。
PD07 in the phage composition is placed for 60min at 40-60 ℃, the activity is stable, and the activity is stable when the pH is 5-9; the PYC04 is placed at 40-60 ℃ for 60min, the activity is stable, and the activity is stable when the pH value is 5-11.
Further, the phage composition further comprises one or more of escherichia coli phage PD07 and a mutant of salmonella phage PYC04. Since bacteriophages are very susceptible to mutation during replication, it is preferred that mutants of such bacteriophages are also within the scope of the present application. Homology can be determined appropriately by computer programs well known in the art, with mutants of PD07 and PYC04 being at least 90% identical to the native sequence of the phage; and the mutant has substantially the same function of killing pathogenic bacteria as the original phage. More preferably, the mutants are 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the native sequence of the respective phage. Among them, the sequences of PD07 and PYC04 can be sequenced by a known method according to the biological materials deposited in the present invention. Mutants of bacteriophage PD07 and PYC04 may be point, deletion or addition mutations, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases being changed relative to the original bacteriophage sequence. It is not necessary for the skilled person to invent in the inventive step for the selection of mutants with similar properties from the phages provided according to the invention.
Furthermore, the sterilization composition also comprises auxiliary materials; the auxiliary material is one or more of SM buffer solution, sodium alginate, sucrose, maltodextrin and glucose. Preferably, the germicidal composition further comprises bacteriophages of specific pathogenic bacteria of different species of bacteria.
Specifically, the dosage form of the phage composition is powder, aqua, lyophilized agent, gel, cream and ointment.
The invention also provides application of the phage composition in a medicine for preventing or treating chicken salpingitis. The application method comprises the following steps: the phage composition is used as a therapeutic drug to be added into livestock and poultry feed with the salpingitis of the laying hens, or the phage composition is sprayed on the body surfaces of the livestock and poultry, or is drenched into the livestock and poultry, or is injected into the muscles of the livestock and poultry, or is drunk, wherein the optimal drinking water mode is used for preventing and treating the salpingitis of the laying hens, and the laying rate and the laying quality of the laying hens are improved.
The invention also provides a bactericide for preventing and treating chicken salpingitis, which comprises the phage composition.
The application of the phage composition in prevention and control of chicken colibacillosis and salmonellosis is characterized by reducing the morbidity and mortality of salpingitis of chicken flocks caused by escherichia coli and salmonellosis, reducing diarrhea caused by escherichia coli and salmonellosis of chicken flocks, and improving the laying rate and the egg quality.
The invention also provides an environmental disinfectant, the effective component of which is the phage composition consisting of the phage PD07, the PYC04 or the two phages; preferably, the concentration of each bacteriophage is 5X 10 8 PFU/ml or more. The application method comprises the following steps: the phage composition is used as an environmental disinfectant in slaughterhouses, livestock and poultry product processing workshops, appliances and breeding environments, and can prevent the pollution of escherichia coli and salmonella in the environments. For example, including but not limited to, disinfection and decontamination of water distribution systems, medical facilities, aquaculture facilities, public and private facilities, or other environmental surfaces by immersion in a liquid, spraying, use in conjunction with an aqueous carrier, etc., can be effective in controlling the growth and activity of target bacteria. The liquid soaking, spraying forms include but are not limited to detergents, disinfectants, detergents, and the like.
Preferably, the environmental disinfectant also contains other active ingredients for the inhibition or elimination of viruses, bacteria in the environment.
The invention also provides a feed additive, which comprises the phage PD07, PYC04 or a phage composition consisting of the two phages; preferably, the concentration of each bacteriophage in the feed is at least 1X 10 8 PFU/g。
In addition, the invention also provides a detection kit, which comprises the phage composition. The phage composition can be used to prepare test kits for detecting host pathogens specifically infected by the phage composition or for controlling diseases caused by infection of host pathogens by the phage composition, based on the present disclosure and common knowledge in the art.
The invention has the following beneficial effects:
1. two strains of phage are screened, namely escherichia coli phage PD07 and salmonella phage PYC04, and can be used for preventing and treating diseases caused by escherichia coli or salmonella infection, such as chicken salpingitis.
2. The invention also provides a phage composition for treating salpingitis of bacterial chicken, which is a component for effectively preventing and treating salpingitis by compounding the Escherichia coli phage PD07 and the salmonella phage PYC04.
3. The phage related to the invention is obtained from nature, has large quantity and wide source, is easy to separate and obtain, and belongs to natural organisms. Therefore, the composition for treating the salpingitis of the chicken by taking the lytic coliphage and the salmonella phage as main components and the composition for treating and preventing the salpingitis of the chicken can reduce the cost, provide a green novel biological agent, have no pungent smell and have no environmental pollution problem.
4. The phage composition designed by the invention has wide application, can be used as an environmental disinfectant, can also be used as an aqueous solution or a freeze-dried preparation medicament, is mixed with feed and drunk along with water, is used for preventing and treating diseases caused by escherichia coli and salmonella infection, and can quickly, thoroughly, safely and residue-free kill escherichia coli and salmonella in sick chickens and even drug-resistant escherichia coli and salmonella.
Drawings
FIG. 1 is an electron microscope picture of Escherichia coli phage PD 07;
FIG. 2 is an electron microscope picture of Salmonella phage PYC04;
FIG. 3 is a plaque picture of Escherichia coli bacteriophage PD 07;
FIG. 4 is a photograph of a plaque of Salmonella phage PYC04;
FIG. 5 is a picture of the pH stability of Escherichia coli bacteriophage PD 07;
FIG. 6 is a pH stability graph of Salmonella phage PYC04;
FIG. 7 is a picture of the temperature stability of Escherichia coli bacteriophage PD 07;
FIG. 8 is a temperature stability picture of Salmonella phage PYC04;
FIG. 9 is a photograph of one-step growth curve of Escherichia coli phage PD 07;
FIG. 10 is a photograph of a one-step growth curve of Salmonella phage PYC04.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Example 1 isolation and preparation of phages
The samples of the fecal sewage, chicken intestinal tracts, livers and the like in the invention are collected from a plurality of chicken farms in Shandong province.
1. Isolation and identification of pathogenic bacteria
From a certain egg chicken farm in Shandong, sick chicken with salpingitis are collected for dissection, and pathogenic bacteria are separated from the oviduct. Respectively culturing in SS culture medium, performing microscopic morphological identification on the obtained pathogenic bacteria, and performing identification through corresponding PCR.
As a result: separating off-white black-heart microcolonies on an SS culture medium, wherein the edges of the microcolonies are smooth and convex, and identifying the microcolonies as salmonella through salmonella specific primer PCR; colonies with red color and slightly light surrounding color are also obtained on the SS culture medium and are identified as escherichia coli through escherichia coli specific primer PCR.
2. Isolation of coliphage from Salmonella phage
Taking 50mL of the dung sewage of the chicken farm or 5g of chicken intestines and livers, adding 500 mu L of pathogenic bacteria, adding NB culture medium to 200mL, and soaking and incubating overnight at 37 ℃. The next day, 5-8mL of liquid is taken out, centrifuged at 11000rpm for 10min, and the supernatant is filtered through a 0.22 μm sterile microporous membrane to obtain phage stock solution. Separating bacteriophage by using a double-layer plate method, diluting a bacteriophage stock solution by a multiple ratio, uniformly mixing 120 mu L of each diluent with 120 mu L of pathogenic bacteria proliferation solution, incubating at 37 ℃ for 5min, placing on upper-layer agar (the agar concentration is 0.7%) which is insulated at about 60 ℃, rapidly pouring on a plate with the lower-layer agar (the agar concentration is 1.5%) after uniformly mixing, shaking uniformly and flatly placing until a culture medium is solidified, and placing at 37 ℃ for inverted culture for 3-6 h to obtain a double-layer plate with the formed plaques. Adopting different pathogenic bacteria (salmonella or escherichia coli), respectively separating to obtain two phages according to the method, and naming the obtained escherichia coli phage as PD07 and the obtained salmonella phage as PYC04.
3. Purification and propagation of coliphage PD07 and Salmonella phage PYC04
Picking single plaques in the double-layer culture medium for forming plaques, placing the single plaques in 1mL of NB broth at 37 ℃, and shaking at 170rpm for 30min to obtain phage leachate. Taking 120 mu L of phage leachate, uniformly mixing with 120 mu L of corresponding pathogenic bacteria proliferation liquid (incubating for 5min at 37 ℃), placing the mixture on the upper layer agar (the agar concentration is 0.7%) which is kept at about 60 ℃, quickly pouring the mixture on the lower layer agar (the agar concentration is 1.5%) after uniformly mixing, shaking the mixture evenly and flatly placing the mixture on a culture medium for solidification, placing the culture medium at 37 ℃ for inverted culture for 3-6 h, and obtaining the double-layer plate for forming the plaques again. On the plaque-forming double-layer medium, a single plaque is picked by a sterilized forceps and placed in 1mL of LB broth, and the mixture is shaken at 170rpm for 30min to obtain a phage extract. Repeating the steps for 3 times to obtain purified phage leachate, taking 100 mu L of purified phage and 100 mu L of pathogenic bacterium proliferation liquid to culture in 5mL of liquid NB culture medium at 37 ℃ under the oscillation of 170rpm until the liquid becomes clear, centrifuging the clear liquid at 11000rpm for 10min, taking the supernatant, and filtering by using a sterile microporous filter membrane of 0.22 mu m to obtain the phage proliferation liquid.
Coli phage PD07 can form clear plaques on double agar medium plates, without halo-rings around, with well-defined edges, about 1mm in diameter (see FIG. 3).
The Salmonella phage PYC04 formed a medium and clear plaque with a halo-ring, regular edge, and a diameter of about 2mm on a double agar medium plate (see FIG. 4).
Example 2 morphological Properties of Escherichia coli phage PD07 and Salmonella phage PYC04
Phage PD07 was observed under a transmission electron microscope (see FIG. 1). The head of the phage is in a polyhedral structure and has a slender tail, the length of the head of the phage is about 86.67nm, the transverse diameter of the head of the phage is about 106.67nm, and the tail of the phage is about 120nm. The morphology of the bacteriophage PD07, defined by the International Committee for Virus Classification (ICTV), corresponds to a characteristic of the Myoviridae family, belonging to the Myoviridae family.
The phage PYC04 was observed under a transmission electron microscope (see FIG. 2). The head of the phage is in a polyhedral structure and has a slender tail, the length of the head of the phage is about 66.67nm, the transverse diameter of the head of the phage is about 60nm, and the tail of the phage is about 133.33nm. The form of the bacteriophage PYC04, defined by the international committee for virus classification (ICTV), corresponds to a characteristic of the long-tailed bacteriophages, family, and belongs to the long-tailed bacteriophages.
Example 3 Whole genome analysis of phages PD07 and PYC04
Respectively extracting genomes of the Escherichia coli phage PD07 and the salmonella phage PYC04, and performing whole genome sequencing to obtain:
the genome size of the phage PD07 is 40629bp, the phage PD07 is a double-stranded DNA virus, the proportions of the whole genome sequences A, G, C and T4 bases are 23.81%, 23.38%, 26.51% and 26.38%, and the content of G + C% is 49.89%. The genome size of the phage PYC04 is 41843bp, and is circular double-stranded DNA, the proportions of the 4 basic groups of the genome sequence A, G, C and T are respectively 25.03%, 24.94%, 24.56% and 25.47%, and the content of G + C% is 49.50%.
Through RAST prediction analysis, the phage PD07 genome contains 271 Open Reading Frames (ORFs), and the phage PYC04 genome contains 62 Open Reading Frames (ORFs); respectively comparing 271 ORFs of the phage PD07 and 62 ORFs of the phage PYC04 protein sequences by using online tools BLASTp and a Conserved Domain Database (CDD), wherein the phage PD07 contains 24 known encoded functional proteins, and the rest ORFs are hypothetical proteins; the phage PYC04 contains 17 known encoded functional proteins, and the remaining ORFs are hypothetical proteins.
In the genome of phage PD 07: (1) the positions of two tail fiber protein gene sequences (shown as sequence 1 and sequence 3 in the sequence table) relevant to recognition of a phage host are 35790-38147, 38384-38950; the expressed protein sequences are shown in sequence 2 and sequence 4 respectively; (2) the positions of highly conserved terminal enzyme big subunit (terminase large subunit) protein genes (see sequence 5 in a sequence table) are 23199 to 24962; the expressed protein sequence is shown in sequence 6; (3) the sequence of the DNA polymerase (DNA polymerase) gene is shown in sequence 7 in the sequence table, the position is 6872-9043, and the expressed protein sequence is shown in sequence 8; (4) the gene sequence of lyase (lysozyme) related to the cracking ability is shown in a sequence 9 in the sequence table, the position of the gene is 24959-25408, and the expressed protein sequence is shown in a sequence 10.
In the genome of bacteriophage PYC 04: (1) the positions of three tail fiber protein gene sequences (see sequence 11, sequence 13 and sequence 15 in the sequence table) related to the recognition of a phage host are 2487-4517, 4530-7088 and 14375-15544; the expressed protein sequences are shown in sequence 12, sequence 14 and sequence 16 respectively; (2) the positions of highly conserved terminal enzyme big subunit (terminase large subunit) protein genes (shown as a sequence 17 in a sequence table) are 25256-26527; the expressed protein sequence is shown in sequence 18; (3) the position of the sequence of the DNA polymerase (DNA polymerase) gene (shown as sequence 19 in the sequence table) is 38144-40414; the expressed protein sequence is shown in sequence 20; (4) the positions of the lyase (lysozyme) gene sequence (shown as sequence 21 in the sequence table) related to the cracking capability are 28792-29280; the expressed protein sequence is shown in sequence 22.
The specific information of the above genes is shown in Table 1 below.
TABLE 1 Gene sequence information Table
Figure BDA0003193359100000091
The genomes of both phages were analyzed by the software tRNAscan-SE without tRNA genes. Both genomes were analyzed by PHASTER to be free of lysogenically related genes. Selecting a virulence gene and a conventional drug resistance gene from lysogenic phage in pathogenic bacteria, and performing sequence analysis on genomes of the two phages through an online tool CGE server to obtain: the genomes of the two phages do not contain drug resistance genes and virulence genes (namely escherichia coli drug resistance genes TEM, SHV, OXA, tetA, aac (3) -II, aph (3') -II and the like, virulence genes irp1, irp2, fyuA and the like, salmonella drug resistance genes blaTEM, blaPSE, aadA1, aadA2, tetB, sul1, qnrB and the like, virulence genes spvA, spvB, spvC, spvD and the like), and the two phages are proved to have no unfavorable genes, thereby ensuring the safety and effectiveness of the two phages.
Example 4 potency assay for Escherichia coli phage PD07 and Salmonella phage PYC04
Diluting the multiplied phage stock solution by 10 times, and taking 10 times -6 、10 -7 、10 -8 3 dilutions. 120 mu L of the 3 dilutions of phage and 120 mu L of host bacterial suspension are mixed and added into a sterile EP tube, and the mixture is fully mixed. After incubation for 5min at 37 ℃, adding the mixture into preheated and melted 0.7% upper agar, uniformly mixing, quickly pouring the mixture onto a 1.5% lower agar plate, obliquely rotating the plate to ensure uniform distribution, solidifying the agar, then inverting the plate in a constant temperature box at 37 ℃ to culture for 3-6 h, and observing the result. And (3) making 2 parallels for each dilution gradient, taking the dilution of the plaque between 30 and 300 as the standard during counting, taking the average value of 2 parallels of each dilution, and calculating the titer of the phage.
Phage titer (pfu/mL) = average number of plaques × dilution × 10.
The results show that: the phage titer of PD07 was 4.5X 10 10 PFU/mL, phage titer of PYC04 2.5X 10 11 PFU/mL。
Example 5 pH stability of Escherichia coli phage PD07 and Salmonella phage PYC04
Taking sterile test tubes, adding 4.5mL of NB broth with different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13) into each of three tubes, then placing the test tubes into a water bath kettle at 37 ℃, adding 500 mu L of PD07 phage proliferation liquid after the temperature is stabilized, and uniformly mixing the solutions in the water bath at 37 ℃ for 1h, 2h and 3h. Immediately after the reaction, an appropriate amount of 1mol/L HCl or NaOH was added to the mixture to adjust the pH of the mixture to about 7, 10-fold dilution was performed, and the titer was measured using an appropriate dilution gradient, with 2 replicates per pH tube. And drawing a PD07 bacteriophage pH value stability curve by taking the pH value as an abscissa and the logarithm value of the bacteriophage titer as an ordinate.
Adding 4.5mL of NB broth with different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13) into a sterile test tube, placing the test tube into a water bath kettle at 37 ℃, adding 500 mu L of PYC04 phage proliferation solution after the temperature is stable, and uniformly mixing in water bath at 37 ℃ for 1h, 2h and 3h. Immediately after the reaction, an appropriate amount of 1mol/L HCl or NaOH was added to the mixture to adjust the pH of the mixture to about 7, 10-fold dilution was performed, and the titer was measured using an appropriate dilution gradient, with 2 replicates per pH tube. And drawing a PYC04 bacteriophage pH value stability curve by taking the pH value as an abscissa and the logarithm value of the bacteriophage titer as an ordinate.
As shown in the test results of fig. 5-6, PD07 was stable at pH 5-9, began to decline in potency at pH 10 for 2h, and declined by 7 orders of magnitude or inactivated at pH 12 or pH 13 for 1 h; the PYC04 has stable titer at pH 5-11, and the titer is reduced by 6 orders of magnitude or inactivated after the PYC04 acts for 1 hour at pH 12 or pH 13.
Example 6 Heat stability of Escherichia coli phage PD07 and Salmonella phage PYC04
Packaging 100 μ L PD07 proliferation solution into sterile EP tube, and respectively treating in water bath at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C and 80 deg.C for 20min, 40min and 60min. Each temperature was set to 2 replicates. After the action is finished, sampling, immediately placing the sample in an ice bath for cooling, diluting by 10 times, and taking a proper dilution gradient to measure the titer. And drawing a PD07 phage thermal stability curve by taking the temperature as an abscissa and taking the logarithm value of the phage titer as an ordinate.
Packaging 100ml of PHC 04 proliferation solution in sterile EP tube, and respectively treating in water bath at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 20min, 40min and 60min. Each temperature was set to 2 replicates. After the action is finished, sampling, immediately placing the sample in an ice bath for cooling, diluting by 10 times, and taking a proper dilution gradient to measure the titer. And drawing a PYC04 phage thermal stability curve by taking the temperature as an abscissa and taking a logarithm value of the phage titer as an ordinate.
As shown in the test results of fig. 7-8: the titer of the phage PD07 is stable in the temperature range of 40-60 ℃, the titer is reduced by 4 orders of magnitude when the phage PD07 acts for 20min at 70 ℃; the phage is inactivated by 20min at 80 ℃. The titer of the phage PYC04 is stable in the temperature range of 40-60 ℃, the titer is reduced by 3 orders of magnitude after 20min at 70 ℃, the phage is inactivated after 20min at 80 ℃.
Example 7 one-step growth Curve assay of Escherichia coli phage PD07 and Salmonella phage PYC04
1mL of each of the PD07 proliferation solution and the fresh E.coli proliferation solution having an multiplicity of infection of 10 was mixed well (at this time, the mixture was incubated at 37 ℃ for 5min, centrifuged at 12000rpm for 30s, the supernatant was aspirated as much as possible with a micropipette, washed 1 time with 5mL of NB broth (centrifuged at 12000rpm for 30 s), and the supernatant was discarded. Suspending and precipitating with preheated NB broth (total volume is 5 mL), mixing well, quickly placing in 37 deg.C shaking table at 170rpm, shaking and culturing, taking out 150 μ L at 0 time and every 10min, centrifuging at 10000rpm for 1min, measuring titer, making 2 parallels, averaging the results,
1mL of each of the PYC04 proliferation solution and the fresh Salmonella proliferation solution having an infection multiplicity of 10 was mixed well (timing was started), incubated at 37 ℃ for 5min, centrifuged at 12000rpm for 30s, the supernatant was aspirated as much as possible using a micropipette, washed 1 time with 5mL of NB broth (centrifuged at 12000rpm for 30 s), and the supernatant was discarded. Suspending and precipitating with preheated NB broth (total volume is 5 mL), mixing well, rapidly placing in shaking table at 37 deg.C, shaking at 170rpm, taking out 150 μ L at 0 time and every 10min, centrifuging at 10000rpm for 1min, measuring titer, making 2 parallels, averaging the results,
and drawing a one-step growth curve by taking the infection time as an abscissa and the titer of the phage in an infection system as an ordinate to respectively obtain the latent period, the burst period and the burst amount of the PD07 phage and the PYC04 phage.
Burst = stationary phase phage concentration/initial bacteria concentration
And (3) testing results: as shown in FIGS. 9 and 10, the titer of each of 10 phages PD07 and PYC04 remained substantially unchanged within 0-40 min from the initial stage of infection 6 PFU/mL and 10 7 PFU/mL; and in 40-50 min, the outbreak period is entered, the titer of the phage is increased sharply, and after 60min, the titer of the phage is stabilized and reaches 10min 9 PFU/mL, calculated to give burst volumes of phage PD07 and PYC04 of 90 and 94, respectively.
Example 8 preparation of a composition of phages PD07 and PYC04
Respectively taking out the titer of 1 × 10 10 Stock solutions of PFU/ml phages PD07 and PYC04 were mixed homogeneously in equal volumes into the solution to a concentration of 1X 10 9 PFU/mL of phage composition.
Example 9 assay of the lysis Rate of bacteriophages
15 strains of escherichia coli and 15 strains of salmonella stored in a laboratory are selected, and the lysis spectra of the phages PD07 and PYC04 are detected by adopting a double-layer plate method. The strains are pathogenic bacteria which are separated and identified in the oviduct of a sick chicken suffering from salpingitis in chicken farms in Shandong, guangxi, henan, anhui, jiangsu and other places within 2017 to 2019. The information of the host bacteria used for detection and the lysis results are shown in Table 2 below.
TABLE 2 phage lysis results
Figure BDA0003193359100000131
Figure BDA0003193359100000141
Note: +: cracking, and brightening plaques; -: not cracked.
The experimental results show that: aiming at 15 clinically separated escherichia coli and 15 salmonella, 10 escherichia coli can be cracked by the phage PD07, and the cracking rate is up to 66.67%; the phage PYC04 can crack 12 salmonella strains, and the cracking rate is as high as 80%; the cocktail composition of 2 phage has better cracking effect on mixed infection of escherichia coli and salmonella, and overcomes the limitation of single phage cracking spectrum.
Example 9 safety test of phages
30 layers were divided into a test group (15) and a control group (15). The test group had 1ml of the phage composition orally administered to each chicken, and the control group had an equivalent amount of sterile physiological saline orally administered. The chickens were kept for 14 days to observe their clinical signs and dissected to observe visceral lesions.
The results showed that the test group of laying hens did not die after 14 days of the composition treatment, and no damage to the respiratory and digestive tracts nor abnormal activity was observed. The internal organs of the chickens in the dissected test group were free of lesions. The results show that the phage composition is safe and has no toxic or side effect.
EXAMPLE 10 test of therapeutic Effect of phages on salpingitis
Bacterial liquid: the escherichia coli and the salmonella are separated from the oviduct of a chicken clinically suffering from salpingitis, 5 strains of pathogenic escherichia coli and 5 strains of salmonella proliferating bacteria liquid which are separated and identified in different areas are respectively selected, and the same amount of bacteria liquid is uniformly mixed.
The method for molding salpingitis comprises the following steps: 45 laying hens of 200 days old are selected and evenly divided into a test group, a control group and a blank group of 3 groups, wherein each group comprises 15 laying hens. The experimental group and the control group were subjected to salpingitis modeling.
Fixing two legs with ring finger, index finger, little finger and thumb of left hand respectively, making palm tightly close to the tail end of breastbone of laying hen, standing the hand upright, making the cloaca of hen upward, making chicken head downward, making right thumb and 4 fingers form splayed soft part crossing two sides of cloaca, pressing downward slightly, pushing upward with left hand supporting the breast of hen, turning over the oviduct orifice, and injecting mold-making bacteria solution into uterus of oviduct by another person. When 80% of chickens have listlessness, white purulent secretion is discharged from the oviduct, congestion and swelling of cloaca, anus pasting and the like, administration treatment is started.
Administration: experimental group oral administration phage composition 1ml (potency 1X 10) 8 pfu/ml) for 5 consecutive days, and the control group was given an equivalent dose of sterile physiological saline for 10 days. And observing the egg laying condition within 10 days, and recording the egg laying amount, the egg quality and the total number of escherichia coli and salmonella in the oviduct. The results of the experiment are shown in Table 3 below.
TABLE 3 therapeutic test of phages on salpingitis
Figure BDA0003193359100000151
Figure BDA0003193359100000161
Note: fallopian tube swab bacterial distribution: the more the number of the "+" is, the more the bacteria are divided, the more serious;
degree of anatomical lesion of fallopian tube: "-" indicates smooth surface of fallopian tube, no bleeding, normal follicle; "+" indicates that the fallopian tube is slightly edematous, without necrosis or atrophy, and the dominant follicle is evident; "+ +" indicates rough surface of fallopian tube, abundant secretion, congestion and degeneration of follicle, thickening of fallopian tube wall, edema; "++++" follicular hyperemia degeneration, edema of fallopian tubes, yolk-like substance and even secondary peritonitis, necrotic areas or atrophy on the surface of fallopian tubes with bad smell.
The experimental results show that: compared with the control group, the laying rate and the egg qualification rate of the phage treatment group are obviously improved, and the pathogenic bacteria and the pathological change degree in the oviduct are reduced. This demonstrates that the phage composition has a superior effect on salpingitis.
EXAMPLE 11 in situ testing of phage for treatment of bacterial salpingitis Chicken flocks
In a chicken house of Qingdao Shandong, 8000 feathers and 300 days old, serious salpingitis is caused, and the laying rate of the chicken house is reduced and is increasedDirty eggs, blood eggs and chickens die sporadically, the belly of the dead chicken is enlarged, and a large amount of escherichia coli and a small amount of salmonella are contained in the oviduct through laboratory detection. The chicken house is subjected to a treatment containing 1 × 10 8 pfu/mL of the phage composition in drinking water. After 7 days of continuous drinking water treatment, the egg production, the dirty eggs, the blood egg production and the like of the chicken house are recorded, and 5 chickens are randomly dissected to observe the pathological changes of the oviduct.
Further calculations can be made in conjunction with the results of table 4: after 7 days of drinking water treatment with the phage composition, the number of broken eggs in the chicken farm was reduced by 96%, the number of fecal eggs was reduced by 92%, the number of sand shell eggs was reduced by 89%, the number of blood eggs was reduced by 93%, and the number of dead panning was reduced by 91%, and the egg production rate was increased by 11%. The randomly dissected chicken oviduct has no pathological changes such as hydrops, congestion, edema and the like, and the wall surface of the oviduct is smooth without abnormal secretion. The above results illustrate that: the salpingitis of the chicken coop is obviously improved after the phage composition is used for treatment.
TABLE 4 bacteriophage composition efficacy test results for layer chicken
Figure BDA0003193359100000162
Figure BDA0003193359100000171
The phage composition has efficient and specific killing effect on pathogenic bacteria in laying hen salpingitis, and has excellent acid resistance and heat resistance, so that the phage composition has treatment and prevention effects on laying hen salpingitis, and the laying rate and the laying quality of laying hens are improved. In addition, the phage composition can be used as an environmental disinfectant to eliminate escherichia coli and salmonella in slaughterhouses, livestock and poultry product processing workshops and appliances, and breeding environments. In addition, the phage used in the invention has no toxic or side effect on the laying hens, so the phage is safe and efficient.
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Sequence listing
<110> Qingdao Nonbert Biotech, inc
<120> a phage composition and uses thereof
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atgccgctta tcacacaaag cataaaaaat cttaaaggtg gcattagcca gcagcctgat 60
atactgaggt tctcagacca aggcgaggaa caggttaact gctggtcatc cgagagtgat 120
ggactccaga agcgcccacc tacagtcttc aagagacgtc ttaacatcga cgttgggagc 180
aaccctaagt tccatctgat taaccgtgac gagcatgagc agtattacat cgtgttcaat 240
gggtccaaca ttcaggtagt tgacttgagt ggtaatcaat actcagtgtc cggtgtggta 300
gattacgtta agtcctccaa cccacgagat gacatccgtg tcgttaccgt ggcagactat 360
acgttcgtcg ttaaccgtaa ggttgtcgtc aaaggtggaa gcgagaagtc acactctggt 420
tataacagca aagcacgagc tttaattaac ctgcgtggtg gacaatatgg tcgaaccctt 480
aaggtgggaa tcaacggtgg ggttaaggtc tcgcataagc taccagcagg taataatgcc 540
gagaatgacc cacctaaggt ggacgctcaa gccattggtg cagctctgag agaccttctt 600
gttgctgcct atcctaactt cacgtttgac cttgggtctg gcttcctgtt aatcactgcc 660
ccatcaggga ctgacattaa ctcagtggag acggaggacg gctacgctaa ccagctaata 720
agcccagtcc tagacactgt gcagacaatc tctaaactac ctcttgccgc tcctaatggg 780
tatatcatta agattcaagg cgagacaaac agtagcgccg atgaatacta cgtgatatat 840
gactccaaca ctaagacgtg gaaggagaca gtggagccgg gagttgtcac tgggttcgat 900
aacaccacaa tgccacacgc tctggtacga caatctgatg gctcctttga gttcaagact 960
ctggattggt ccaagcgtgg tgctggtaac gatgacacaa accctatgcc tagcttcgtg 1020
gatgctgcga ttaatgatgt gttcttctac aggaacagac ttgggttctt gtcgggtgag 1080
aacgtaatca tgagccgttc agccagctac tttgcgttct tccctaagag tgtggcgaca 1140
ctgagcgatg atgaccctat tgatgtagct gtaagccacc ctcgtatctc aatccttaag 1200
tatgccgttc cgtttagcga gcagctacta ctttggtcag acgaggtgca gttcgtaatg 1260
acaagctctg gtgtccttac ctcgaagtct atccagcttg atgtaggctc agagtttgcc 1320
ttgggtgaca atgccagacc gttcgctgta ggacgctcag tcttcttctc agcgcctcgt 1380
gggtcattca ccagcattaa gcgatacttc gctgtagcag atgtgtctga cgtgaaggat 1440
gccgatgata ccactggcca cgtactgtcc tatattccta atggggtgtt tgacattcaa 1500
ggtacaggga ctgagaacta catctgcgtc aactctacag gtgcatacaa ccgaatctac 1560
atctacaagt tcttgtttaa ggatggtgta cagcttcaag cctcttggtc gcactgggag 1620
ttccctaaag ctgataagat tctggcgtct gcgtcgattg gctctaccat gttcattgtt 1680
cgtcagcacc aaggcggcgt ggaccttgag caccttaagt tcatcaagga ggcaactgac 1740
ttcccgtcag agccgtatag actacacgtt gactccaagg tgtctatggt cattccaatt 1800
ggctcatacg acgctgacac ctataagact acggttgaca ttggttctgc ttatggcggt 1860
aacgctccgt ctcccggccg gtactatctg attgacagtc agggtgctta tttggacctt 1920
ggggacttga ccagtgtctc tactgtgatt accctcaacg gcgattggtc aggacgtaca 1980
gtgttcatcg gacggtccta tctgatgtcc tacaagttct cacggttcct gattaagatt 2040
gaagacgaga gtggcactca gtcagaagac actggacgcc tacagcttcg tcgggcttgg 2100
gtcaactaca aggacactgg cgctctgaga ctcattgtca gaaacggtga gcgggagttc 2160
gtaaacacct ttaacgggta caaccttggt cagcaagcca taggtgctac aaacattggt 2220
gacgggcagt atcgcttcgc tatgaatggt aacgcattaa ccacgagtct aaccttagag 2280
tccgactatc ctaccccagt gtccatcgtt gggtgcggct gggaggcgtc atacgctaag 2340
aaagctcgtt ccgtctaa 2358
<210> 2
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<213> Tail tubular protein B of phage PD07 (tail tubular protein B of phase PD 07)
<400> 2
Met Pro Leu Ile Thr Gln Ser Ile Lys Asn Leu Lys Gly Gly Ile Ser
1 5 10 15
Gln Gln Pro Asp Ile Leu Arg Phe Ser Asp Gln Gly Glu Glu Gln Val
20 25 30
Asn Cys Trp Ser Ser Glu Ser Asp Gly Leu Gln Lys Arg Pro Pro Thr
35 40 45
Val Phe Lys Arg Arg Leu Asn Ile Asp Val Gly Ser Asn Pro Lys Phe
50 55 60
His Leu Ile Asn Arg Asp Glu His Glu Gln Tyr Tyr Ile Val Phe Asn
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Gly Ser Asn Ile Gln Val Val Asp Leu Ser Gly Asn Gln Tyr Ser Val
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Ser Gly Val Val Asp Tyr Val Lys Ser Ser Asn Pro Arg Asp Asp Ile
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Arg Val Val Thr Val Ala Asp Tyr Thr Phe Val Val Asn Arg Lys Val
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Val Val Lys Gly Gly Ser Glu Lys Ser His Ser Gly Tyr Asn Ser Lys
130 135 140
Ala Arg Ala Leu Ile Asn Leu Arg Gly Gly Gln Tyr Gly Arg Thr Leu
145 150 155 160
Lys Val Gly Ile Asn Gly Gly Val Lys Val Ser His Lys Leu Pro Ala
165 170 175
Gly Asn Asn Ala Glu Asn Asp Pro Pro Lys Val Asp Ala Gln Ala Ile
180 185 190
Gly Ala Ala Leu Arg Asp Leu Leu Val Ala Ala Tyr Pro Asn Phe Thr
195 200 205
Phe Asp Leu Gly Ser Gly Phe Leu Leu Ile Thr Ala Pro Ser Gly Thr
210 215 220
Asp Ile Asn Ser Val Glu Thr Glu Asp Gly Tyr Ala Asn Gln Leu Ile
225 230 235 240
Ser Pro Val Leu Asp Thr Val Gln Thr Ile Ser Lys Leu Pro Leu Ala
245 250 255
Ala Pro Asn Gly Tyr Ile Ile Lys Ile Gln Gly Glu Thr Asn Ser Ser
260 265 270
Ala Asp Glu Tyr Tyr Val Ile Tyr Asp Ser Asn Thr Lys Thr Trp Lys
275 280 285
Glu Thr Val Glu Pro Gly Val Val Thr Gly Phe Asp Asn Thr Thr Met
290 295 300
Pro His Ala Leu Val Arg Gln Ser Asp Gly Ser Phe Glu Phe Lys Thr
305 310 315 320
Leu Asp Trp Ser Lys Arg Gly Ala Gly Asn Asp Asp Thr Asn Pro Met
325 330 335
Pro Ser Phe Val Asp Ala Ala Ile Asn Asp Val Phe Phe Tyr Arg Asn
340 345 350
Arg Leu Gly Phe Leu Ser Gly Glu Asn Val Ile Met Ser Arg Ser Ala
355 360 365
Ser Tyr Phe Ala Phe Phe Pro Lys Ser Val Ala Thr Leu Ser Asp Asp
370 375 380
Asp Pro Ile Asp Val Ala Val Ser His Pro Arg Ile Ser Ile Leu Lys
385 390 395 400
Tyr Ala Val Pro Phe Ser Glu Gln Leu Leu Leu Trp Ser Asp Glu Val
405 410 415
Gln Phe Val Met Thr Ser Ser Gly Val Leu Thr Ser Lys Ser Ile Gln
420 425 430
Leu Asp Val Gly Ser Glu Phe Ala Leu Gly Asp Asn Ala Arg Pro Phe
435 440 445
Ala Val Gly Arg Ser Val Phe Phe Ser Ala Pro Arg Gly Ser Phe Thr
450 455 460
Ser Ile Lys Arg Tyr Phe Ala Val Ala Asp Val Ser Asp Val Lys Asp
465 470 475 480
Ala Asp Asp Thr Thr Gly His Val Leu Ser Tyr Ile Pro Asn Gly Val
485 490 495
Phe Asp Ile Gln Gly Thr Gly Thr Glu Asn Tyr Ile Cys Val Asn Ser
500 505 510
Thr Gly Ala Tyr Asn Arg Ile Tyr Ile Tyr Lys Phe Leu Phe Lys Asp
515 520 525
Gly Val Gln Leu Gln Ala Ser Trp Ser His Trp Glu Phe Pro Lys Ala
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Asp Lys Ile Leu Ala Ser Ala Ser Ile Gly Ser Thr Met Phe Ile Val
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Arg Gln His Gln Gly Gly Val Asp Leu Glu His Leu Lys Phe Ile Lys
565 570 575
Glu Ala Thr Asp Phe Pro Ser Glu Pro Tyr Arg Leu His Val Asp Ser
580 585 590
Lys Val Ser Met Val Ile Pro Ile Gly Ser Tyr Asp Ala Asp Thr Tyr
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Lys Thr Thr Val Asp Ile Gly Ser Ala Tyr Gly Gly Asn Ala Pro Ser
610 615 620
Pro Gly Arg Tyr Tyr Leu Ile Asp Ser Gln Gly Ala Tyr Leu Asp Leu
625 630 635 640
Gly Asp Leu Thr Ser Val Ser Thr Val Ile Thr Leu Asn Gly Asp Trp
645 650 655
Ser Gly Arg Thr Val Phe Ile Gly Arg Ser Tyr Leu Met Ser Tyr Lys
660 665 670
Phe Ser Arg Phe Leu Ile Lys Ile Glu Asp Glu Ser Gly Thr Gln Ser
675 680 685
Glu Asp Thr Gly Arg Leu Gln Leu Arg Arg Ala Trp Val Asn Tyr Lys
690 695 700
Asp Thr Gly Ala Leu Arg Leu Ile Val Arg Asn Gly Glu Arg Glu Phe
705 710 715 720
Val Asn Thr Phe Asn Gly Tyr Asn Leu Gly Gln Gln Ala Ile Gly Ala
725 730 735
Thr Asn Ile Gly Asp Gly Gln Tyr Arg Phe Ala Met Asn Gly Asn Ala
740 745 750
Leu Thr Thr Ser Leu Thr Leu Glu Ser Asp Tyr Pro Thr Pro Val Ser
755 760 765
Ile Val Gly Cys Gly Trp Glu Ala Ser Tyr Ala Lys Lys Ala Arg Ser
770 775 780
Val
785
<210> 3
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<212> DNA
<213> Tail tube protein A of bacteriophage PD07 (tail tubular protein A of phase PD 07)
<400> 3
atggctcact acattccact gaatgctaac gatgacttag atgccatcaa cgatatgtta 60
gctgctatcg gtgaaccagc agtcctacag cttgacgagg ggaacgctga cgtctcgaac 120
gctcaacgta tactgcatcg tgtcaatcgt caggtccaag ctaaaggctg gaactttaac 180
atcaacgaaa atgctgtcct gacacctgat gtccaagaca acaggattcg attcctaccg 240
tcataccttc gtgtaatgac tgccggggcc accagctact acagcaacat gggtggatac 300
ctgtacgacc tgtccactca gtcaaccact ttcaccgacc ctataacggt ggagcttgtt 360
gagatgaaac cattctctga gatgccagtg gtattcaggg actacatcgt taccaaggcc 420
agccgtgagt tcaacgctaa gttcttcggt agccaagagt cagagctata ccttcgtgag 480
caggaagcag aactctatca gcaggttatg gagtacgaga tggacactgg tcgctacaac 540
atgatgtctg acatcgggag ggactaa 567
<210> 4
<211> 188
<212> PRT
<213> Tail tubular protein A of phage PD07 (tail tubular protein A of phase PD 07)
<400> 4
Met Ala His Tyr Ile Pro Leu Asn Ala Asn Asp Asp Leu Asp Ala Ile
1 5 10 15
Asn Asp Met Leu Ala Ala Ile Gly Glu Pro Ala Val Leu Gln Leu Asp
20 25 30
Glu Gly Asn Ala Asp Val Ser Asn Ala Gln Arg Ile Leu His Arg Val
35 40 45
Asn Arg Gln Val Gln Ala Lys Gly Trp Asn Phe Asn Ile Asn Glu Asn
50 55 60
Ala Val Leu Thr Pro Asp Val Gln Asp Asn Arg Ile Arg Phe Leu Pro
65 70 75 80
Ser Tyr Leu Arg Val Met Thr Ala Gly Ala Thr Ser Tyr Tyr Ser Asn
85 90 95
Met Gly Gly Tyr Leu Tyr Asp Leu Ser Thr Gln Ser Thr Thr Phe Thr
100 105 110
Asp Pro Ile Thr Val Glu Leu Val Glu Met Lys Pro Phe Ser Glu Met
115 120 125
Pro Val Val Phe Arg Asp Tyr Ile Val Thr Lys Ala Ser Arg Glu Phe
130 135 140
Asn Ala Lys Phe Phe Gly Ser Gln Glu Ser Glu Leu Tyr Leu Arg Glu
145 150 155 160
Gln Glu Ala Glu Leu Tyr Gln Gln Val Met Glu Tyr Glu Met Asp Thr
165 170 175
Gly Arg Tyr Asn Met Met Ser Asp Ile Gly Arg Asp
180 185
<210> 5
<211> 1764
<212> DNA
<213> terminal enzyme Large subunit of bacteriophage PD07 (terminating enzyme Large subunit of phase PD 07)
<400> 5
ttgagtaaag acttagtggc gcgtcaggcg ctaatgactg cccgtatgaa ggcagacttc 60
gtgttcttcc tgttcgttct atggaaagct ctgtcactcc cagtcccgac tcgctgtcag 120
attgacatgg cgaagaaact atcggctggg gacaacaggc gtttcatcct acaggcgttc 180
cgtggtatcg ggaagtcctt cattacgtgt gcattcgtgg tctggaaact atggaacaat 240
ccagacttga agttcatgat tgtgtcggcc tcaaaggaac gagccgatgc taactccatc 300
ttcatcaagc gtatcatcga cctcatgcct cagcttcagg aactcaaacc taagcaggga 360
cagagggatg cggttatcag cttcgacgtt gggccagcaa agccagacca ctcaccttca 420
gttaagtccg ttggtatcac tggtcagttg actggtagtc gtgctgacat cctgattgcc 480
gatgacgttg aggttccgaa caactcagcg actcaggctg caagagaccg cctgtcagag 540
cttgtgaaag agttcgacgc tatcctgaag ccgggaggta caatcatata tctgggtact 600
cctcagaacg agatgacctt gtatcgtgag ctagaaggcc gtggatacac cactactatc 660
tggcctgctc gttatccacg cgacaggaag gactggcagt cttacggaga ccgtctggct 720
cctatgcttc aggcagagct tgaagaggac cctgaggcct tctactggcg tccgaccgat 780
gaagttcgat tcgacgatac tgacctgaag gaacgtgagc tgtcctacgg taaagctggc 840
ttcgctctac agttcatgct taacccgaac ctgagtgatg ccgagaaata ccctctgaag 900
ctccgtgacc ttatcgtagc ggacttagac ccagcgtcta gcccaatggt ctaccaatgg 960
ttgccgaacc ctcataacaa gcgtgaggac gttcctaacg ttggcctcat gggtgactcg 1020
taccacacgt atcagactgt aggttctgcg ttcagctcat acacccagaa gattctggtc 1080
attgacccta gtggtcgtgg taaggatgaa actggttatg cggtattgta ccagctcaac 1140
ggctacatct tcgctatgga agttggtggt atgcgtggtg gctacgaaga ctctactctg 1200
gaagctctgg ctaagattgg tcataagtgg aaggtcaacg aatacgtcat tgagggtaac 1260
ttcggtgatg gtatgtacct tgagctattc aagcctgtag cggcccgtat ccatcctgct 1320
gctgtaactg aagtgaagag taagggtcag aaagaactcc gcatctgtga cgttctggag 1380
cctatcatgg gatctcaccg acttatcgtt aacgctgctg ctatcgtcca agactaccag 1440
acagcctctg acaaggatgg tgttcgtaac cctatctact ctctcttcta ccagatgacc 1500
cgtatctctc gtgaacgtgg agccttggca cacgatgacc gacttgatgc gctggctatc 1560
ggtgtacagt tcttcgttga gtcgatggct aaggatgcca acaaaggcga acgtgaagtc 1620
actgaggagt ggctggagga acagatggag aacccacgga aaggcttcga gtccatccac 1680
actgagttct gggacaatgg ggtccgggta actcacgata cggacgacga gctgggacta 1740
gggtcctacg ttacgttcca ctag 1764
<210> 6
<211> 587
<212> PRT
<213> terminal enzyme Large subunit of phage PD07 (terminase large subunit of phase PD 07)
<400> 6
Met Ser Lys Asp Leu Val Ala Arg Gln Ala Leu Met Thr Ala Arg Met
1 5 10 15
Lys Ala Asp Phe Val Phe Phe Leu Phe Val Leu Trp Lys Ala Leu Ser
20 25 30
Leu Pro Val Pro Thr Arg Cys Gln Ile Asp Met Ala Lys Lys Leu Ser
35 40 45
Ala Gly Asp Asn Arg Arg Phe Ile Leu Gln Ala Phe Arg Gly Ile Gly
50 55 60
Lys Ser Phe Ile Thr Cys Ala Phe Val Val Trp Lys Leu Trp Asn Asn
65 70 75 80
Pro Asp Leu Lys Phe Met Ile Val Ser Ala Ser Lys Glu Arg Ala Asp
85 90 95
Ala Asn Ser Ile Phe Ile Lys Arg Ile Ile Asp Leu Met Pro Gln Leu
100 105 110
Gln Glu Leu Lys Pro Lys Gln Gly Gln Arg Asp Ala Val Ile Ser Phe
115 120 125
Asp Val Gly Pro Ala Lys Pro Asp His Ser Pro Ser Val Lys Ser Val
130 135 140
Gly Ile Thr Gly Gln Leu Thr Gly Ser Arg Ala Asp Ile Leu Ile Ala
145 150 155 160
Asp Asp Val Glu Val Pro Asn Asn Ser Ala Thr Gln Ala Ala Arg Asp
165 170 175
Arg Leu Ser Glu Leu Val Lys Glu Phe Asp Ala Ile Leu Lys Pro Gly
180 185 190
Gly Thr Ile Ile Tyr Leu Gly Thr Pro Gln Asn Glu Met Thr Leu Tyr
195 200 205
Arg Glu Leu Glu Gly Arg Gly Tyr Thr Thr Thr Ile Trp Pro Ala Arg
210 215 220
Tyr Pro Arg Asp Arg Lys Asp Trp Gln Ser Tyr Gly Asp Arg Leu Ala
225 230 235 240
Pro Met Leu Gln Ala Glu Leu Glu Glu Asp Pro Glu Ala Phe Tyr Trp
245 250 255
Arg Pro Thr Asp Glu Val Arg Phe Asp Asp Thr Asp Leu Lys Glu Arg
260 265 270
Glu Leu Ser Tyr Gly Lys Ala Gly Phe Ala Leu Gln Phe Met Leu Asn
275 280 285
Pro Asn Leu Ser Asp Ala Glu Lys Tyr Pro Leu Lys Leu Arg Asp Leu
290 295 300
Ile Val Ala Asp Leu Asp Pro Ala Ser Ser Pro Met Val Tyr Gln Trp
305 310 315 320
Leu Pro Asn Pro His Asn Lys Arg Glu Asp Val Pro Asn Val Gly Leu
325 330 335
Met Gly Asp Ser Tyr His Thr Tyr Gln Thr Val Gly Ser Ala Phe Ser
340 345 350
Ser Tyr Thr Gln Lys Ile Leu Val Ile Asp Pro Ser Gly Arg Gly Lys
355 360 365
Asp Glu Thr Gly Tyr Ala Val Leu Tyr Gln Leu Asn Gly Tyr Ile Phe
370 375 380
Ala Met Glu Val Gly Gly Met Arg Gly Gly Tyr Glu Asp Ser Thr Leu
385 390 395 400
Glu Ala Leu Ala Lys Ile Gly His Lys Trp Lys Val Asn Glu Tyr Val
405 410 415
Ile Glu Gly Asn Phe Gly Asp Gly Met Tyr Leu Glu Leu Phe Lys Pro
420 425 430
Val Ala Ala Arg Ile His Pro Ala Ala Val Thr Glu Val Lys Ser Lys
435 440 445
Gly Gln Lys Glu Leu Arg Ile Cys Asp Val Leu Glu Pro Ile Met Gly
450 455 460
Ser His Arg Leu Ile Val Asn Ala Ala Ala Ile Val Gln Asp Tyr Gln
465 470 475 480
Thr Ala Ser Asp Lys Asp Gly Val Arg Asn Pro Ile Tyr Ser Leu Phe
485 490 495
Tyr Gln Met Thr Arg Ile Ser Arg Glu Arg Gly Ala Leu Ala His Asp
500 505 510
Asp Arg Leu Asp Ala Leu Ala Ile Gly Val Gln Phe Phe Val Glu Ser
515 520 525
Met Ala Lys Asp Ala Asn Lys Gly Glu Arg Glu Val Thr Glu Glu Trp
530 535 540
Leu Glu Glu Gln Met Glu Asn Pro Arg Lys Gly Phe Glu Ser Ile His
545 550 555 560
Thr Glu Phe Trp Asp Asn Gly Val Arg Val Thr His Asp Thr Asp Asp
565 570 575
Glu Leu Gly Leu Gly Ser Tyr Val Thr Phe His
580 585
<210> 7
<211> 2172
<212> DNA
<213> DNA polymerase of phage PD07 (DNA polymerase of phase PD 07)
<400> 7
atgtttgacc ttaagagtat ctggggttcg gacatcgaga caaacggtct ccttgataca 60
gtctcccagt tccactgcgg ggtcctgatt aacgccgagt cgaatgagac ccttaagtat 120
ggggtagctc cgatggtcgg tatcgtcggt ggcttcaaag agtatgtgca gaaggtggaa 180
gagattgccg catcgtctga tggtatgctg gtattccaca acggtatcaa ctatgacgtc 240
cctgctattg acaagctgaa acgcctgtac tttgggaaac gttttaactt cccgaaacac 300
aagatgattg ataccttggt gctgggtcgc ttgatgtatc caaacattaa gttctcagac 360
atgggagcgg tgaaagctgg tcgtctgccc cctcagatga tgggacgaca atctcttgag 420
gcttggggtt atcgtctcgg tgagatgaag ggtgagtaca aacacgatta cgttgccaag 480
tgcaaggctg aaggtatcga atataaggct ggggacgaat ggttgttccc gtctcaggag 540
atgctggact ataacgttca agacgttgtg gtcacactgg cgttgttcaa gaagttcctg 600
actgacaagt attacttcca gtctgaacag ttcgctttcg accagattta tgcgttgcgt 660
ctggaacatg atgctgcgtg gacctgtgcg aagatggaac gtaacggcta tccgatgaac 720
accgagatgg tcgaaggctt atatcgtgaa ctcaccgtca aacgtgcaga gtttctggac 780
aagctgcgtt cgactttcgg gagctggtat gcaccaaagg gaggcaagga gttcttcagg 840
catccacgga caggtaagga ccttccgaag tatccgagag tcgtgtatcc caaggtcggt 900
ggcatcttta agaagcctaa gaacaaagct caacgcttag gtcttgaacc ttgtgaactc 960
gatacgcgag acacgatgga gggagcaccg ttcacgccaa tcacttacgt tgagtttaac 1020
ccgggaagcg gagatcactt agcgaaagtc ctgatggagc gtgggtggga gcctgtggac 1080
ttcactgaca ccgggaaacc tgtagtcgat gacgagacgt tagaacacgt taagttacca 1140
gacgcagagg ctcaggcttg cgtagagctg gtccgtgagt atctggtagt ccagaagcgc 1200
atcggtcagg cggctgaagg taagaacgca tggttgaaac ttgtaggtcc agacggacgt 1260
atgcacggtt caatcaaccc atgcggggca gtaaccggac gtgcgaccca tagttcacca 1320
aacatggctc aagtcccagc taatggtgct ccatatggtg agacttgccg tggcgctttc 1380
ggtgcagcgt ggaacaagaa ggatggtaag ccagaccctt ggattcaagt aggtgtggat 1440
gcctcaggtc ttgagcttcg ttgtctgggg aaccgagcgg ctccgtttga tggtggtgcg 1500
tatgcgaaga ctgtggtcga aggtgacatc cactgggcca acgcagtaaa cgctgggtta 1560
gcacctaacg tcccacgcga taagtcgaac cacgaccatg atgctttccg taacaacgct 1620
aagacgttca tctatgcgtt cctgtatggt gcaggggccg ctaagattgg actgatagtc 1680
ggcggcggta agaaggaagg ttcagctctc atgaagaaat tcattgaggg tacaccagcc 1740
atcaaagacc tcagggaagc tgtgagtaat acgttaatct cagactctaa gtgggtggac 1800
ggagagaaca tcgtcaagtg gaaacgccgt tggttgcgtg gacttgatgg tcgccgtatc 1860
cacatccggt cgccacactc agcactgaac gcattacttc aaggtgatgg tgcggtagtc 1920
tgtaagcact ggattgtgga gactgaacgt atgctcgaag aggcagggta tgttcacggc 1980
tgggaaggtg actttgcgta catggcttgg gtgcatgacg aattgcagat tgcgtgtcgt 2040
actcaggaga ttgccgaaga agtcgtcaag attgctcaac ttgctatgcg taaggtgggt 2100
gagttctata attttaaatg tgttcttgat acggagggta agattgggcc cacgtggaaa 2160
gaatgtcatt aa 2172
<210> 8
<211> 723
<212> PRT
<213> DNA polymerase of phage PD07 (DNA polymerase of phase PD 07)
<400> 8
Met Phe Asp Leu Lys Ser Ile Trp Gly Ser Asp Ile Glu Thr Asn Gly
1 5 10 15
Leu Leu Asp Thr Val Ser Gln Phe His Cys Gly Val Leu Ile Asn Ala
20 25 30
Glu Ser Asn Glu Thr Leu Lys Tyr Gly Val Ala Pro Met Val Gly Ile
35 40 45
Val Gly Gly Phe Lys Glu Tyr Val Gln Lys Val Glu Glu Ile Ala Ala
50 55 60
Ser Ser Asp Gly Met Leu Val Phe His Asn Gly Ile Asn Tyr Asp Val
65 70 75 80
Pro Ala Ile Asp Lys Leu Lys Arg Leu Tyr Phe Gly Lys Arg Phe Asn
85 90 95
Phe Pro Lys His Lys Met Ile Asp Thr Leu Val Leu Gly Arg Leu Met
100 105 110
Tyr Pro Asn Ile Lys Phe Ser Asp Met Gly Ala Val Lys Ala Gly Arg
115 120 125
Leu Pro Pro Gln Met Met Gly Arg Gln Ser Leu Glu Ala Trp Gly Tyr
130 135 140
Arg Leu Gly Glu Met Lys Gly Glu Tyr Lys His Asp Tyr Val Ala Lys
145 150 155 160
Cys Lys Ala Glu Gly Ile Glu Tyr Lys Ala Gly Asp Glu Trp Leu Phe
165 170 175
Pro Ser Gln Glu Met Leu Asp Tyr Asn Val Gln Asp Val Val Val Thr
180 185 190
Leu Ala Leu Phe Lys Lys Phe Leu Thr Asp Lys Tyr Tyr Phe Gln Ser
195 200 205
Glu Gln Phe Ala Phe Asp Gln Ile Tyr Ala Leu Arg Leu Glu His Asp
210 215 220
Ala Ala Trp Thr Cys Ala Lys Met Glu Arg Asn Gly Tyr Pro Met Asn
225 230 235 240
Thr Glu Met Val Glu Gly Leu Tyr Arg Glu Leu Thr Val Lys Arg Ala
245 250 255
Glu Phe Leu Asp Lys Leu Arg Ser Thr Phe Gly Ser Trp Tyr Ala Pro
260 265 270
Lys Gly Gly Lys Glu Phe Phe Arg His Pro Arg Thr Gly Lys Asp Leu
275 280 285
Pro Lys Tyr Pro Arg Val Val Tyr Pro Lys Val Gly Gly Ile Phe Lys
290 295 300
Lys Pro Lys Asn Lys Ala Gln Arg Leu Gly Leu Glu Pro Cys Glu Leu
305 310 315 320
Asp Thr Arg Asp Thr Met Glu Gly Ala Pro Phe Thr Pro Ile Thr Tyr
325 330 335
Val Glu Phe Asn Pro Gly Ser Gly Asp His Leu Ala Lys Val Leu Met
340 345 350
Glu Arg Gly Trp Glu Pro Val Asp Phe Thr Asp Thr Gly Lys Pro Val
355 360 365
Val Asp Asp Glu Thr Leu Glu His Val Lys Leu Pro Asp Ala Glu Ala
370 375 380
Gln Ala Cys Val Glu Leu Val Arg Glu Tyr Leu Val Val Gln Lys Arg
385 390 395 400
Ile Gly Gln Ala Ala Glu Gly Lys Asn Ala Trp Leu Lys Leu Val Gly
405 410 415
Pro Asp Gly Arg Met His Gly Ser Ile Asn Pro Cys Gly Ala Val Thr
420 425 430
Gly Arg Ala Thr His Ser Ser Pro Asn Met Ala Gln Val Pro Ala Asn
435 440 445
Gly Ala Pro Tyr Gly Glu Thr Cys Arg Gly Ala Phe Gly Ala Ala Trp
450 455 460
Asn Lys Lys Asp Gly Lys Pro Asp Pro Trp Ile Gln Val Gly Val Asp
465 470 475 480
Ala Ser Gly Leu Glu Leu Arg Cys Leu Gly Asn Arg Ala Ala Pro Phe
485 490 495
Asp Gly Gly Ala Tyr Ala Lys Thr Val Val Glu Gly Asp Ile His Trp
500 505 510
Ala Asn Ala Val Asn Ala Gly Leu Ala Pro Asn Val Pro Arg Asp Lys
515 520 525
Ser Asn His Asp His Asp Ala Phe Arg Asn Asn Ala Lys Thr Phe Ile
530 535 540
Tyr Ala Phe Leu Tyr Gly Ala Gly Ala Ala Lys Ile Gly Leu Ile Val
545 550 555 560
Gly Gly Gly Lys Lys Glu Gly Ser Ala Leu Met Lys Lys Phe Ile Glu
565 570 575
Gly Thr Pro Ala Ile Lys Asp Leu Arg Glu Ala Val Ser Asn Thr Leu
580 585 590
Ile Ser Asp Ser Lys Trp Val Asp Gly Glu Asn Ile Val Lys Trp Lys
595 600 605
Arg Arg Trp Leu Arg Gly Leu Asp Gly Arg Arg Ile His Ile Arg Ser
610 615 620
Pro His Ser Ala Leu Asn Ala Leu Leu Gln Gly Asp Gly Ala Val Val
625 630 635 640
Cys Lys His Trp Ile Val Glu Thr Glu Arg Met Leu Glu Glu Ala Gly
645 650 655
Tyr Val His Gly Trp Glu Gly Asp Phe Ala Tyr Met Ala Trp Val His
660 665 670
Asp Glu Leu Gln Ile Ala Cys Arg Thr Gln Glu Ile Ala Glu Glu Val
675 680 685
Val Lys Ile Ala Gln Leu Ala Met Arg Lys Val Gly Glu Phe Tyr Asn
690 695 700
Phe Lys Cys Val Leu Asp Thr Glu Gly Lys Ile Gly Pro Thr Trp Lys
705 710 715 720
Glu Cys His
<210> 9
<211> 450
<212> DNA
<213> lyase protein Gp18.5 of Phage PD07 (phase Rz-like lysine protein Gp18.5 of phase PD 07)
<400> 9
atgctcaaat ttttaaagag agcggctccg tggctacttg cagcagtgat gtttgctggc 60
ggctaccaca ccgctaacaa taagtgggag gctaaggtca atgcagaata cacctcgaat 120
cttaaggcat cggaggatac aagacttgct gtccaagctg aagtcaacaa ggtgtccaaa 180
cggtttcagg acgaaatgtc ctcgctggaa ggcagcactg ataggattat tgctgacctt 240
cagtctgaca ataagcggtt gcgcatccga gtcaaaccaa cgagtggaac cacgcaaagt 300
gacggtcgat gcatcattga tgatttcgcc gaacttgacg aacgagatgc taaacgtctt 360
atcgccatcg gagtgaaagg agacaaatgg attaaggcac ttcaggacac tgtgagagcc 420
ttacagcaag ataaggaggt gagaccttga 450
<210> 10
<211> 149
<212> PRT
<213> lyase protein Gp18.5 of Phage PD07 (phase Rz-like lysine protein Gp18.5 of phase PD 07)
<400> 10
Met Leu Lys Phe Leu Lys Arg Ala Ala Pro Trp Leu Leu Ala Ala Val
1 5 10 15
Met Phe Ala Gly Gly Tyr His Thr Ala Asn Asn Lys Trp Glu Ala Lys
20 25 30
Val Asn Ala Glu Tyr Thr Ser Asn Leu Lys Ala Ser Glu Asp Thr Arg
35 40 45
Leu Ala Val Gln Ala Glu Val Asn Lys Val Ser Lys Arg Phe Gln Asp
50 55 60
Glu Met Ser Ser Leu Glu Gly Ser Thr Asp Arg Ile Ile Ala Asp Leu
65 70 75 80
Gln Ser Asp Asn Lys Arg Leu Arg Ile Arg Val Lys Pro Thr Ser Gly
85 90 95
Thr Thr Gln Ser Asp Gly Arg Cys Ile Ile Asp Asp Phe Ala Glu Leu
100 105 110
Asp Glu Arg Asp Ala Lys Arg Leu Ile Ala Ile Gly Val Lys Gly Asp
115 120 125
Lys Trp Ile Lys Ala Leu Gln Asp Thr Val Arg Ala Leu Gln Gln Asp
130 135 140
Lys Glu Val Arg Pro
145
<210> 11
<211> 2031
<212> DNA
<213> spike protein of phase PYC04 (tailspike protein of phase PYC 04)
<400> 11
atgtctagtg gttgcggtga tgtattgtca cttaacgatt tacaggtagc taaaaaacac 60
cagattttcg aagccgaggt gatcaccggc aaacagggtg gtgtagcggg tggcgcagat 120
atcgactacg ctactaacca ggtaaccggg cagacgcaga agacgctgcc cgcggtcttg 180
cgtgacgccg gtttctctcc ggcgtctttt aacttcacaa ccggcggaac cctgggagct 240
gacgacgcgg ataaagcggt tctttggcct attgaggacg gcggagacgg taactattac 300
gtatggcgcg ggtcactccc taaagttatc cctgcggcgt cgacacctct tacgacgggc 360
ggcatttcgg attccgcttg ggtcgcgttc ggggatatca ccttccgtgc ggaagcggat 420
aagaaattta aatactccgt taagctgtcc gactttacta cgttacaaca attggcagac 480
gccgccgttg atagcgttct tatcgaccgc gattacactt tcagtaataa cgagaccgtt 540
aacttcagcg ggaagaccct gaccatcgac tgtaaagcga agtttattgg tgatggtatg 600
ttaatatggg agcaactcgg cgaagggtcc gttgtgaacc agccgcatat gcagacacaa 660
accacgccgt acacggtgta tagattcgac gacaacggta actgggtgac taacccatca 720
acggtgctgg cgtcggtagt ccaaaggatg gataaggggt ataagccaaa cattaacgat 780
ttagatattt gggacgacct tcctgataat gtaaaaaatc aggttgctgg tgcaactttg 840
cgaataatga gcggcgataa tatcatcgtg gagaacccag aagctacatt tggtgggtat 900
ctgttcacac tctgtaatcg tattctcgta aaaaatccac gtaattttat tgcctgggag 960
tctggtatta catttgagaa ccatcaaaca accgcatggg gtacaggtaa ctgggtggtc 1020
ggtggtgaga taaaatacgg ttctggttct gcagttttat ttatcagaaa tgacggtggt 1080
gatgatcaca atggaggggt aagggattta atatcatatc gcgttggcga atctggtgtt 1140
aagacatatc agaatgaaat cgggggacgg tcagccagga actaccgttt ggtgttcgac 1200
aatataacta caatccagtg ttattatgat gggattgatg ttaatgctga cacgggggcg 1260
ccaaccgaac gtgtagacga ttacactctt gctgagtacc catggttcca tttacctact 1320
cagcacatca tccgcaatat cattacacgt gactgtatgg gtatcggcgc gtggtgggat 1380
gggcaaaaaa atatcattga taatgttgta acctacgaag cccataaaga ggggattttc 1440
gacaggggta ctaacaatga tattactaat gtaacagtta taggtgcaaa caaggattta 1500
actaacctaa atcagcttac atgcgaaggc ggcagcagat tgcgcggagt gatgattcac 1560
gcatactcta cacaagggta tgccgtatac gcgccgggtt ccgaaatatc gtctgtttcg 1620
tgcgcgggtt ctggtacgaa aaagatactt tgtacgtatg ttgctgatat tcagggtggg 1680
aacataaacg tacagcatgg tgataacgac atgaccctgg ctatgcgccc ggcaatgggg 1740
ggcacaataa atccatcatt acttatgaca gcaagttgcc aagtagcatc gccgggtggt 1800
gaagctagta ttgttaagct ttcagcaata cagggaggtg tacgagtagg tgagctccaa 1860
cttaaccgct taggctttaa gcatatgagc atacctgttg catcttcgca attaccagat 1920
agtgctttag aacataactc atctataggt ttcttcttcg gcagtgatgg ggtgttgcga 1980
atccttgcca agaagccaga cgggacttac gtaacctaca ccttatccta a 2031
<210> 12
<211> 676
<212> PRT
<213> spike protein of phase PYC04 (tailspike protein of phase PYC 04)
<400> 12
Met Ser Ser Gly Cys Gly Asp Val Leu Ser Leu Asn Asp Leu Gln Val
1 5 10 15
Ala Lys Lys His Gln Ile Phe Glu Ala Glu Val Ile Thr Gly Lys Gln
20 25 30
Gly Gly Val Ala Gly Gly Ala Asp Ile Asp Tyr Ala Thr Asn Gln Val
35 40 45
Thr Gly Gln Thr Gln Lys Thr Leu Pro Ala Val Leu Arg Asp Ala Gly
50 55 60
Phe Ser Pro Ala Ser Phe Asn Phe Thr Thr Gly Gly Thr Leu Gly Ala
65 70 75 80
Asp Asp Ala Asp Lys Ala Val Leu Trp Pro Ile Glu Asp Gly Gly Asp
85 90 95
Gly Asn Tyr Tyr Val Trp Arg Gly Ser Leu Pro Lys Val Ile Pro Ala
100 105 110
Ala Ser Thr Pro Leu Thr Thr Gly Gly Ile Ser Asp Ser Ala Trp Val
115 120 125
Ala Phe Gly Asp Ile Thr Phe Arg Ala Glu Ala Asp Lys Lys Phe Lys
130 135 140
Tyr Ser Val Lys Leu Ser Asp Phe Thr Thr Leu Gln Gln Leu Ala Asp
145 150 155 160
Ala Ala Val Asp Ser Val Leu Ile Asp Arg Asp Tyr Thr Phe Ser Asn
165 170 175
Asn Glu Thr Val Asn Phe Ser Gly Lys Thr Leu Thr Ile Asp Cys Lys
180 185 190
Ala Lys Phe Ile Gly Asp Gly Met Leu Ile Trp Glu Gln Leu Gly Glu
195 200 205
Gly Ser Val Val Asn Gln Pro His Met Gln Thr Gln Thr Thr Pro Tyr
210 215 220
Thr Val Tyr Arg Phe Asp Asp Asn Gly Asn Trp Val Thr Asn Pro Ser
225 230 235 240
Thr Val Leu Ala Ser Val Val Gln Arg Met Asp Lys Gly Tyr Lys Pro
245 250 255
Asn Ile Asn Asp Leu Asp Ile Trp Asp Asp Leu Pro Asp Asn Val Lys
260 265 270
Asn Gln Val Ala Gly Ala Thr Leu Arg Ile Met Ser Gly Asp Asn Ile
275 280 285
Ile Val Glu Asn Pro Glu Ala Thr Phe Gly Gly Tyr Leu Phe Thr Leu
290 295 300
Cys Asn Arg Ile Leu Val Lys Asn Pro Arg Asn Phe Ile Ala Trp Glu
305 310 315 320
Ser Gly Ile Thr Phe Glu Asn His Gln Thr Thr Ala Trp Gly Thr Gly
325 330 335
Asn Trp Val Val Gly Gly Glu Ile Lys Tyr Gly Ser Gly Ser Ala Val
340 345 350
Leu Phe Ile Arg Asn Asp Gly Gly Asp Asp His Asn Gly Gly Val Arg
355 360 365
Asp Leu Ile Ser Tyr Arg Val Gly Glu Ser Gly Val Lys Thr Tyr Gln
370 375 380
Asn Glu Ile Gly Gly Arg Ser Ala Arg Asn Tyr Arg Leu Val Phe Asp
385 390 395 400
Asn Ile Thr Thr Ile Gln Cys Tyr Tyr Asp Gly Ile Asp Val Asn Ala
405 410 415
Asp Thr Gly Ala Pro Thr Glu Arg Val Asp Asp Tyr Thr Leu Ala Glu
420 425 430
Tyr Pro Trp Phe His Leu Pro Thr Gln His Ile Ile Arg Asn Ile Ile
435 440 445
Thr Arg Asp Cys Met Gly Ile Gly Ala Trp Trp Asp Gly Gln Lys Asn
450 455 460
Ile Ile Asp Asn Val Val Thr Tyr Glu Ala His Lys Glu Gly Ile Phe
465 470 475 480
Asp Arg Gly Thr Asn Asn Asp Ile Thr Asn Val Thr Val Ile Gly Ala
485 490 495
Asn Lys Asp Leu Thr Asn Leu Asn Gln Leu Thr Cys Glu Gly Gly Ser
500 505 510
Arg Leu Arg Gly Val Met Ile His Ala Tyr Ser Thr Gln Gly Tyr Ala
515 520 525
Val Tyr Ala Pro Gly Ser Glu Ile Ser Ser Val Ser Cys Ala Gly Ser
530 535 540
Gly Thr Lys Lys Ile Leu Cys Thr Tyr Val Ala Asp Ile Gln Gly Gly
545 550 555 560
Asn Ile Asn Val Gln His Gly Asp Asn Asp Met Thr Leu Ala Met Arg
565 570 575
Pro Ala Met Gly Gly Thr Ile Asn Pro Ser Leu Leu Met Thr Ala Ser
580 585 590
Cys Gln Val Ala Ser Pro Gly Gly Glu Ala Ser Ile Val Lys Leu Ser
595 600 605
Ala Ile Gln Gly Gly Val Arg Val Gly Glu Leu Gln Leu Asn Arg Leu
610 615 620
Gly Phe Lys His Met Ser Ile Pro Val Ala Ser Ser Gln Leu Pro Asp
625 630 635 640
Ser Ala Leu Glu His Asn Ser Ser Ile Gly Phe Phe Phe Gly Ser Asp
645 650 655
Gly Val Leu Arg Ile Leu Ala Lys Lys Pro Asp Gly Thr Tyr Val Thr
660 665 670
Tyr Thr Leu Ser
675
<210> 13
<211> 2559
<212> DNA
<213> Tail associated protein of phase PYC04 (purified tail associated protein of phase PYC 04)
<400> 13
ttggcgctag taatccacta cacacgaaac gaagacggca cgtttgatgt caagcgctac 60
cgcgataatc cgatgaactt cgtcgtgaac catgttccgg acggggttcc ggtacgtgtt 120
ttcatcgacg aaatcggaga agataacgac gtaacagaag acttcgaagc actgaaagaa 180
aacgcgactt tccacattgt ggaatcagct ggtggtggcg ccattaaagg cgtcatgaag 240
atttttagcg ttatccttaa accgctggcg aaactattat cgccgtccgt gaaaggtgcg 300
tcctcgaacc ttgcgaactc gcaggcggat tccccaaaca acagtcttac tgaccgtaac 360
aacaaggcgc gcccatacga gcgcagttac gacatctgcg ggacggtgca aacaataccc 420
aataacctta tgtctactta taaggtgttt aacgctgccg gtaaaattgt agagtacggc 480
tactacgacg ccgggcgtgg ccacctcgac atacacccgg atggcataac ggacggtgat 540
acccgggtat cggatataac aggtacgtcg gttgccgtgt acgcgccgta tacgtcacca 600
aataatacat ctacacccca ggtcatggta ggggacccca tagagcaggg cctgtacatc 660
accgtagaat ctaacgaagt ggatggcgtg gttttgaaag cacctaacgg cctgggtatt 720
tctttctctt acatgtcggg gtatccgtct ttgtcaggaa acattggcac tatatacgac 780
cctacaggcg gctcggattt ttctggagta ctggtgccta atgatacatt ttcgctggtg 840
tcagcgtgga caaatacaga cgttgacctc tccggcggcg gatatcaggt agtcagcgtg 900
tccgaaggga ctgttacctt tatagtacct ggtggtctca ttggtaggtg gcaagaaata 960
agaccgggtt cgtttttccg cggtgacgga gaggcctcgc tgcaaccaga caacacgtat 1020
gagaaaacct taaccgattg ggtttcaata aaccgcaccg aggttgagcg catcgtggcc 1080
aatatcgccg ctgcgaacgg catgtataaa gacaacggca aatcgaaaac actggcgttt 1140
gtcaccgctg agatacagta ccagctactc gatgaaaata gcgttcctta cgggccaata 1200
tacacggcgc aaggaactgt gtccgggcgt acaccggact acaacggcgt cactatttac 1260
gccgacctgc cgattgcgtc tcgcgtaagg gtcagggccc gcagggttac agaccttgat 1320
ttcaattttg aagggtctgt ggttgatgaa ataacgtacg ttaacttata cgggcaaaca 1380
cgcgataaca ctccgcacta cggcaacaga acaaccgtac actcgatgcg caagcagacg 1440
ccgcgcgctg cggaggttaa gcaaccacag ttacgtatga ttgccaccga aatggtgtac 1500
aaatacctcg gtaatggtgt tttcgaagac acgatgactc ccaatacaca agccgtgcaa 1560
tctcttatcc gcctggcgcg tgacccagat gtgggcggtt taaacctgac agtacgcaac 1620
atggataagt tacttgctgt gcagaacgag gtcgaagcgt attttggtga caaacaggct 1680
ggagaatttt gttacacgtt tgatgactat aaaaccacta tgcaggatat agttagtact 1740
atagccgacg ctatcttctg taccccatac aggcgtgggg cggacatcct tctcgatttt 1800
gaacgccctc gcatgggccc tgagatggtg ttcacccacc gaagtaaggc cggtacttcc 1860
gaaaaatgga ccagaacctt taacgatgct caggtgttcg atagtcttaa attctcgtac 1920
atagacccaa aaacaaacgt taaagaaaca ataacaatac cagaaactgg tggggttaaa 1980
actgagactt atgactcaaa aggaatccgc aactataagc aggctttctg ggcagcgcac 2040
cgccgtcacc agaagaacat tttaaagaaa atttcagtgt cgtttaccgc cacggaagag 2100
ggtatctttg ctttgccaaa tcgtgccatt agtgtcgtta agggctcacg tatggctaca 2160
tacgatggct acataaccgc ggtaaacggg ctcaccgtag aactgtcaca accagttaag 2220
ttcacctcgg gagatgacca ttctttgatt ctcaagttac gtgacggcgg cgtgcaaagt 2280
gttaatgtag cccctggagc gcacgaccgg caggtaatta tgacatcagt accgcaagaa 2340
gccatttaca caggtaatag tgctttgaaa actgaatttt cattcggcaa cgaagcaagg 2400
cataatgctc agatgattct tgtttctaca gtagaccctg gcgatgaccg aacagtcaaa 2460
ataaccgggt ttaactatga caaggatttc tataagttcg acaacgtacc gcctttcggt 2520
cgtgcgttct ctaacggatt cgataacggt tttaactaa 2559
<210> 14
<211> 852
<212> PRT
<213> Tail associated protein of phase PYC04 (purified tail associated protein of phase PYC 04)
<400> 14
Met Ala Leu Val Ile His Tyr Thr Arg Asn Glu Asp Gly Thr Phe Asp
1 5 10 15
Val Lys Arg Tyr Arg Asp Asn Pro Met Asn Phe Val Val Asn His Val
20 25 30
Pro Asp Gly Val Pro Val Arg Val Phe Ile Asp Glu Ile Gly Glu Asp
35 40 45
Asn Asp Val Thr Glu Asp Phe Glu Ala Leu Lys Glu Asn Ala Thr Phe
50 55 60
His Ile Val Glu Ser Ala Gly Gly Gly Ala Ile Lys Gly Val Met Lys
65 70 75 80
Ile Phe Ser Val Ile Leu Lys Pro Leu Ala Lys Leu Leu Ser Pro Ser
85 90 95
Val Lys Gly Ala Ser Ser Asn Leu Ala Asn Ser Gln Ala Asp Ser Pro
100 105 110
Asn Asn Ser Leu Thr Asp Arg Asn Asn Lys Ala Arg Pro Tyr Glu Arg
115 120 125
Ser Tyr Asp Ile Cys Gly Thr Val Gln Thr Ile Pro Asn Asn Leu Met
130 135 140
Ser Thr Tyr Lys Val Phe Asn Ala Ala Gly Lys Ile Val Glu Tyr Gly
145 150 155 160
Tyr Tyr Asp Ala Gly Arg Gly His Leu Asp Ile His Pro Asp Gly Ile
165 170 175
Thr Asp Gly Asp Thr Arg Val Ser Asp Ile Thr Gly Thr Ser Val Ala
180 185 190
Val Tyr Ala Pro Tyr Thr Ser Pro Asn Asn Thr Ser Thr Pro Gln Val
195 200 205
Met Val Gly Asp Pro Ile Glu Gln Gly Leu Tyr Ile Thr Val Glu Ser
210 215 220
Asn Glu Val Asp Gly Val Val Leu Lys Ala Pro Asn Gly Leu Gly Ile
225 230 235 240
Ser Phe Ser Tyr Met Ser Gly Tyr Pro Ser Leu Ser Gly Asn Ile Gly
245 250 255
Thr Ile Tyr Asp Pro Thr Gly Gly Ser Asp Phe Ser Gly Val Leu Val
260 265 270
Pro Asn Asp Thr Phe Ser Leu Val Ser Ala Trp Thr Asn Thr Asp Val
275 280 285
Asp Leu Ser Gly Gly Gly Tyr Gln Val Val Ser Val Ser Glu Gly Thr
290 295 300
Val Thr Phe Ile Val Pro Gly Gly Leu Ile Gly Arg Trp Gln Glu Ile
305 310 315 320
Arg Pro Gly Ser Phe Phe Arg Gly Asp Gly Glu Ala Ser Leu Gln Pro
325 330 335
Asp Asn Thr Tyr Glu Lys Thr Leu Thr Asp Trp Val Ser Ile Asn Arg
340 345 350
Thr Glu Val Glu Arg Ile Val Ala Asn Ile Ala Ala Ala Asn Gly Met
355 360 365
Tyr Lys Asp Asn Gly Lys Ser Lys Thr Leu Ala Phe Val Thr Ala Glu
370 375 380
Ile Gln Tyr Gln Leu Leu Asp Glu Asn Ser Val Pro Tyr Gly Pro Ile
385 390 395 400
Tyr Thr Ala Gln Gly Thr Val Ser Gly Arg Thr Pro Asp Tyr Asn Gly
405 410 415
Val Thr Ile Tyr Ala Asp Leu Pro Ile Ala Ser Arg Val Arg Val Arg
420 425 430
Ala Arg Arg Val Thr Asp Leu Asp Phe Asn Phe Glu Gly Ser Val Val
435 440 445
Asp Glu Ile Thr Tyr Val Asn Leu Tyr Gly Gln Thr Arg Asp Asn Thr
450 455 460
Pro His Tyr Gly Asn Arg Thr Thr Val His Ser Met Arg Lys Gln Thr
465 470 475 480
Pro Arg Ala Ala Glu Val Lys Gln Pro Gln Leu Arg Met Ile Ala Thr
485 490 495
Glu Met Val Tyr Lys Tyr Leu Gly Asn Gly Val Phe Glu Asp Thr Met
500 505 510
Thr Pro Asn Thr Gln Ala Val Gln Ser Leu Ile Arg Leu Ala Arg Asp
515 520 525
Pro Asp Val Gly Gly Leu Asn Leu Thr Val Arg Asn Met Asp Lys Leu
530 535 540
Leu Ala Val Gln Asn Glu Val Glu Ala Tyr Phe Gly Asp Lys Gln Ala
545 550 555 560
Gly Glu Phe Cys Tyr Thr Phe Asp Asp Tyr Lys Thr Thr Met Gln Asp
565 570 575
Ile Val Ser Thr Ile Ala Asp Ala Ile Phe Cys Thr Pro Tyr Arg Arg
580 585 590
Gly Ala Asp Ile Leu Leu Asp Phe Glu Arg Pro Arg Met Gly Pro Glu
595 600 605
Met Val Phe Thr His Arg Ser Lys Ala Gly Thr Ser Glu Lys Trp Thr
610 615 620
Arg Thr Phe Asn Asp Ala Gln Val Phe Asp Ser Leu Lys Phe Ser Tyr
625 630 635 640
Ile Asp Pro Lys Thr Asn Val Lys Glu Thr Ile Thr Ile Pro Glu Thr
645 650 655
Gly Gly Val Lys Thr Glu Thr Tyr Asp Ser Lys Gly Ile Arg Asn Tyr
660 665 670
Lys Gln Ala Phe Trp Ala Ala His Arg Arg His Gln Lys Asn Ile Leu
675 680 685
Lys Lys Ile Ser Val Ser Phe Thr Ala Thr Glu Glu Gly Ile Phe Ala
690 695 700
Leu Pro Asn Arg Ala Ile Ser Val Val Lys Gly Ser Arg Met Ala Thr
705 710 715 720
Tyr Asp Gly Tyr Ile Thr Ala Val Asn Gly Leu Thr Val Glu Leu Ser
725 730 735
Gln Pro Val Lys Phe Thr Ser Gly Asp Asp His Ser Leu Ile Leu Lys
740 745 750
Leu Arg Asp Gly Gly Val Gln Ser Val Asn Val Ala Pro Gly Ala His
755 760 765
Asp Arg Gln Val Ile Met Thr Ser Val Pro Gln Glu Ala Ile Tyr Thr
770 775 780
Gly Asn Ser Ala Leu Lys Thr Glu Phe Ser Phe Gly Asn Glu Ala Arg
785 790 795 800
His Asn Ala Gln Met Ile Leu Val Ser Thr Val Asp Pro Gly Asp Asp
805 810 815
Arg Thr Val Lys Ile Thr Gly Phe Asn Tyr Asp Lys Asp Phe Tyr Lys
820 825 830
Phe Asp Asn Val Pro Pro Phe Gly Arg Ala Phe Ser Asn Gly Phe Asp
835 840 845
Asn Gly Phe Asn
850
<210> 15
<211> 1170
<212> DNA
<213> Tail protein of phase PYC04 (reactive tail protein of phase PYC 04)
<400> 15
atggcgttac aaccatataa aggcgcgatg accgcgcagt tttacgttct tgagacgacg 60
ccgggggtaa cacccactaa cccggtatgg caaccactgc gcaacactgg gggtattccc 120
gccgtaaccc gcgacgccct catctctaat gaactggacg gcagccgtga aacatcatct 180
atccgcaccg gtaaccgtca ggtaactggt gaatatgcta ttgaacttag cgcggaaagc 240
caggatgagt tgctggccgg cgcaatgacc agttcctggg tagcaggttc cactaaatcg 300
ggaatcagcg ttaccgtaga cccggtggcg aaaactttca cacgcgcaga cggtagcttt 360
gtgacagacg gcgttgaagt aggcgacctg gtgcaattcg atggtttatc cggaaataac 420
gacaaagcgt tcatggtcac ggctgtcaca gctacagtta taaccggtgc gggtatccag 480
cataccctta ccgccgaatc cgacgtccag gccaatttgc gtatcgcgga taaactggaa 540
actggtaact tgtgtaaaac ctattcaatc ctgacatggt tgaaaggtaa atgcggaaat 600
cctgattcgt acatcataac tcgtggtgtt gaatttaccg ggttcaccat cgaacaagcc 660
gttaacgcga tggttacagg ttcattcccg ttcattggct tgaatcagga aatcctacaa 720
gcaccgccga gtggttcaga tttcacgacc aattttagcg cccgtccgtt tgcatcggtt 780
gacgtatccg cctatgacgg cgccgctccg cttaaactta tcgacacgtt caccattact 840
aacgacaaca gcgcgtccgc acagttcgag ttaggaaata acagcgtggc atttgtcgaa 900
cgtggccgcg cggctaacac cttctcgttg gcgggtaagc tgtacgacat gacgttattg 960
aataaattcc tgaacgaaac gcaaatggag gtatcttctg ttctgaacgg cccggacggt 1020
gccatgagtt tcaccttaaa acgcgcttcg ttgacatcag caactccgga aatcggtggt 1080
cccgaatctg tcaccctgtc tcttgagggc caggcaaccg gcaaccagtt ccagtcttca 1140
attgttatcc agcgcattaa gtacgcctaa 1170
<210> 16
<211> 389
<212> PRT
<213> Tail protein of phase PYC04 (reactive tail protein of phase PYC 04)
<400> 16
Met Ala Leu Gln Pro Tyr Lys Gly Ala Met Thr Ala Gln Phe Tyr Val
1 5 10 15
Leu Glu Thr Thr Pro Gly Val Thr Pro Thr Asn Pro Val Trp Gln Pro
20 25 30
Leu Arg Asn Thr Gly Gly Ile Pro Ala Val Thr Arg Asp Ala Leu Ile
35 40 45
Ser Asn Glu Leu Asp Gly Ser Arg Glu Thr Ser Ser Ile Arg Thr Gly
50 55 60
Asn Arg Gln Val Thr Gly Glu Tyr Ala Ile Glu Leu Ser Ala Glu Ser
65 70 75 80
Gln Asp Glu Leu Leu Ala Gly Ala Met Thr Ser Ser Trp Val Ala Gly
85 90 95
Ser Thr Lys Ser Gly Ile Ser Val Thr Val Asp Pro Val Ala Lys Thr
100 105 110
Phe Thr Arg Ala Asp Gly Ser Phe Val Thr Asp Gly Val Glu Val Gly
115 120 125
Asp Leu Val Gln Phe Asp Gly Leu Ser Gly Asn Asn Asp Lys Ala Phe
130 135 140
Met Val Thr Ala Val Thr Ala Thr Val Ile Thr Gly Ala Gly Ile Gln
145 150 155 160
His Thr Leu Thr Ala Glu Ser Asp Val Gln Ala Asn Leu Arg Ile Ala
165 170 175
Asp Lys Leu Glu Thr Gly Asn Leu Cys Lys Thr Tyr Ser Ile Leu Thr
180 185 190
Trp Leu Lys Gly Lys Cys Gly Asn Pro Asp Ser Tyr Ile Ile Thr Arg
195 200 205
Gly Val Glu Phe Thr Gly Phe Thr Ile Glu Gln Ala Val Asn Ala Met
210 215 220
Val Thr Gly Ser Phe Pro Phe Ile Gly Leu Asn Gln Glu Ile Leu Gln
225 230 235 240
Ala Pro Pro Ser Gly Ser Asp Phe Thr Thr Asn Phe Ser Ala Arg Pro
245 250 255
Phe Ala Ser Val Asp Val Ser Ala Tyr Asp Gly Ala Ala Pro Leu Lys
260 265 270
Leu Ile Asp Thr Phe Thr Ile Thr Asn Asp Asn Ser Ala Ser Ala Gln
275 280 285
Phe Glu Leu Gly Asn Asn Ser Val Ala Phe Val Glu Arg Gly Arg Ala
290 295 300
Ala Asn Thr Phe Ser Leu Ala Gly Lys Leu Tyr Asp Met Thr Leu Leu
305 310 315 320
Asn Lys Phe Leu Asn Glu Thr Gln Met Glu Val Ser Ser Val Leu Asn
325 330 335
Gly Pro Asp Gly Ala Met Ser Phe Thr Leu Lys Arg Ala Ser Leu Thr
340 345 350
Ser Ala Thr Pro Glu Ile Gly Gly Pro Glu Ser Val Thr Leu Ser Leu
355 360 365
Glu Gly Gln Ala Thr Gly Asn Gln Phe Gln Ser Ser Ile Val Ile Gln
370 375 380
Arg Ile Lys Tyr Ala
385
<210> 17
<211> 1272
<212> DNA
<213> terminal enzyme Large subunit of phase PYC04 (terminating enzyme Large subet of phase PYC 04)
<400> 17
gtgaacgttg atatcacagc tacggaaccg cagggcgcgt tccttaatct gcattgtaag 60
ttcccggcct tcgtcgcggg cttcggcaca ggtaaatcgg aagtcatgtg caactccgcc 120
ctgttagaca gcatggaagg tggtagtgat tcacttatcg ccatgtatga accgacatac 180
gacctggttc gccttatcct cgctccgcgt atggaagaga agttgtctga ttggggtatt 240
cgctacaagt ataataaatc cgacaacatc atttatacct catccgggca attcggggat 300
tttgtccttc gcacattgga taatccagca cgaattgttg gctacgaatc gttccgcgca 360
aaaatagacg agttggacac gttaaataaa gaccacgccg agcacgcctg gaacaaagtt 420
atcgcccgta accgtcagtt gccgcgtaca tatcgtccaa ttaccccgaa gcccgctaat 480
acagtttcgg tatttacgac gccagaaggc ttccgttttg tgcacgacag atgggctgta 540
aaaaagaaac caggctatga aatgattcag gcctcgacaa catccaatcc ctttctaccg 600
gaagattatg tgcagtcgtt gcgggatacg tatccaggcc agttaattga tgcctacatc 660
ggcggcgaat ttgtcaacct gacatccggc agcgtgtatt atgcttacga ccgacgtaag 720
aacagcagcc gggagactat acaaccgggt gaaactttgt atattgggca ggactttaac 780
gtcgggcata tggctagcac cgtgtatgtt cagcgtgagt atgtctggca cgcggtagcc 840
gagctggtgg atatgttcga caccccggat gtggtcaggg agattaccga gcggtggaaa 900
cgacacggcc accacatcgt cgtgtacccg gacgccagcg gcaagaaccg taaatcgact 960
gatgccagta cgtcagatat tgcccaatta cagaacgcgg gtttcgagat acgtgcgaaa 1020
tcagttaacc ctgcggttaa agaccgtgta gcatcggtga ataaagcgct ggagtctggt 1080
agattaatgg tcaacgagca ggcttgcccg gttacggcac gttgcctgga gcaacaggct 1140
tacgataaaa acggtatacc ggataagaca agcggcaacg accaccagaa cgacgcgaca 1200
ggatacccta tcgcctacga aatgccgttg gttaaacctg tttcccatat cccggttact 1260
tttgcacttt aa 1272
<210> 18
<211> 423
<212> PRT
<213> terminal enzyme Large subunit of phase PYC04 (terminase large subunit of phase PYC 04)
<400> 18
Met Asn Val Asp Ile Thr Ala Thr Glu Pro Gln Gly Ala Phe Leu Asn
1 5 10 15
Leu His Cys Lys Phe Pro Ala Phe Val Ala Gly Phe Gly Thr Gly Lys
20 25 30
Ser Glu Val Met Cys Asn Ser Ala Leu Leu Asp Ser Met Glu Gly Gly
35 40 45
Ser Asp Ser Leu Ile Ala Met Tyr Glu Pro Thr Tyr Asp Leu Val Arg
50 55 60
Leu Ile Leu Ala Pro Arg Met Glu Glu Lys Leu Ser Asp Trp Gly Ile
65 70 75 80
Arg Tyr Lys Tyr Asn Lys Ser Asp Asn Ile Ile Tyr Thr Ser Ser Gly
85 90 95
Gln Phe Gly Asp Phe Val Leu Arg Thr Leu Asp Asn Pro Ala Arg Ile
100 105 110
Val Gly Tyr Glu Ser Phe Arg Ala Lys Ile Asp Glu Leu Asp Thr Leu
115 120 125
Asn Lys Asp His Ala Glu His Ala Trp Asn Lys Val Ile Ala Arg Asn
130 135 140
Arg Gln Leu Pro Arg Thr Tyr Arg Pro Ile Thr Pro Lys Pro Ala Asn
145 150 155 160
Thr Val Ser Val Phe Thr Thr Pro Glu Gly Phe Arg Phe Val His Asp
165 170 175
Arg Trp Ala Val Lys Lys Lys Pro Gly Tyr Glu Met Ile Gln Ala Ser
180 185 190
Thr Thr Ser Asn Pro Phe Leu Pro Glu Asp Tyr Val Gln Ser Leu Arg
195 200 205
Asp Thr Tyr Pro Gly Gln Leu Ile Asp Ala Tyr Ile Gly Gly Glu Phe
210 215 220
Val Asn Leu Thr Ser Gly Ser Val Tyr Tyr Ala Tyr Asp Arg Arg Lys
225 230 235 240
Asn Ser Ser Arg Glu Thr Ile Gln Pro Gly Glu Thr Leu Tyr Ile Gly
245 250 255
Gln Asp Phe Asn Val Gly His Met Ala Ser Thr Val Tyr Val Gln Arg
260 265 270
Glu Tyr Val Trp His Ala Val Ala Glu Leu Val Asp Met Phe Asp Thr
275 280 285
Pro Asp Val Val Arg Glu Ile Thr Glu Arg Trp Lys Arg His Gly His
290 295 300
His Ile Val Val Tyr Pro Asp Ala Ser Gly Lys Asn Arg Lys Ser Thr
305 310 315 320
Asp Ala Ser Thr Ser Asp Ile Ala Gln Leu Gln Asn Ala Gly Phe Glu
325 330 335
Ile Arg Ala Lys Ser Val Asn Pro Ala Val Lys Asp Arg Val Ala Ser
340 345 350
Val Asn Lys Ala Leu Glu Ser Gly Arg Leu Met Val Asn Glu Gln Ala
355 360 365
Cys Pro Val Thr Ala Arg Cys Leu Glu Gln Gln Ala Tyr Asp Lys Asn
370 375 380
Gly Ile Pro Asp Lys Thr Ser Gly Asn Asp His Gln Asn Asp Ala Thr
385 390 395 400
Gly Tyr Pro Ile Ala Tyr Glu Met Pro Leu Val Lys Pro Val Ser His
405 410 415
Ile Pro Val Thr Phe Ala Leu
420
<210> 19
<211> 2271
<212> DNA
<213> DNA polymerase of phase PYC04 (DNA polymerase of phase PYC 04)
<400> 19
atgaatctgc tttatctcga tactgaaaca ttctcagaag ccgatttgaa aaaagtcggt 60
tcctatgcct atgccgaaca tccgaccacc gaaattgtta tttgcaccta cgctttcgat 120
gaaggccccg tgcaggtatg ggatgccacc gacgggagcg atatgccgcg tgatttgcgc 180
cgggcgatgc tgaagctgca aaaaccagac agcaatctca aactggtagg ccaaaacttc 240
cttatgttcg accgaccagt tattaagcat tgctggggat tcgaactcct ggtagaaaac 300
attatagaca ctatgatagt cgcgttccga catgccctcc cgggttcact ggccgcgctg 360
tgtgaggttt taaacattga cgcaagcatg gctaaggata aacgcggcaa ggcgctcata 420
cagcggttca gtaagcctac gcctaagaac tataagattc gacgatatac cgccaatacc 480
cacccaaaag agtgggcaga atttatcgca tacgcaaaaa gcgacattac gtctatgcgt 540
gaagtgtata agaaaatgcc gaagtggggg aattctgagt tcgaagaccg cgtgctgtgg 600
ttagaccaag taattaacga ccgcggattt aaggttgacg ttgcccttgc tgaggccgcg 660
attgaagcag tgacgcgcca caaggaagag ctacaggaag aagcccaacg caaatacggc 720
ggttcactaa ccggcaaaga cttcttgcct attttacagg aactggcgcc ggcgcaccgc 780
attcacaacg cacagaagtc aacactaaac gatttgctgg cagatgagga tttaccggac 840
gatgcgcgta ctattattga gatgcgtctc ggggctgcat ccactgcatc gacgaaatac 900
gcgccgttgc tgttaggccg ttcttccgat gaccgccgtc gtggttgtct gcaatacggt 960
ggagcgaagc ggacattgcg atgggccggg aaaggttttc agccacagaa cctggcgcgc 1020
ggatattatc acgatgaccc gaagaataaa aagaagaaag tacgttacga ctggatgtca 1080
gataaggact ggtggagagt aacacaccca ttgtcctacg gaattgacct gttgcttaaa 1140
ggtcgggcgc atcgccgttt tgatgtagcc aaactaacgg cgtctacggt gcgtagctgc 1200
atcatcccgg aggccgggca taagtttgtt gttgcggact actctaacgt tgaaggacgc 1260
ggtctcgcgt tcctgtccgg ggaagaaaca gcacttgata ctttccgctc gggcctcgac 1320
atatactgcg taacagcagg aaagatgttc ggcatggacc cggatgatat taagaaaaac 1380
ttcaatgata tcagacagat aggaaaggct tgcgagttgg gtctcggcta tgagggtggt 1440
gtcggagcgt tcgttacatt cgccaaaaac ttaggattag accttatcga aatggctaaa 1500
acaatggccg gaactttccc cgaccatatc tgggccgcta ccgctcgcgg gtatgagtgg 1560
gcacggatac aggaggccaa gcgaccgccg cggccaggtg aaaaggatga ccgtccatcg 1620
tatattctcg ataagaaagt atggcgtaca tgtgatgcta ttaaacgtat gtggcgtgaa 1680
tcgcacccgc aaaccgtagc tttctggcac gaccttaaag acggtatcct tgccgctgta 1740
cggaatcctg gccgcgattt ctgggcgggc gcaaatatcc gaagcaacgg tgaaagagct 1800
atacgcatat ggcggacgac ggaaaccgat tcatccggtc gaaacatccc ggggtggtgg 1860
ctatgcatgg agctgccgtc gggccgcata ttgtcgtatc cagggatagg tgtcagcgtg 1920
acaaaggaaa ccgacgaaga tgggcgggta aacaccaatg taaggattaa gtaccagggc 1980
gagaaccagt taacacggca gtggaccact ctatacacac acggcgggaa ggcttgcgag 2040
aacatcgttc aggcgttttg tcgtgactta ttggcttatg caatgcttaa tgttgaggct 2100
ggagggtatc ctatcgttct ttcggttcat gatgaactgg tatgtgagac cccggataca 2160
ccagattaca cggtagcgga actgggaaaa ctaatgtgtg cattgccaga atgggctgat 2220
ggttttcctc ttgtagcgga aggtgcggag ttaaaacggt atgctaagta a 2271
<210> 20
<211> 756
<212> PRT
<213> DNA polymerase of phase PYC04 (DNA polymerase of phase PYC 04)
<400> 20
Met Asn Leu Leu Tyr Leu Asp Thr Glu Thr Phe Ser Glu Ala Asp Leu
1 5 10 15
Lys Lys Val Gly Ser Tyr Ala Tyr Ala Glu His Pro Thr Thr Glu Ile
20 25 30
Val Ile Cys Thr Tyr Ala Phe Asp Glu Gly Pro Val Gln Val Trp Asp
35 40 45
Ala Thr Asp Gly Ser Asp Met Pro Arg Asp Leu Arg Arg Ala Met Leu
50 55 60
Lys Leu Gln Lys Pro Asp Ser Asn Leu Lys Leu Val Gly Gln Asn Phe
65 70 75 80
Leu Met Phe Asp Arg Pro Val Ile Lys His Cys Trp Gly Phe Glu Leu
85 90 95
Leu Val Glu Asn Ile Ile Asp Thr Met Ile Val Ala Phe Arg His Ala
100 105 110
Leu Pro Gly Ser Leu Ala Ala Leu Cys Glu Val Leu Asn Ile Asp Ala
115 120 125
Ser Met Ala Lys Asp Lys Arg Gly Lys Ala Leu Ile Gln Arg Phe Ser
130 135 140
Lys Pro Thr Pro Lys Asn Tyr Lys Ile Arg Arg Tyr Thr Ala Asn Thr
145 150 155 160
His Pro Lys Glu Trp Ala Glu Phe Ile Ala Tyr Ala Lys Ser Asp Ile
165 170 175
Thr Ser Met Arg Glu Val Tyr Lys Lys Met Pro Lys Trp Gly Asn Ser
180 185 190
Glu Phe Glu Asp Arg Val Leu Trp Leu Asp Gln Val Ile Asn Asp Arg
195 200 205
Gly Phe Lys Val Asp Val Ala Leu Ala Glu Ala Ala Ile Glu Ala Val
210 215 220
Thr Arg His Lys Glu Glu Leu Gln Glu Glu Ala Gln Arg Lys Tyr Gly
225 230 235 240
Gly Ser Leu Thr Gly Lys Asp Phe Leu Pro Ile Leu Gln Glu Leu Ala
245 250 255
Pro Ala His Arg Ile His Asn Ala Gln Lys Ser Thr Leu Asn Asp Leu
260 265 270
Leu Ala Asp Glu Asp Leu Pro Asp Asp Ala Arg Thr Ile Ile Glu Met
275 280 285
Arg Leu Gly Ala Ala Ser Thr Ala Ser Thr Lys Tyr Ala Pro Leu Leu
290 295 300
Leu Gly Arg Ser Ser Asp Asp Arg Arg Arg Gly Cys Leu Gln Tyr Gly
305 310 315 320
Gly Ala Lys Arg Thr Leu Arg Trp Ala Gly Lys Gly Phe Gln Pro Gln
325 330 335
Asn Leu Ala Arg Gly Tyr Tyr His Asp Asp Pro Lys Asn Lys Lys Lys
340 345 350
Lys Val Arg Tyr Asp Trp Met Ser Asp Lys Asp Trp Trp Arg Val Thr
355 360 365
His Pro Leu Ser Tyr Gly Ile Asp Leu Leu Leu Lys Gly Arg Ala His
370 375 380
Arg Arg Phe Asp Val Ala Lys Leu Thr Ala Ser Thr Val Arg Ser Cys
385 390 395 400
Ile Ile Pro Glu Ala Gly His Lys Phe Val Val Ala Asp Tyr Ser Asn
405 410 415
Val Glu Gly Arg Gly Leu Ala Phe Leu Ser Gly Glu Glu Thr Ala Leu
420 425 430
Asp Thr Phe Arg Ser Gly Leu Asp Ile Tyr Cys Val Thr Ala Gly Lys
435 440 445
Met Phe Gly Met Asp Pro Asp Asp Ile Lys Lys Asn Phe Asn Asp Ile
450 455 460
Arg Gln Ile Gly Lys Ala Cys Glu Leu Gly Leu Gly Tyr Glu Gly Gly
465 470 475 480
Val Gly Ala Phe Val Thr Phe Ala Lys Asn Leu Gly Leu Asp Leu Ile
485 490 495
Glu Met Ala Lys Thr Met Ala Gly Thr Phe Pro Asp His Ile Trp Ala
500 505 510
Ala Thr Ala Arg Gly Tyr Glu Trp Ala Arg Ile Gln Glu Ala Lys Arg
515 520 525
Pro Pro Arg Pro Gly Glu Lys Asp Asp Arg Pro Ser Tyr Ile Leu Asp
530 535 540
Lys Lys Val Trp Arg Thr Cys Asp Ala Ile Lys Arg Met Trp Arg Glu
545 550 555 560
Ser His Pro Gln Thr Val Ala Phe Trp His Asp Leu Lys Asp Gly Ile
565 570 575
Leu Ala Ala Val Arg Asn Pro Gly Arg Asp Phe Trp Ala Gly Ala Asn
580 585 590
Ile Arg Ser Asn Gly Glu Arg Ala Ile Arg Ile Trp Arg Thr Thr Glu
595 600 605
Thr Asp Ser Ser Gly Arg Asn Ile Pro Gly Trp Trp Leu Cys Met Glu
610 615 620
Leu Pro Ser Gly Arg Ile Leu Ser Tyr Pro Gly Ile Gly Val Ser Val
625 630 635 640
Thr Lys Glu Thr Asp Glu Asp Gly Arg Val Asn Thr Asn Val Arg Ile
645 650 655
Lys Tyr Gln Gly Glu Asn Gln Leu Thr Arg Gln Trp Thr Thr Leu Tyr
660 665 670
Thr His Gly Gly Lys Ala Cys Glu Asn Ile Val Gln Ala Phe Cys Arg
675 680 685
Asp Leu Leu Ala Tyr Ala Met Leu Asn Val Glu Ala Gly Gly Tyr Pro
690 695 700
Ile Val Leu Ser Val His Asp Glu Leu Val Cys Glu Thr Pro Asp Thr
705 710 715 720
Pro Asp Tyr Thr Val Ala Glu Leu Gly Lys Leu Met Cys Ala Leu Pro
725 730 735
Glu Trp Ala Asp Gly Phe Pro Leu Val Ala Glu Gly Ala Glu Leu Lys
740 745 750
Arg Tyr Ala Lys
755
<210> 21
<211> 489
<212> DNA
<213> lyase of phase PYC04 (lysozyme of phase PYC 04)
<400> 21
atgtcaaacc gaaacatcag tgataacgga ttacacttca ccgccgcgtt cgaggggttc 60
cggggaaccg cctaccgcgc gacaccttca gaaaaatact ttactattgg ctacggccac 120
tacggtgctg atgtgaaaga aggccagaag attaccgaag gccagggcct cctgcttctg 180
cataaggata tggctaaggc cgtagctgcg gtggacgccg ttgcgcatcc gacgctcaat 240
cagtcacagt tcgatgcgat gtgcgacctg gtgtataacg ctggcgccgg tgtgattgcg 300
gcttctaccg gaacagggca ggccctgcgt aagggcgacg ttgccacact gcggaataag 360
ttaactcagt tccattatca gaacggcaaa tcactcctcg gattacgtcg tcgtgccgcc 420
ggtcgtgttg cgctgttcga tggcatgctg tggcaacagg ccgaagccat cggccgcggc 480
gcaaagtag 489
<210> 22
<211> 162
<212> PRT
<213> lyase of phase PYC04 (lysozyme of phase PYC 04)
<400> 22
Met Ser Asn Arg Asn Ile Ser Asp Asn Gly Leu His Phe Thr Ala Ala
1 5 10 15
Phe Glu Gly Phe Arg Gly Thr Ala Tyr Arg Ala Thr Pro Ser Glu Lys
20 25 30
Tyr Phe Thr Ile Gly Tyr Gly His Tyr Gly Ala Asp Val Lys Glu Gly
35 40 45
Gln Lys Ile Thr Glu Gly Gln Gly Leu Leu Leu Leu His Lys Asp Met
50 55 60
Ala Lys Ala Val Ala Ala Val Asp Ala Val Ala His Pro Thr Leu Asn
65 70 75 80
Gln Ser Gln Phe Asp Ala Met Cys Asp Leu Val Tyr Asn Ala Gly Ala
85 90 95
Gly Val Ile Ala Ala Ser Thr Gly Thr Gly Gln Ala Leu Arg Lys Gly
100 105 110
Asp Val Ala Thr Leu Arg Asn Lys Leu Thr Gln Phe His Tyr Gln Asn
115 120 125
Gly Lys Ser Leu Leu Gly Leu Arg Arg Arg Ala Ala Gly Arg Val Ala
130 135 140
Leu Phe Asp Gly Met Leu Trp Gln Gln Ala Glu Ala Ile Gly Arg Gly
145 150 155 160
Ala Lys

Claims (10)

1. The phage composition is characterized by comprising a salmonella phage PYC04 and an escherichia coli phage PD07, wherein the preservation number of the escherichia coli phage PD07 is CGMCC No18865; the preservation number of the salmonella bacteriophage PYC04 is CGMCC No18867.
2. The phage composition of claim 1, wherein the ratio of viable numbers of coliphage to salmonella phage is 1; the content of each bacteriophage is not less than 1 × 10 8 pfu/mL。
3. The phage composition of claim 1, further comprising an adjuvant; the auxiliary material is one or more of SM buffer solution, sodium alginate, sucrose, maltodextrin and glucose.
4. Use of a phage composition according to any of claims 1-3 in the manufacture of a medicament for the prevention and treatment of salpingitis in chickens.
5. A bactericide for preventing and treating salpingitis in chickens, comprising the phage composition of any one of claims 1 to 3.
6. An environmental disinfectant, characterized in that the active ingredient comprises the phage composition according to any of claims 1 to 3.
7. An environmental disinfectant according to claim 6 wherein each bacteriophage is present in a concentration of 5 x 10 8 PFU/ml or more.
8. A feed additive comprising the phage composition of any of claims 1 to 3.
9. The feed additive of claim 8 wherein the concentration of each bacteriophage in the feed is at least 1 x 10 8 PFU/g。
10. A test kit comprising the phage composition of any of claims 1-3.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103289963A (en) * 2012-12-12 2013-09-11 青岛农业大学 Bacteriophage with environment disinfection capability and applications thereof
RU2496874C1 (en) * 2012-06-05 2013-10-27 Федеральное бюджетное учреждение науки Государственный научный центр прикладной микробиологии и биотехнологии (ФБУН ГНЦ ПМБ) STRAIN OF BACTERIOPHAGE Escherichia coli ECD4, HAVING LYTIC ACTIVITY IN RESPECT TO BACTERIA Escherichia coli OF SEROTYPE O104:H4
CN108359644A (en) * 2018-02-07 2018-08-03 青岛诺安百特生物技术有限公司 A kind of wide range salmonella bacteriophage and its application
CN110129283A (en) * 2019-05-24 2019-08-16 青岛诺安百特生物技术有限公司 A kind of short-tail coliphage and its application

Family Cites Families (2)

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CN108546685B (en) * 2018-04-20 2021-01-05 华中农业大学 Salmonella enteritidis bacteriophage LPSE28 and application thereof in food
CN110241091B (en) * 2019-05-29 2021-01-05 华中农业大学 Salmonella dublin phage D1-2 and application thereof in liquid egg samples

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2496874C1 (en) * 2012-06-05 2013-10-27 Федеральное бюджетное учреждение науки Государственный научный центр прикладной микробиологии и биотехнологии (ФБУН ГНЦ ПМБ) STRAIN OF BACTERIOPHAGE Escherichia coli ECD4, HAVING LYTIC ACTIVITY IN RESPECT TO BACTERIA Escherichia coli OF SEROTYPE O104:H4
CN103289963A (en) * 2012-12-12 2013-09-11 青岛农业大学 Bacteriophage with environment disinfection capability and applications thereof
CN108359644A (en) * 2018-02-07 2018-08-03 青岛诺安百特生物技术有限公司 A kind of wide range salmonella bacteriophage and its application
CN110129283A (en) * 2019-05-24 2019-08-16 青岛诺安百特生物技术有限公司 A kind of short-tail coliphage and its application

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