CN110563815A - Pseudomonas aeruginosa bacteriophage K8 putative protein GP075, and mutant strain, mutant protein and application thereof - Google Patents

Abstract

The invention relates to a pseudomonas aeruginosa bacteriophage K8 hypothetical protein GP075, the nucleotide sequence of which is SEQ No.1, and the amino acid sequence of which is SEQ No. 10. The protein GP075 is supposed to be mutated, the mutated protein has the function of structural protein, the phage mutant strain is additionally provided with additional receptor recognition protein on the basis of original recognition of lipopolysaccharide, can recognize lipopolysaccharide O-antigen defective host cells or host cells only containing core oligosaccharide structures (core oligosaccharides), and has wider host range and stronger capability of cracking and adsorbing the host cells, so that the phage mutant strain is expected to be applied to phage preparations and can prevent and treat various infections caused by pseudomonas aeruginosa.

Description

A hypothetical protein GP075 of Pseudomonas aeruginosa phage K8 and its mutant strain and mutation protein and application

technical field

The invention belongs to the technical field of bioengineering, in particular to a hypothetical protein GP075 of Pseudomonas aeruginosa phage K8 and its mutant strain, mutant protein and application.

Background technique

Pseudomonas aeruginosa is an opportunistic pathogen that easily infects immunocompromised, debilitated and cystic fibrosis patients, resulting in higher morbidity and mortality in cystic fibrosis patients. As the natural killer of bacteria, phage can specifically kill bacteria, and the study of the interaction between phage and host will help to develop phage preparations for the treatment of bacterial infections. In recent years, the research on Pseudomonas aeruginosa and its phages has been increasing day by day, and the infection mechanism of various phages of Pseudomonas aeruginosa has become more and more clear. Most of the isolated Pseudomonas aeruginosa phage recognition receptors are lipopolysaccharide O- Antigens, the most studied are genes related to LPS synthesis, mainly including wbpL, wbpR, wbpO, algC, wbpV, galU, wbpT, wzy, wapH, migA, ssg and wbpS. At the same time, most phages use their tails to recognize cell receptors, and under the right conditions, the interaction of the tails and the receptors induces a structural rearrangement of the tails, which eventually transduces to the head-tail junction, triggering its opening, and finally making the phage The DNA is released, completing the phage infection process. On the basis of existing research, the phage receptor binding protein of Pseudomonas aeruginosa is tail filament protein, but there are few studies on hypothetical proteins with unknown functions as receptor binding proteins.

In recent years, with the abuse of antibiotics, the number of antibiotic-resistant bacteria has been increasing, and the problems of various infectious diseases caused by resistant bacteria need to be solved urgently. For example, Pseudomonas aeruginosa is an important conditional pathogen that causes nosocomial infections. As we all know, phages can infect host bacteria, and have strong host specificity, and do not affect other non-host normal cells. Therefore, the study of phages Therapy, as an alternative to antibiotics, is particularly important. Currently the most researched cocktail therapy, that is, mixed phage therapy (referring to a treatment method that uses a variety of phages in combination to kill a variety of bacteria), as early as 2014, the European Union launched the Phagoburn project to target clinically common aeruginosa. "Cocktail therapy" has had a good effect on various infections caused by Monomonas, Escherichia, Proteus, Klebsiella, and Staphylococcus, and the safety of cocktail therapy has been evaluated. On the other hand, while using phage preparations to treat bacterial infections, the combination of phage preparations and antibiotics can also be considered to achieve therapeutic effects.

In the bactericidal experiments of phages in vivo and in vitro, the same phage has different bactericidal efficiency for different host bacteria, which is related to the process of phage infection of host bacteria affected by the gene expression level of host bacteria, and the mechanism of phage lysis host depends on host bacteria, Thereby increasing the complexity of phage lysing host bacteria. Knowing more about the host genes associated with phage infection will benefit the therapeutic efficiency of phage and provide therapeutic options for various clinical infections in the future.

Through searching, no patent publications related to the patent application of the present invention have been found.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a pseudomonas aeruginosa phage K8 hypothetical protein GP075 and its mutant strain, mutant protein and application, the hypothetical protein GP075 is mutated, and the mutated protein has structural protein The function of this type of phage mutant strains is based on the original recognition of lipopolysaccharide, adding an additional receptor recognition protein, which can recognize lipopolysaccharide O-antigen-deficient host cells or host cells that only contain core oligosaccharides (core oligosaccharides) , and has a wider host range, stronger ability to lyse and adsorb host cells, and is expected to be applied to phage preparations to prevent and treat various infections caused by Pseudomonas aeruginosa.

The technical solution adopted by the present invention to solve its technical problems is:

A hypothetical protein GP075 of Pseudomonas aeruginosa phage K8, the nucleotide sequence of which is SEQ No.1, and the amino acid sequence of which is SEQ No.10.

A Pseudomonas aeruginosa phage K8 mutant strain K8-D7, said mutant strain K8-D7 can infect lipopolysaccharide O-antigen-deficient host cells, its sequence is amino acid 105, 106 in the hypothetical protein GP075 as described above An amino acid sequence that completely repeats 106 to 112 is inserted between them; the nucleotide sequence of the mutant K8-D7 is SEQ No.2, and its amino acid sequence is SEQ No.11.

A mutant protein GP075-14 containing the hypothetical protein GP075 as described above, its sequence is to insert two completely repeated amino acid sequences from 106 to 112 between amino acids 105 and 106 of the GP075, and the mutant protein GP075-14 The nucleotide sequence is SEQ No.3, and the amino acid sequence is SEQ No.12.

A mutant protein GP075-21 containing the hypothetical protein GP075 as claimed in claim 1, its sequence is GP075 amino acid 105, 106 inserting 3 completely repeated amino acid sequences from 106 to 102, the mutant protein GP075-21 The nucleotide sequence is SEQ No.4, and the amino acid sequence is SEQ No.13.

A Pseudomonas aeruginosa phage K8 mutant strain K8-E126K, said mutant strain K8-E126K can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is the mutation E126K in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant K8-E126K is SEQ No.5, and its amino acid sequence is SEQ No.14.

A Pseudomonas aeruginosa phage K8 mutant strain K8-S142L, said mutant strain K8-S142L can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is the mutation S142L in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant K8-S142L is SEQ No.6, and its amino acid sequence is SEQ No.15.

A Pseudomonas aeruginosa phage K8 mutant strain K8-L189R, said mutant strain K8-L189R is capable of infecting lipopolysaccharide O-antigen-deficient host cells, and its sequence is a mutation L189R in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant K8-L189R is SEQ No.7, and its amino acid sequence is, for example, SEQ No.16.

A Pseudomonas aeruginosa bacteriophage K8 mutant strain K8-P197L, said mutant strain K8-P197L can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is mutated P197L in the above-mentioned hypothetical protein GP075, so The nucleotide sequence of the mutant K8-P197L is SEQ No.8, and its amino acid sequence is SEQ No.17.

A Pseudomonas aeruginosa phage K8 mutant strain K8-T239A, said mutant strain K8-T239A can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is the mutation T239A in the above-mentioned putative protein GP075, so The nucleotide sequence of the mutant K8-T239A is SEQ No.9, and its amino acid sequence is SEQ No.18.

A Pseudomonas aeruginosa phage K8 mutant strain K8-X, the mutant strain K8-X can infect lipopolysaccharide O-antigen-deficient host cells, and GP075 has no mutation.

Use of the Pseudomonas aeruginosa bacteriophage K8 mutant strain K8-X as described above in the growth inhibition of Pseudomonas aeruginosa O-antigen-deficient strains, or in the inhibition of spontaneous mutations in Pseudomonas aeruginosa.

Advantage and positive effect that the present invention obtains are:

1. During the research of the present invention, it was found that the phage K8 mutant population contained a variety of GP075 mutant phages, and the isolated phage K8 mutant strains mainly included K8-D7, K8-E126K, K8-S142L, K8-L189R, K8-P197L , K8-T239A, and in the process of studying the diversity of GP075 mutations, two mutant proteins, GP075-14 and GP075-21, were found with a relatively high proportion. The putative protein GP075 in the above phages has a mutation, and the mutated protein has the function of a structural protein. On the basis of the original recognition of lipopolysaccharide, this type of phage mutant strain has added an additional receptor recognition protein, which can recognize lipopolysaccharide O-antigen-deficient Host cells or host cells containing only core oligosaccharides have a wider host range, stronger ability to lyse and adsorb host cells, and are expected to be used in phage preparations to prevent and treat Pseudomonas aeruginosa. Various infections.

2. The K8 mutant strain isolated in the present invention can recognize the new receptor core oligosaccharide on the host cell, while retaining the original recognition of the original receptor O-antigen, has dual receptor recognition ability, and greatly improves the recognition of aeruginosa. The bactericidal ability of Pseudomonas has the ability to inhibit the spontaneous mutation of Pseudomonas aeruginosa, and can be used in the development and application of phage preparations to solve the infection problem caused by resistant Pseudomonas aeruginosa.

3. In the present invention, the new function of the phage K8 hypothetical protein GP075 mutant is excavated, and the function of this kind of protein mutant is redefined through this study, and it plays an important role in the process of phage infection of host cells. The bacteriophage containing the GP075 mutant has strong bactericidal activity, and can be used as a phage preparation for Pseudomonas aeruginosa to guide clinical medication for Pseudomonas aeruginosa infection and achieve the effect of treating Pseudomonas aeruginosa infection.

4. In the present invention, the genetic background of bacteriophage K8 is clear, and on this basis, strive to find a new mutant phage with O-antigen-deficient recognition, so that the follow-up research will be more scientific. Using O-antigen-deficient host cells as indicator bacteria to isolate phage K8 mutant strains, the research is more purposeful.

5. The present invention provides a class of hypothetical protein GP075 mutants whose functions are related to phage infection. This type of mutants is an important structural protein for phages to recognize the core oligosaccharides of host cells; the present invention provides multiple strains of broad-spectrum Pseudomonas aeruginosa phages, Infect host cells deficient in LPS O-antigen or containing only core oligosaccharides.

6. The significance of the present invention is that in recent years, Pseudomonas aeruginosa has become the main pathogenic strain of hospital-acquired diseases, but in the process of using antibiotics for treatment, Pseudomonas aeruginosa gradually develops resistance to antibiotics and evolves multidrug resistance. In order to better treat the infection caused by Pseudomonas aeruginosa, while actively developing new antibiotics, scientists are constantly looking for alternatives to antibiotics. As the killer of bacteria, phages can recognize and kill bacteria and have a good therapeutic effect on infections caused by bacteria. No virulence factors were found in all the proteins with known functions in the entire genome of phage K8 and its phage mutant strains, which laid a good foundation for future phage therapy and provided safety guarantees during the use of phage preparations in the future.

7. The present invention uses Pseudomonas aeruginosa phage K8 as the starting phage. Previous studies have confirmed that the recognition receptor of wild-type Pseudomonas aeruginosa phage K8 is lipopolysaccharide O-antigen. In the absence of host cell O-antigen, phage K8 cannot infect this host cell, and there is no research on the function of GP075 at the present stage. A number of spontaneous mutant strains of K8 were isolated, the most common feature of which is the ability to infect lipopolysaccharide O-antigen-deficient host cells, and the whole genome of phage K8-T239A was sequenced, and it was found that only the 239th position of protein GP075 in this phage was composed of Threonine T was mutated to alanine A, and the GP075 of other phages was sequenced and found that the gene had different mutations. In the present invention, this is used as a starting point to study the new function of GP075 and its application in inhibiting the growth of Pseudomonas aeruginosa.

Description of drawings

Fig. 1 is the basic property analysis diagram of the interaction between phage K8 mutant strain and host cell in the present invention; Wherein, A is the host range diagram of bacteriophage K8 and mutant strain K8-T239A, B is the adsorption rate diagram of PAK and mutant strain, and C is the host Bacterial LPS structure diagram;

Fig. 2 is the mutated gene identification figure of bacteriophage K8 mutant strain among the present invention;

Fig. 3 is the granule functional protein analysis diagram of LC-MS detection K8 phage mutant strain among the present invention;

Fig. 4 is the antibacterial and bactericidal ability application figure of bacteriophage K8 mutant strain in the present invention; Wherein, A is phage K8 and mutant strain K8-T239A inhibition PAK growth curve figure, B is phage K8 and mutant strain K8-T239A inhibition SK75 growth curve Figure C is the screening map of PAK-resistant phage K8 and mutant strain K8-T239A, D is the screening map of SK75-resistant phage K8-T239A, and E is the spontaneous mutation rate map of host strains resistant to phage K8 and mutant strain K8-T239A.

Detailed ways

The embodiments of the present invention will be described in detail below. It should be noted that the embodiments are illustrative, not restrictive, and cannot limit the protection scope of the present invention.

The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

A hypothetical protein GP075 of Pseudomonas aeruginosa phage K8, wild-type GP075 protein, its nucleotide sequence is SEQ No.1, and its amino acid sequence is SEQ No.10.

A Pseudomonas aeruginosa phage K8 mutant strain K8-D7, said mutant strain K8-D7 can infect lipopolysaccharide O-antigen-deficient host cells, its sequence is amino acid 105, 106 in the hypothetical protein GP075 as described above An amino acid sequence that completely repeats 106 to 112 is inserted between them; the nucleotide sequence of the mutant K8-D7 is SEQ No.2, and its amino acid sequence is SEQ No.11.

A mutant protein GP075-14 containing the hypothetical protein GP075 as described above, its sequence is to insert two completely repeated amino acid sequences from 106 to 112 between amino acids 105 and 106 of the GP075, and the mutant protein GP075-14 The nucleotide sequence is SEQ No.3, and the amino acid sequence is SEQ No.12.

A mutant protein GP075-21 containing the hypothetical protein GP075 as claimed in claim 1, its sequence is GP075 amino acid 105, 106 inserting 3 completely repeated amino acid sequences from 106 to 102, the mutant protein GP075-21 The nucleotide sequence is SEQ No.4, and the amino acid sequence is SEQ No.13.

A Pseudomonas aeruginosa phage K8 mutant strain K8-E126K, said mutant strain K8-E126K can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is the mutation E126K in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant K8-E126K is SEQ No.5, and its amino acid sequence is SEQ No.14.

A Pseudomonas aeruginosa phage K8 mutant strain K8-S142L, said mutant strain K8-S142L can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is the mutation S142L in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant K8-S142L is SEQ No.6, and its amino acid sequence is SEQ No.15.

A Pseudomonas aeruginosa phage K8 mutant strain K8-L189R, said mutant strain K8-L189R is capable of infecting lipopolysaccharide O-antigen-deficient host cells, and its sequence is a mutation L189R in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant K8-L189R is SEQ No.7, and its amino acid sequence is, for example, SEQ No.16.

A Pseudomonas aeruginosa bacteriophage K8 mutant strain K8-P197L, said mutant strain K8-P197L can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is mutated P197L in the above-mentioned hypothetical protein GP075, so The nucleotide sequence of the mutant K8-P197L is SEQ No.8, and its amino acid sequence is SEQ No.17.

A Pseudomonas aeruginosa phage K8 mutant strain K8-T239A, said mutant strain K8-T239A can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is the mutation T239A in the above-mentioned putative protein GP075, so The nucleotide sequence of the mutant K8-T239A is SEQ No.9, and its amino acid sequence is SEQ No.18.

A Pseudomonas aeruginosa phage K8 mutant strain K8-X, the mutant strain K8-X can infect lipopolysaccharide O-antigen-deficient host cells, and GP075 has no mutation.

Use of the Pseudomonas aeruginosa bacteriophage K8 mutant strain K8-X as described above in the growth inhibition of Pseudomonas aeruginosa O-antigen-deficient strains, or in the inhibition of spontaneous mutations in Pseudomonas aeruginosa.

The present invention mainly isolates a plurality of Pseudomonas aeruginosa bacteriophage K8 mutant strains, determines the host range of this type of bacteriophage, and identifies the specific mutation site of the mutant phage through whole genome sequencing and GP075 in vitro amplicon sequencing, and the specific mutation site of the type of bacteriophage The practical application value is to evaluate the antibacterial and bactericidal ability.

The specific isolation and identification steps of Pseudomonas aeruginosa phage K8 mutant strain of the present invention can be as follows:

(1) Construction of a spontaneous mutation library of Pseudomonas aeruginosa phage K8;

(2) using lipopolysaccharide O-antigen-deficient host cells to isolate and obtain a phage K8 mutant;

(3) Phage genome extraction, GP075 gene amplification in vitro;

(4) Phage whole genome sequencing and GP075 amplicon sequencing identification;

(5) The bacteriophage K8 mutant acts on O-antigen-deficient host cells;

(6) Inhibitory effect of phage K8 mutant strain on spontaneous mutation of host cells.

Specifically, the relevant material steps of the present invention are as follows:

1. Experimental materials

The strains and phages used in the experiment are shown in Table 1.

Table 1 Bacterial strains and phages used in the present invention

2. Isolation of bacteriophage K8 mutant strain

Subgroups of mutant strains of bacteriophage K8, using receptor-deficient Pseudomonas aeruginosa SK2 (wbpV), SK15 (wbpO), SK45 (wbpR) as indicator bacteria 200 μL (OD ₆₀₀ =0.6) and 100 μL K8 phage (10 ⁹ ) Subpopulations, mutant phage capable of infecting O-antigen-deficient strains were isolated using the double-layer plate method. Incubate the double-layer plate at 37°C for 4 hours until clear plaques appear. The plaques that appear at this time are formed by mutated phages. Pick a single plaque and place it in fresh LB culture medium. After purification for three times, mutant phages with uniform size and the same degree of transparency were obtained. At this time, the phage was the K8 mutant strain.

3. Host range analysis of phage mutant strains

Take 200 μL logarithmic phase indicator bacteria (OD ₆₀₀ =0.6) and mix with 3mL melted soft agar (0.5%, mass concentration m/v), and quickly pour into solidified LB solid (the content of agar is 1.5%, mass concentration m/v) The upper layer of the culture medium, let stand at room temperature for about 15 minutes, after the soft agar is dry, 1 μL of 100-fold diluted phage lysate (10 ⁸ pfu/mL), vertically spot on the soft agar layer of the double-layer plate, 37°C , Static culture for 4h, observe whether there is a transparent phage circle formation.

4. Whole genome sequencing of bacteriophage K8 mutant strain

Extract the genomic DNA of bacteriophage K8-T239A, analyze the purity and concentration of phage DNA with a nucleic acid analyzer, and meet the three indicators, that is, the concentration of DNA samples is not less than 10ng/uL, and the total amount is not less than 5ug; OD260/OD280 should be between 1.8 and 2.0 Between; OD260/OD230 greater than 1.8. Send the samples that meet the indicators to the sequencing company.

Phage whole-genome DNA sequencing was performed using the sequencing platform Illumina Hiseq 2500. Perform image recognition (Base calling) and preliminary quality analysis on the sequencing results, use the next-generation sequencing data quality filtering software Trimmomatic (v0.30) to remove low-quality and linker sequences, etc., and use the preprocessed read mapping (reads mapping) The software Velvet_v1.2.10 was used for assembly and splicing. Use the online software NCBI ( https://www.ncbi.nlm.nih.gov/ ) and select Align two sequences in BLAST to compare with the whole genome sequence of phage K8 to find the mutations in the phage mutant strain.

5. Gene gp075 amplicon sequencing

Using the extracted phage mutants as templates, primers GP075-F: 5'-ATATCACCGTAACTACGGT-3', GP075-R: 5'-GTTGACTGTAATCAGCCATT-3' were used to amplify the gene gp075 fragment in vitro, and sanger sequencing was performed, and the sequencing results were For the base sequence in the sequence, first use the software Chromas version2.4 to observe the reliability of the sequencing results (no overlapping peaks, etc.), and use the TranslationsProtein in the software Primer Premier 5.exe to sequence the reliable sequence to translate the base sequence into an amino acid sequence. Align two sequences in the online software NCBI ( https://www.ncbi.nlm.nih.gov/ ) Protein BLAST were compared with the amino acid sequence of the protein GP075 to analyze the changes of the putative protein GP075 in a single mutant phage.

6. Adsorption rate

The host bacteria cultivated overnight were inoculated into 5 mL liquid LB medium according to the transfer amount of 3%, cultivated to logarithmic phase (OD ₆₀₀ =0.6), 650 μL host bacteria and 650 μL (MOI=0.001) phage were drawn, mixed evenly, and statically Put it in the adsorption for 1 min, immediately draw 100 μL, and record it as the total number of phage infection centers at this time, then centrifuge at 13,000 rpm for 30 s, discard the supernatant, add 1200 μL liquid LB to mix well, absorb 100 μL again, and record it as the phage infection after the first washing For the number of centers, according to the above-mentioned washing method, wash once every minute, wash 6 times in total, and use the double-layer plate method to measure the number of infected centers remaining after each washing by using the double-layer plate method. The formula for calculating the phage adsorption rate is as follows:

A—the adsorption rate of phage (%);

S—the titer (pfu/mL) of phage in the supernatant after centrifugation;

C—the titer (pfu/mL) of phage in the control group that does not add bacterium;

7. Extraction of strain LPS (Lipopolysaccharides)

Refer to the experimental method (Merino S, Gonzalez V, Tomás J M. The Polymerization of Aeromonas hydrophila AH-3O-Antigen LPS: Concerted Action of WecP and Wzy [J]. PlosOne, 2015, 10(7): e0131905.) The operation is as follows:

(1) Cultivation of strains: pick and purify a single colony, inoculate in 5mL LB liquid medium in a sterile environment, 37°C, 220rpm, culture overnight, transfer 3% transfer amount to 100mL LB liquid the next day In culture medium, culture to logarithmic phase (OD600=0.6), 4°C, 7000rpm, centrifuge for 10min, discard supernatant, collect bacterial cells, resuspend bacterial cells with 5mL distilled water, mix well, and set aside.

(2) Hot phenol water method: add an equal volume of water-saturated phenol to the above-mentioned resuspended bacteria liquid, 68°C, 120rpm, water bath shaker, act for 30min, then ice bath for 30min, 4°C, 7000rpm, centrifuge for 10min, collect water Phase, repeat the above operation twice.

(3) Dialysis: absorb the above-mentioned collected water phase into a dialysis bag, stir the distilled water for dialysis, change the distilled water every 4 hours, and dialyze for 20 hours until the water-saturated phenol is completely removed by dialysis.

(4) Concentrate the aqueous phase: Concentrate the aqueous phase in the dialysis bag with 30% PEG8000 for about 30-60 minutes until the volume of the concentrated solution is about 1 mL.

(5) Ethanol precipitation: suck out the concentrated solution, add 2 times the volume of absolute ethanol and 1/10 volume of sodium acetate solution, and settle overnight at -20°C.

(6) Washing of LPS: After settling, centrifuge at 12000 rpm for 10 min, discard the supernatant, and obtain LPS. Finally, wash twice with 75% ethanol, discard the supernatant, blot the excess liquid as much as possible, and place it at room temperature for 30 minutes. After the LPS sample is dried, add an appropriate amount of sterile water and store at -20°C.

Eight, LC-MS detection of phage protein particles

Referring to the description of the experimental method (Yang H, Liang L, Lin S, & Jia S (2010) Isolation and characterization of a virulent bacteriophage AB1 of Acinetobacterbaumannii. BMC microbiology 10:131.), use SDS-PAGE to separate the phage particle protein and obtain the isolated After the final phage particle protein glue, the protein is cut into small fragments by specific enzymatic hydrolysis method, and then the relative molecular mass of each product peptide is detected by mass spectrometry, and the mass number of the obtained proteolysis peptide is retrieved in the corresponding database. Find matching protein fragments.

The separated SDS-PAGE was detected according to the following mass spectrometry method (Hellman J. Polyacrylamide enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF [J]. Proteomics, 2007, 7(19): 3441-4.), For structural protein detection, each protein group sample was subjected to in-gel digestion, and the enzymatic digestion was performed approximately according to trypsin: protein = 1:40;

Each 1D gel lane was divided into 4 parts, which were digested separately.

LC-MS detection analysis

(1) Liquid phase conditions: Phase A: water (0.1% formic acid), phase B: Acetonitrile (0.1% formic acid), flow rate 300nL/min; gradient: 0-5min, 2% B; 5-80min, 2% -25 %B; 85-100min, 25%-35%B; 100-105min, 35%-95%; 105-120min, 95%B; C18 column.

(2) Mass spectrometry conditions: voltage 2.2kv, MS scanning range 350-1550m/z, detector orbitrap; MS2 is fragmented by HCD and detected by ion trap.

LC-MS data analysis

(3) Mascot v2.5 software (Matrixscience, Boston, USA) was used to search the provided database for protein identification, and MaxQuant software was used for comparative analysis.

(4) Information of the used high-resolution mass spectrometer (LC-MS): Obitrap Fusion (Thermofisher, San Jose, USA).

Data filtering principles:

(1) pep_expect value ≤ 0.01 is more credible

(2) The identified protein requires more than two independent characteristic fragments

(3) Other data are for reference.

9. Application of phage antibacterial experiment

Phage inhibition curve test method: the host bacteria cultivated overnight were inoculated in 96-well bacterial culture dishes according to 3% transfer amount, and the diluted phage (MOI=0.001) was added, and the control group did not add phage (MOI=0). Each group was repeated three times. Cultivate at 37°C and 160 rpm, measure OD ₆₀₀ every 0.5 h with a multi-functional microplate reader, and measure the growth rate of the host bacteria in the presence or absence of phage until 5.5 h.

1. Pseudomonas aeruginosa resistant phage spontaneous mutation assay

The spontaneous mutation frequency of wild-type Pseudomonas aeruginosa strains resistant to phages was determined by the turbidity method (Zhang Kebin, Chen Zhijin, Jin Xiaolin, et al. Isolation and identification of Pseudomonas aeruginosa phages and determination of phage-resistant mutation frequencies[J] ]. Microbiology Bulletin, 2002, 29(1): 40-45.). The overnight cultured host bacteria PAK and RO2-15 were serially diluted to 10 ⁻¹⁰ . Take 100 μL of samples from each of the 10 ^-6 , 10 ^-7 , and 10 ^-8 dilution tubes, and use the colony counting method to measure the number of bacteria/mL. At the same time, add 10 μL of phage stock solution (10 ¹⁰ pfu/mL) to each dilution tube, mix well, and shake culture at 37°C. After 4-6 hours, it can be seen that each tube gradually becomes clear, indicating that the bacteria have been lysed, and continue to culture for 20 hours. , it can be found that the high-concentration bacterial solution tubes gradually return to turbidity until no new turbidity returns appear on the next day. Interpret the result at this time: the reciprocal of the number of bacteria contained in the lowest concentration tube that can return to turbidity is the frequency of tolerance mutations. 10 parallel experiments were performed for each group of experiments.

2. Results and Discussion

In the present invention, multiple strains of bacteriophage K8 mutants are isolated from O-antigen-deficient host cells. In the present invention, K8-T239A is used as a typical representative for the following research.

(1) Isolation and determination of basic properties of phage K8 mutant strain

In the present invention, multiple strains of bacteriophage K8 mutants are isolated from O-antigen-deficient host cells. In the present invention, K8-T239A is used as a typical representative for the following research. Pseudomonas aeruginosa PAK transposon Tn5G insertion mutants SK98(ssg), SK75(wzy), SK2(wbpV), M21(galU), SK15(wbpO), P2-25(wapH), SK45(wbpR), The spotting assays were all resistant to K8, among which SK75, SK2, SK15, and SK45 were sensitive to phage K8-T239A to form a plaque transparent circle, and SK98, M21, and P2-25 were resistant to phage K8-T239A (Figure 1A). According to the results of the adsorption capacity of PAK and 7 insertion mutants with phage K8 and K8-T239A, it was found that PAK, SK75, SK2, SK15, and SK45 had significant differences in the adsorption capacity of K8 and K8-T239A, that is, the adsorption capacity of K8-T239A was higher than that of K8 Strong, but all mutants had lower adsorption capacity than PAK (Fig. 1B).

The phenotype analysis of the mutant genes of the strains found that the wild-type PAK had both LPS OSA and Coreoligosaccharide, and the mutant strains SK75, SK2, SK15, and SK45 whose deletion genes were wzy, wbpV, wbpO, and wbpR were presumed to be LPS OSA deletions , SK98, M21, P2-25 indel genes were ssg, galU, wapH presumed that the LPS of these three bacterial strains were completely deleted (Fig. 1C). Tricine-SDS-PAGE analyzed the lipopolysaccharide components of PAK and 7 insertion mutant strains, and found that LPSCore oligosaccharides existed in the strains sensitive to phage K8-T239A, PAK, SK75, SK2, SK15, and SK45, and resistant to phage K8-T239A SK98, M21, and P2-25 strains completely lacked lipopolysaccharide components (Fig. 1C). The results of Tricine-SDS-PAGE were completely consistent with the previous genotype speculation. Combined with the results of spotting assay, it is not difficult to speculate that K8-T239A recognizes the new receptor as LPS Core oligosaccharide, but at the same time retains the recognition of the original receptor LPSOSA.

(2) Phage Whole Genome Sequencing and Amplicon Sequencing Analysis

Whole-genome sequencing found that compared with K8, phage K8-T239A had only one point mutation in the entire genome sequence. Further analysis found that the point mutation appeared at the 715th position of the gene gp075 base level from A→G (adenine was mutated to Guanine), amino acid level 239 T→A (threonine is mutated to alanine), this protein is annotated as a hypothetical protein; at the same time, amplicon sequencing was carried out on the gene gp075 of other isolated phage mutants, and 25 strains SVN (point mutation, Single nucleotide site variation) occurred in GP075. The mutation occurred at four different positions on the gene gp075. According to the amino acid level analysis, the 126th position of E126K was mutated from E glutamic acid to K lysine (such as sequence 14), S142L The 142nd position is mutated from S serine to L leucine (such as sequence 15), the 189th position of L189R is mutated from L leucine to R arginine (such as sequence 16), and the 197th position of P197L is mutated from P proline to L leucine (such as sequence 17); 11 strains of GP075 have no mutations, and it is speculated that the mutations occurred in other hypothetical proteins or structural proteins; in addition, there is an insertion repeat sequence CGGTGCTCCATGGTACTCGGT, located between the 314th and 315th positions of the gene gp075, the 21bp The repeat sequence is exactly the same as the original sequence 315 to 335 of the gene gp075. What is more interesting is that the beginning and end of the repeat sequence and the adjacent 4 base sequences are all CGGT (such as nucleic acid sequence 2). This feature of the original sequence makes the insertion site between 314 and 315, but the encoded amino acid does not have any frameshift mutation, and there is a repeat sequence of 7 amino acids with the 106th to 112th of GP075, that is, GAPWY S V (such as Sequence 11), (Fig. 2).

(3) LC-MS detection of granule proteins of phage mutant strains

The structural proteins in phage K8 and K8-T239A particles were separated by SDS-PAGE, and the structural proteins separated on the gel were further identified by LC-MS. Analysis of identification results showed that there were 13 proteins in K8-T239A, and only 12 proteins in phage K8 particles. After comparison, it was found that phage K8 particles had less hypothetical protein GP075 than phage K8-T239A, and the other 12 identified proteins were consistent. Including 8 functional proteins, respectively GP035, GP057, GP062, GP063, GP068, GP074, GP076, GP078, the corresponding protein functions are DNA ligase (DNA ligase), major capsid protein (majorcapsid protein), hypothetical structural protein ( putative structural protein, putative structural protein, putative tape measure protein, putative baseplate related protein, putative tail fiber protein, putative tail fiber protein ); the four hypothetical proteins are encoded by GP053, GP056, GP071, and GP110, respectively (Fig. 3). This result indicates that the GP075 protein mutant has a new function and can function as a structural protein that has no annotated function.

(4) Antibacterial application of bacteriophage

One of the basic properties of phages is to infect host cells and lyse host cells. The co-cultivation of phages and host cells can inhibit the growth of host cells. In the growth curve of wild-type Pseudomonas aeruginosa PAK, no phage control group was added, cultured to 5.5h, OD600 reached 0.7, co-cultured with phage K8, K8-T239A respectively, PAK almost stopped growing after 2.0h, phage K8 , K8-T239A had a strong inhibitory effect on the growth of PAK (Fig. 4A). The same amount of K8, K8-T239A was co-cultured with PAK at different dilutions (10-fold gradient dilution), and the turbidity of the bacterial solution at each dilution was observed. The minimum turbidity concentration of PAK can be used to calculate the spontaneous mutation rate of PAK-resistant phages. The results show that , PAK and K8 were co-cultured, only one tube was clear when the PAK concentration was 7.0×10 ⁵ cfu/ml, but when the bacterial concentration was 7.0×10 ⁴ cfu/ml, the co-culture solution was all clear, indicating the minimum turbidity concentration of PAK The mean value is 7.0×10 ⁵ cfu/ml, and the mean frequency of PAK-resistant K8 spontaneous mutation is 2.91×10 ^-6 ; K8-T239A is co-cultured with PAK, the minimum turbidity concentration is 7.0×10 ⁸ cfu/ml, and the calculated PAK tolerance The average spontaneous mutation frequency of K8-T239A was 2.83×10 ^-9 , and the spontaneous mutation frequency of PAK-resistant K8-T239A was much lower than that of K8, and there was a significant difference (Fig. 4B, 4E).

SK75 growth curve, the growth status of SK75 without adding phage was used as a control. After culturing for 5.5 hours, OD600 reached 0.7. After adding phage K8, the growth status of SK75 was not inhibited, and OD600 reached 0.69; adding phage K8-TT239A and SK75 together After culturing for 3.0 hours, the growth of SK75 was inhibited, and then almost stopped growing, so it can be seen that K8-T239A has an inhibitory effect on the growth of SK75 (Figure 4C). SK75 was co-cultured with K8-T239A. When the bacterial concentration of SK75 was 1.04×10 ² cfu/ml, there were still two tubes of turbidity. When the bacterial concentration was 1.04×10cfu/ml, the co-culture solution was completely clear, indicating that the minimum turbidity concentration of SK75 was mainly concentrated at 1.04 ×10 ³ cfu/ml, the calculated average frequency of SK75 resistant K8-T239A spontaneous mutation was 9.93×10 ^-4 (Fig. 4D, Fig. 4E). Phage K8 and K8-T239A effectively inhibited the growth of PAK. When co-cultured, the spontaneous mutation rate of PAK resistant to K8-T239A was 1000 times lower than that of K8; at the same time, K8-T239A inhibited the growth of SK75, and K8-T239A could inhibit the growth of SK75 to a certain extent. The frequency of spontaneous mutations suppressing SK75 is 10 ⁶ higher than that of PAK resistant K8-T239A. The above results further confirm that K8-T239A can recognize dual receptors and has good application value.

Although the embodiments of the present invention are disclosed for the purpose of illustration, those skilled in the art will understand that various alternatives, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims, therefore However, the scope of the present invention is not limited to the contents disclosed in the embodiments and drawings.

sequence listing

Sequence 1 Nucleic acid sequence of Pseudomonas aeruginosa bacteriophage K8 gene gp075

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 2 Nucleic acid sequence of Pseudomonas aeruginosa bacteriophage K8-D7 gene gp075

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 3 Nucleic acid sequence of protein GP075-14

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 4 Nucleic acid sequence of protein GP075-21

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 5 Nucleic acid sequence of Pseudomonas aeruginosa bacteriophage K8-E126K gene gp075

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAAAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 6 Pseudomonas aeruginosa phage K8-S142L gene gp075 nucleic acid sequence

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTTAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 7 Pseudomonas aeruginosa phage K8-L142R gene gp075 nucleic acid sequence

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCGAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 8 Pseudomonas aeruginosa phage K8-P142L gene gp075 nucleic acid sequence

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCTTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 9 Nucleic acid sequence of Pseudomonas aeruginosa phage K8-T239A gene gp075

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGGCCAACCTAGTGACATAA

SEQUENCE 10 GP075 Amino Acid Sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILTNLVT

Sequence 11 GP075-7 amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILTNLVT

Sequence 12 GP075-14 amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPWYSVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILTNLVT

Sequence 13 GP075-21 amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPWYSVGAPWYSVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILTNLVT

Sequence 14 GP075-E126K amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEKYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILTNLVT

Sequence 15 GP075-S142L amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNLTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILTNLVT

Sequence 16 GP075-L189R amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDRGGAGALLPKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILTNLVT

Sequence 17 GP075-P197L amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLLKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILTNLVT

Sequence 18 GP075-T239A amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVNYTYTEFQAGRVFATEGFPGGQGVGDLNDPTVGGILANLVT。

sequence listing

<110> Tianjin University of Science and Technology

<120> A hypothetical protein GP075 of Pseudomonas aeruginosa phage K8 and its mutant strain, mutant protein and application

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 732

<212>DNA

<213> Nucleotide sequence of hypothetical protein GP075 (Unknown)

<400> 1

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccaactgg taacgccaga 360

gagccgagcg acgaagagta tcggatgatc ctgaaagcaa agatcatcaa gaacagaaca 420

aactcaaccc cagagcaagt tatcgaagct tataaatttg tattcggggt tcctgaagta 480

ttcctagagg agtacgctcc cgctgctgtc cgtatcggca tcggtaagat tctaacgaac 540

gtagagcgta gtcttctatt cgacctaggt ggtgcaggtg cattgcttcc taagactatc 600

ggggttaact acacatacac tgagttccaa gctggccggg tatttgctac agaaggcttc 660

cccggaggac aaggcgttgg agacctaaat gatcccactg ttggtggaat tctgaccaac 720

ctagtgacat aa 732

<210> 2

<211> 753

<212>DNA

<213> Nucleotide sequence of K8-D7 (Unknown)

<400> 2

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccatggta ctcggtcggt 360

gctccaactg gtaacgccag agagccgagc gacgaagagt atcggatgat cctgaaagca 420

aagatcatca agaacagaac aaactcaacc ccagagcaag ttatcgaagc ttataaattt 480

gtattcgggg ttcctgaagt attcctagag gagtacgctc ccgctgctgt ccgtatcggc 540

atcggtaaga ttctaacgaa cgtagagcgt agtcttctat tcgacctagg tggtgcaggt 600

gcattgcttc ctaagactat cggggttaac tacacataca ctgagttcca agctggccgg 660

gtatttgcta cagaaggctt ccccggagga caaggcgttg gagacctaaa tgatcccact 720

gttggtggaa ttctgaccaa cctagtgaca taa 753

<210> 3

<211> 774

<212>DNA

<213> Nucleotide sequence of GP075-14 (Unknown)

<400> 3

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccatggta ctcggtcggt 360

gctccatggt actcggtcgg tgctccaact ggtaacgcca gagagccgag cgacgaagag 420

tatcggatga tcctgaaagc aaagatcatc aagaacagaa caaactcaac cccagagcaa 480

gttatcgaag cttataaatt tgtattcggg gttcctgaag tattcctaga ggagtacgct 540

cccgctgctg tccgtatcgg catcggtaag attctaacga acgtagagcg tagtcttcta 600

ttcgacctag gtggtgcagg tgcattgctt cctaagacta tcggggttaa ctacacatac 660

actgagttcc aagctggccg ggtatttgct acagaaggct tccccggagg acaaggcgtt 720

ggagacctaa atgatcccac tgttggtgga attctgacca acctagtgac ataa 774

<210> 4

<211> 795

<212>DNA

<213> Nucleotide sequence of GP075-21 (Unknown)

<400> 4

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccatggta ctcggtcggt 360

gctccatggt actcggtcgg tgctccatgg tactcggtcg gtgctccaac tggtaacgcc 420

agagagccga gcgacgaaga gtatcggatg atcctgaaag caaagatcat caagaacaga 480

acaaactcaa ccccagagca agttatcgaa gcttataaat ttgtattcgg ggttcctgaa 540

gtattcctag aggagtacgc tcccgctgct gtccgtatcg gcatcggtaa gattctaacg 600

aacgtagagc gtagtcttct attcgaccta ggtggtgcag gtgcattgct tcctaagact 660

atcggggtta actacacata cactgagttc caagctggcc gggtatttgc tacagaaggc 720

ttccccggag gacaaggcgt tggagaccta aatgatccca ctgttggtgg aattctgacc 780

aacctagtga cataa 795

<210> 5

<211> 732

<212>DNA

<213> Nucleotide sequence of K8-E126K (Unknown)

<400> 5

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccaactgg taacgccaga 360

gagccgagcg acgaaaagta tcggatgatc ctgaaagcaa agatcatcaa gaacagaaca 420

aactcaaccc cagagcaagt tatcgaagct tataaatttg tattcggggt tcctgaagta 480

ttcctagagg agtacgctcc cgctgctgtc cgtatcggca tcggtaagat tctaacgaac 540

gtagagcgta gtcttctatt cgacctaggt ggtgcaggtg cattgcttcc taagactatc 600

ggggttaact acacatacac tgagttccaa gctggccggg tatttgctac agaaggcttc 660

cccggaggac aaggcgttgg agacctaaat gatcccactg ttggtggaat tctgaccaac 720

ctagtgacat aa 732

<210> 6

<211> 732

<212>DNA

<213> Nucleotide sequence of K8-S142L (Unknown)

<400> 6

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccaactgg taacgccaga 360

gagccgagcg acgaagagta tcggatgatc ctgaaagcaa agatcatcaa gaacagaaca 420

aacttaaccc cagagcaagt tatcgaagct tataaatttg tattcggggt tcctgaagta 480

ttcctagagg agtacgctcc cgctgctgtc cgtatcggca tcggtaagat tctaacgaac 540

gtagagcgta gtcttctatt cgacctaggt ggtgcaggtg cattgcttcc taagactatc 600

ggggttaact acacatacac tgagttccaa gctggccggg tatttgctac agaaggcttc 660

cccggaggac aaggcgttgg agacctaaat gatcccactg ttggtggaat tctgaccaac 720

ctagtgacat aa 732

<210> 7

<211> 732

<212>DNA

<213> Nucleotide sequence of K8-L189R (Unknown)

<400> 7

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccaactgg taacgccaga 360

gagccgagcg acgaagagta tcggatgatc ctgaaagcaa agatcatcaa gaacagaaca 420

aactcaaccc cagagcaagt tatcgaagct tataaatttg tattcggggt tcctgaagta 480

ttcctagagg agtacgctcc cgctgctgtc cgtatcggca tcggtaagat tctaacgaac 540

gtagagcgta gtcttctatt cgaccgaggt ggtgcaggtg cattgcttcc taagactatc 600

ggggttaact acacatacac tgagttccaa gctggccggg tatttgctac agaaggcttc 660

cccggaggac aaggcgttgg agacctaaat gatcccactg ttggtggaat tctgaccaac 720

ctagtgacat aa 732

<210> 8

<211> 732

<212>DNA

<213> Nucleotide sequence of K8-P197L (Unknown)

<400> 8

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccaactgg taacgccaga 360

gagccgagcg acgaagagta tcggatgatc ctgaaagcaa agatcatcaa gaacagaaca 420

aactcaaccc cagagcaagt tatcgaagct tataaatttg tattcggggt tcctgaagta 480

ttcctagagg agtacgctcc cgctgctgtc cgtatcggca tcggtaagat tctaacgaac 540

gtagagcgta gtcttctatt cgacctaggt ggtgcaggtg cattgcttct taagactatc 600

ggggttaact acacatacac tgagttccaa gctggccggg tatttgctac agaaggcttc 660

cccggaggac aaggcgttgg agacctaaat gatcccactg ttggtggaat tctgaccaac 720

ctagtgacat aa 732

<210> 9

<211> 732

<212>DNA

<213> Nucleotide sequence of K8-T239A (Unknown)

<400> 9

atggctgtca accaatttga cagagaagat tatctggagg tggcccggga acgggtcact 60

gaacagttta aagagaagcc gatctttgat cgcttcctgc aagtgctatt gtctggtaag 120

tttgatatcc agaatgcact ggaagacctc cagactctcc ggtctctgga cacagccacc 180

gggaagcaac tggacattat cggagacatt gtagggcgac cacgcggtct agtgtaccaa 240

gatattttca actattttgg attcgctgga acggagcgtg caggttcttt cggaagcctg 300

tcggacccta cggtcggtgc tccatggtac tcggtcggtg ctccaactgg taacgccaga 360

gagccgagcg acgaagagta tcggatgatc ctgaaagcaa agatcatcaa gaacagaaca 420

aactcaaccc cagagcaagt tatcgaagct tataaatttg tattcggggt tcctgaagta 480

ttcctagagg agtacgctcc cgctgctgtc cgtatcggca tcggtaagat tctaacgaac 540

gtagagcgta gtcttctatt cgacctaggt ggtgcaggtg cattgcttcc taagactatc 600

ggggttaact acacatacac tgagttccaa gctggccggg tatttgctac agaaggcttc 660

cccggaggac aaggcgttgg agacctaaat gatcccactg ttggtggaat tctggccaac 720

ctagtgacat aa 732

<210> 10

<211> 243

<212> PRT

<213> Amino acid sequence of GP075 (Unknown)

<400> 10

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Thr Gly Asn Ala Arg Glu Pro Ser Asp Glu Glu Tyr Arg

115 120 125

Met Ile Leu Lys Ala Lys Ile Ile Lys Asn Arg Thr Asn Ser Thr Pro

130 135 140

Glu Gln Val Ile Glu Ala Tyr Lys Phe Val Phe Gly Val Pro Glu Val

145 150 155 160

Phe Leu Glu Glu Tyr Ala Pro Ala Ala Val Arg Ile Gly Ile Gly Lys

165 170 175

Ile Leu Thr Asn Val Glu Arg Ser Leu Leu Phe Asp Leu Gly Gly Ala

180 185 190

Gly Ala Leu Leu Pro Lys Thr Ile Gly Val Asn Tyr Thr Tyr Thr Glu

195 200 205

Phe Gln Ala Gly Arg Val Phe Ala Thr Glu Gly Phe Pro Gly Gly Gln

210 215 220

Gly Val Gly Asp Leu Asn Asp Pro Thr Val Gly Gly Ile Leu Thr Asn

225 230 235 240

Leu Val Thr

<210> 11

<211> 250

<212> PRT

<213> Amino acid sequence of K8-D7 (Unknown)

<400> 11

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Trp Tyr Ser Val Gly Ala Pro Thr Gly Asn Ala Arg Glu

115 120 125

Pro Ser Asp Glu Glu Tyr Arg Met Ile Leu Lys Ala Lys Ile Ile Lys

130 135 140

Asn Arg Thr Asn Ser Thr Pro Glu Gln Val Ile Glu Ala Tyr Lys Phe

145 150 155 160

Val Phe Gly Val Pro Glu Val Phe Leu Glu Glu Tyr Ala Pro Ala Ala

165 170 175

Val Arg Ile Gly Ile Gly Lys Ile Leu Thr Asn Val Glu Arg Ser Leu

180 185 190

Leu Phe Asp Leu Gly Gly Ala Gly Ala Leu Leu Pro Lys Thr Ile Gly

195 200 205

Val Asn Tyr Thr Tyr Thr Glu Phe Gln Ala Gly Arg Val Phe Ala Thr

210 215 220

Glu Gly Phe Pro Gly Gly Gln Gly Val Gly Asp Leu Asn Asp Pro Thr

225 230 235 240

Val Gly Gly Ile Leu Thr Asn Leu Val Thr

245 250

<210> 12

<211> 257

<212> PRT

<213> Amino acid sequence of GP075-14 (Unknown)

<400> 12

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Trp Tyr Ser Val Gly Ala Pro Trp Tyr Ser Val Gly Ala

115 120 125

Pro Thr Gly Asn Ala Arg Glu Pro Ser Asp Glu Glu Tyr Arg Met Ile

130 135 140

Leu Lys Ala Lys Ile Ile Lys Asn Arg Thr Asn Ser Thr Pro Glu Gln

145 150 155 160

Val Ile Glu Ala Tyr Lys Phe Val Phe Gly Val Pro Glu Val Phe Leu

165 170 175

Glu Glu Tyr Ala Pro Ala Ala Val Arg Ile Gly Ile Gly Lys Ile Leu

180 185 190

Thr Asn Val Glu Arg Ser Leu Leu Phe Asp Leu Gly Gly Ala Gly Ala

195 200 205

Leu Leu Pro Lys Thr Ile Gly Val Asn Tyr Thr Tyr Thr Glu Phe Gln

210 215 220

Ala Gly Arg Val Phe Ala Thr Glu Gly Phe Pro Gly Gly Gln Gly Val

225 230 235 240

Gly Asp Leu Asn Asp Pro Thr Val Gly Gly Ile Leu Thr Asn Leu Val

245 250 255

Thr

<210> 13

<211> 264

<212> PRT

<213> Amino acid sequence of GP075-21 (Unknown)

<400> 13

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Trp Tyr Ser Val Gly Ala Pro Trp Tyr Ser Val Gly Ala

115 120 125

Pro Trp Tyr Ser Val Gly Ala Pro Thr Gly Asn Ala Arg Glu Pro Ser

130 135 140

Asp Glu Glu Tyr Arg Met Ile Leu Lys Ala Lys Ile Ile Lys Asn Arg

145 150 155 160

Thr Asn Ser Thr Pro Glu Gln Val Ile Glu Ala Tyr Lys Phe Val Phe

165 170 175

Gly Val Pro Glu Val Phe Leu Glu Glu Tyr Ala Pro Ala Ala Val Arg

180 185 190

Ile Gly Ile Gly Lys Ile Leu Thr Asn Val Glu Arg Ser Leu Leu Phe

195 200 205

Asp Leu Gly Gly Ala Gly Ala Leu Leu Pro Lys Thr Ile Gly Val Asn

210 215 220

Tyr Thr Tyr Thr Glu Phe Gln Ala Gly Arg Val Phe Ala Thr Glu Gly

225 230 235 240

Phe Pro Gly Gly Gln Gly Val Gly Asp Leu Asn Asp Pro Thr Val Gly

245 250 255

Gly Ile Leu Thr Asn Leu Val Thr

260

<210> 14

<211> 243

<212> PRT

<213> Amino acid sequence of K8-E126K (Unknown)

<400> 14

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Thr Gly Asn Ala Arg Glu Pro Ser Asp Glu Lys Tyr Arg

115 120 125

Met Ile Leu Lys Ala Lys Ile Ile Lys Asn Arg Thr Asn Ser Thr Pro

130 135 140

Glu Gln Val Ile Glu Ala Tyr Lys Phe Val Phe Gly Val Pro Glu Val

145 150 155 160

Phe Leu Glu Glu Tyr Ala Pro Ala Ala Val Arg Ile Gly Ile Gly Lys

165 170 175

Ile Leu Thr Asn Val Glu Arg Ser Leu Leu Phe Asp Leu Gly Gly Ala

180 185 190

Gly Ala Leu Leu Pro Lys Thr Ile Gly Val Asn Tyr Thr Tyr Thr Glu

195 200 205

Phe Gln Ala Gly Arg Val Phe Ala Thr Glu Gly Phe Pro Gly Gly Gln

210 215 220

Gly Val Gly Asp Leu Asn Asp Pro Thr Val Gly Gly Ile Leu Thr Asn

225 230 235 240

Leu Val Thr

<210> 15

<211> 243

<212> PRT

<213> Amino acid sequence of K8-S142L (Unknown)

<400> 15

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Thr Gly Asn Ala Arg Glu Pro Ser Asp Glu Glu Tyr Arg

115 120 125

Met Ile Leu Lys Ala Lys Ile Ile Lys Asn Arg Thr Asn Leu Thr Pro

130 135 140

Glu Gln Val Ile Glu Ala Tyr Lys Phe Val Phe Gly Val Pro Glu Val

145 150 155 160

Phe Leu Glu Glu Tyr Ala Pro Ala Ala Val Arg Ile Gly Ile Gly Lys

165 170 175

Ile Leu Thr Asn Val Glu Arg Ser Leu Leu Phe Asp Leu Gly Gly Ala

180 185 190

Gly Ala Leu Leu Pro Lys Thr Ile Gly Val Asn Tyr Thr Tyr Thr Glu

195 200 205

Phe Gln Ala Gly Arg Val Phe Ala Thr Glu Gly Phe Pro Gly Gly Gln

210 215 220

Gly Val Gly Asp Leu Asn Asp Pro Thr Val Gly Gly Ile Leu Thr Asn

225 230 235 240

Leu Val Thr

<210> 16

<211> 243

<212> PRT

<213> Amino acid sequence of K8-L189R (Unknown)

<400> 16

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Thr Gly Asn Ala Arg Glu Pro Ser Asp Glu Glu Tyr Arg

115 120 125

Met Ile Leu Lys Ala Lys Ile Ile Lys Asn Arg Thr Asn Ser Thr Pro

130 135 140

Glu Gln Val Ile Glu Ala Tyr Lys Phe Val Phe Gly Val Pro Glu Val

145 150 155 160

Phe Leu Glu Glu Tyr Ala Pro Ala Ala Val Arg Ile Gly Ile Gly Lys

165 170 175

Ile Leu Thr Asn Val Glu Arg Ser Leu Leu Phe Asp Arg Gly Gly Ala

180 185 190

Gly Ala Leu Leu Pro Lys Thr Ile Gly Val Asn Tyr Thr Tyr Thr Glu

195 200 205

Phe Gln Ala Gly Arg Val Phe Ala Thr Glu Gly Phe Pro Gly Gly Gln

210 215 220

Gly Val Gly Asp Leu Asn Asp Pro Thr Val Gly Gly Ile Leu Thr Asn

225 230 235 240

Leu Val Thr

<210> 17

<211> 243

<212> PRT

<213> Amino acid sequence of K8-P197L (Unknown)

<400> 17

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Thr Gly Asn Ala Arg Glu Pro Ser Asp Glu Glu Tyr Arg

115 120 125

Met Ile Leu Lys Ala Lys Ile Ile Lys Asn Arg Thr Asn Ser Thr Pro

130 135 140

Glu Gln Val Ile Glu Ala Tyr Lys Phe Val Phe Gly Val Pro Glu Val

145 150 155 160

Phe Leu Glu Glu Tyr Ala Pro Ala Ala Val Arg Ile Gly Ile Gly Lys

165 170 175

Ile Leu Thr Asn Val Glu Arg Ser Leu Leu Phe Asp Leu Gly Gly Ala

180 185 190

Gly Ala Leu Leu Leu Lys Thr Ile Gly Val Asn Tyr Thr Tyr Thr Glu

195 200 205

Phe Gln Ala Gly Arg Val Phe Ala Thr Glu Gly Phe Pro Gly Gly Gln

210 215 220

Gly Val Gly Asp Leu Asn Asp Pro Thr Val Gly Gly Ile Leu Thr Asn

225 230 235 240

Leu Val Thr

<210> 18

<211> 243

<212> PRT

<213> Amino acid sequence of K8-T239A (Unknown)

<400> 18

Met Ala Val Asn Gln Phe Asp Arg Glu Asp Tyr Leu Glu Val Ala Arg

1 5 10 15

Glu Arg Val Thr Glu Gln Phe Lys Glu Lys Pro Ile Phe Asp Arg Phe

20 25 30

Leu Gln Val Leu Leu Ser Gly Lys Phe Asp Ile Gln Asn Ala Leu Glu

35 40 45

Asp Leu Gln Thr Leu Arg Ser Leu Asp Thr Ala Thr Gly Lys Gln Leu

50 55 60

Asp Ile Ile Gly Asp Ile Val Gly Arg Pro Arg Gly Leu Val Tyr Gln

65 70 75 80

Asp Ile Phe Asn Tyr Phe Gly Phe Ala Gly Thr Glu Arg Ala Gly Ser

85 90 95

Phe Gly Ser Leu Ser Asp Pro Thr Val Gly Ala Pro Trp Tyr Ser Val

100 105 110

Gly Ala Pro Thr Gly Asn Ala Arg Glu Pro Ser Asp Glu Glu Tyr Arg

115 120 125

Met Ile Leu Lys Ala Lys Ile Ile Lys Asn Arg Thr Asn Ser Thr Pro

130 135 140

Glu Gln Val Ile Glu Ala Tyr Lys Phe Val Phe Gly Val Pro Glu Val

145 150 155 160

Phe Leu Glu Glu Tyr Ala Pro Ala Ala Val Arg Ile Gly Ile Gly Lys

165 170 175

Ile Leu Thr Asn Val Glu Arg Ser Leu Leu Phe Asp Leu Gly Gly Ala

180 185 190

Gly Ala Leu Leu Pro Lys Thr Ile Gly Val Asn Tyr Thr Tyr Thr Glu

195 200 205

Phe Gln Ala Gly Arg Val Phe Ala Thr Glu Gly Phe Pro Gly Gly Gln

210 215 220

Gly Val Gly Asp Leu Asn Asp Pro Thr Val Gly Gly Ile Leu Ala Asn

225 230 235 240

Leu Val Thr

Claims (10)

1. A pseudomonas aeruginosa bacteriophage K8 hypothetical protein GP075, characterized in that: its nucleotide sequence is SEQ No.1, and its amino acid sequence is SEQ No.10. 2. A Pseudomonas aeruginosa phage K8 mutant strain K8-D7 is characterized in that: said mutant strain K8-D7 can infect lipopolysaccharide O-antigen deficient host cells, and its sequence is as described in claim 1 1 amino acid sequence completely repeated from 106 to 112 is inserted between amino acids 105 and 106 of the hypothetical protein GP075; the nucleotide sequence of the mutant K8-D7 is SEQ No.2, and its amino acid sequence is SEQ No.11 . 3. A mutant protein GP075-14 containing the hypothetical protein GP075 as claimed in claim 1, characterized in that: its sequence is to insert 2 completely repeated amino acids from 106 to 112 between amino acids 105 and 106 of the GP075 Sequence, the nucleotide sequence of the mutant protein GP075-14 is SEQ No.3, and its amino acid sequence is SEQ No.12. 4. A mutant protein GP075-21 containing the hypothetical protein GP075 as claimed in claim 1, characterized in that: its sequence is an amino acid sequence with 3 completely repeated amino acids 106 to 102 inserted between amino acids 105 and 106 of GP075, so The nucleotide sequence of the mutant protein GP075-21 is SEQ No.4, and its amino acid sequence is SEQ No.13. 5. A Pseudomonas aeruginosa phage K8 mutant strain K8-E126K is characterized in that: said mutant strain K8-E126K can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is as described in claim 1 The hypothetical protein GP075 has a mutation E126K, the nucleotide sequence of the mutant K8-E126K is SEQ No.5, and its amino acid sequence is SEQ No.14. 6. A Pseudomonas aeruginosa phage K8 mutant strain K8-S142L, characterized in that: said mutant strain K8-S142L can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is as described in claim 1 The hypothetical protein GP075 has a mutation S142L, the nucleotide sequence of the mutant K8-S142L is SEQ No.6, and its amino acid sequence is SEQ No.15. 7. A Pseudomonas aeruginosa phage K8 mutant strain K8-L189R, characterized in that: said mutant strain K8-L189R can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is as described in claim 1 The hypothetical protein GP075 has a mutation L189R, the nucleotide sequence of the mutant K8-L189R is SEQ No.7, and its amino acid sequence is, for example, SEQ No.16. 8. A Pseudomonas aeruginosa phage K8 mutant strain K8-P197L, characterized in that: said mutant strain K8-P197L can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is as described in claim 1 The hypothetical protein GP075 has a mutation P197L, the nucleotide sequence of the mutant K8-P197L is SEQ No.8, and its amino acid sequence is SEQ No.17. 9. A Pseudomonas aeruginosa phage K8 mutant strain K8-T239A, characterized in that: said mutant strain K8-T239A can infect lipopolysaccharide O-antigen-deficient host cells, and its sequence is as described in claim 1 The hypothetical protein GP075 has a mutation T239A, the nucleotide sequence of the mutant K8-T239A is SEQ No.9, and its amino acid sequence is SEQ No.18. 10. A Pseudomonas aeruginosa bacteriophage K8 mutant strain K8-X, characterized in that: the mutant strain K8-X can infect lipopolysaccharide O-antigen-deficient host cells, in the suppression of Pseudomonas aeruginosa spontaneous mutation For use in this aspect, it is assumed that the protein GP075 is not mutated.