CN110563815B - A pseudomonas aeruginosa phage K8 hypothetical protein GP075 and its mutant strain, mutant protein and application - Google Patents

Abstract

The present invention relates to a hypothetical protein GP075 of Pseudomonas aeruginosa phage K8, whose nucleotide sequence is SEQ No.1 and its amino acid sequence is SEQ No.10. This hypothetical protein GP075 is mutated, and the mutated protein has the function of a structural protein. On the basis of the original recognition of lipopolysaccharide, this phage mutant has added an additional receptor recognition protein, which can recognize lipopolysaccharide O-antigen-deficient host cells Or host cells that only contain 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 various diseases caused by Pseudomonas aeruginosa. Infect.

Description

A kind of pseudomonas aeruginosa phage K8 hypothetical protein GP075 and its mutant strain, mutant protein and application

technical field

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

Background technique

Pseudomonas aeruginosa is an opportunistic pathogen that easily infects immunocompromised, frail, and cystic fibrosis patients, resulting in high morbidity and mortality in cystic fibrosis patients. As the natural nemesis of bacteria, bacteriophage can specifically kill bacteria. The study of the interaction between bacteriophage and the host is helpful for the development of bacteriophage 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. Antigens, most of which are related to LPS synthesis, mainly include wbpL, wbpR, wbpO, algC, wbpV, galU, wbpT, wzy, wapH, migA, ssg and wbpS. At the same time, most phages use their tails to recognize cellular receptors, and under the right conditions, the interaction of the tails and receptors induces structural rearrangements of the tails that eventually travel to the head-to-tail junction, triggering its opening, and ultimately the phage DNA is released to complete the phage infection process. On the basis of the existing research, the phage receptor-binding protein of Pseudomonas aeruginosa is a tail filament protein, and there are few studies on the hypothetical protein of unknown function as a receptor-binding protein.

In recent years, with the abuse of antibiotics, the number of antibiotic-resistant bacteria has been increasing, and the problems of various infections and diseases caused by resistant bacteria need to be solved urgently. For example, Pseudomonas aeruginosa is an important opportunistic pathogen causing nosocomial infection. It is well known that bacteriophages can infect host bacteria, and have strong host specificity and do not affect other non-host normal cells. Therefore, the study of phage Therapy, as an alternative to antibiotics, is particularly important. At present, the most studied cocktail therapy, that is, mixed phage therapy (referring to a treatment method that uses a variety of bacteriophages to kill a variety of bacteria), as early as 2014, the European Union launched the Phagoburn program to target the common clinical symptoms of aeruginosa The "cocktail therapy" has played a good role in various infections caused by Monospora, Escherichia, Proteus, Klebsiella, and Staphylococcus, and the safety of the cocktail therapy was evaluated. On the other hand, when using bacteriophage preparations to treat bacterial infections, it is also possible to consider the combination of bacteriophage preparations and antibiotics to achieve a therapeutic effect.

In the in vivo and in vitro bactericidal experiments of bacteriophage, the bactericidal efficiency of the same bacteriophage is different for different host bacteria, which is related to the influence of the gene expression level of the host bacteria in the process of phage infection of host bacteria. This increases the complexity of phage lysis of host bacteria. Understanding more host genes associated with phage infection will benefit the therapeutic efficiency of phage and provide therapeutic options for multiple clinical infections in the future.

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

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a hypothetical protein GP075 of Pseudomonas aeruginosa phage K8 and its mutants, mutant proteins and applications. The hypothetical protein GP075 is mutated, and the mutant protein has a structural protein On the basis of the original recognition of lipopolysaccharide, this type of phage mutant adds an additional receptor recognition protein, which can recognize lipopolysaccharide O-antigen-deficient host cells or host cells that only contain core oligosaccharides. At the same time, it has a wider host range, stronger ability to lyse and adsorb host cells, and is expected to be used in phage preparations to prevent and treat various infections caused by Pseudomonas aeruginosa.

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

A pseudomonas aeruginosa phage K8 hypothetical protein GP075, 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 capable of infecting lipopolysaccharide O-antigen deficient host cells, the sequence of which is at amino acids 105, 106 of the hypothetical protein GP075 as described above One amino acid sequence that is completely repeated with 106 to 112 is inserted between; the nucleotide sequence of the mutant K8-D7 is SEQ No. 2, and its amino acid sequence is SEQ No. 11.

A mutein GP075-14 containing the above-mentioned hypothetical protein GP075, the sequence of which is to insert 2 amino acid sequences that are completely repeated with 106 to 112 between amino acids 105 and 106 of the GP075, the mutein GP075-14 Its nucleotide sequence is SEQ No.3, and its amino acid sequence is SEQ No.12.

A mutein GP075-21 containing the hypothetical protein GP075 according to claim 1, the sequence of which is that 3 amino acid sequences that are completely repeated with 106 to 102 are inserted between amino acids 105 and 106 of GP075, the mutein GP075-21 Its nucleotide sequence is SEQ No.4, and its amino acid sequence is SEQ No.13.

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

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

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

A Pseudomonas aeruginosa phage K8 mutant strain K8-P197L capable of infecting lipopolysaccharide O-antigen-deficient host cells, the sequence of which is mutated P197L in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant strain K8-P197L is SEQ No.8, and its amino acid sequence is SEQ No.17.

A Pseudomonas aeruginosa phage K8 mutant strain K8-T239A, the mutant strain K8-T239A can infect lipopolysaccharide O-antigen deficient host cells, and its sequence is mutated T239A in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant strain 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 is not mutated.

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

The advantages and positive effects obtained by the present invention are:

1. During the research process of the present invention, it was found that the phage K8 mutant population contains a variety of GP075 mutant phages, and the isolated phage K8 mutants mainly include K8-D7, K8-E126K, K8-S142L, K8-L189R, K8-P197L , K8-T239A, and in the process of studying the mutation diversity of GP075, two mutant proteins, GP075-14 and GP075-21, were found with a higher proportion. The putative protein GP075 in the above phage is mutated, and the mutated protein has the function of a structural protein. On the basis of the original recognition of lipopolysaccharide, this phage mutant has added an additional receptor recognition protein, which can recognize lipopolysaccharide O-antigen deficient Host cells or host cells that only contain core oligosaccharides have a wider host range, stronger ability to lyse and adsorb host cells, and are expected to be used in bacteriophage preparations to prevent and treat diseases caused by 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, and at the same time retains the original recognition of the original receptor O-antigen, has the recognition ability of dual receptors, and greatly improves the recognition of verdigris. The bactericidal ability of Pseudomonas has the ability to inhibit the spontaneous mutation of Pseudomonas aeruginosa, which can be used in the development and application of phage preparations to solve the problem of infection caused by tolerant Pseudomonas aeruginosa.

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

4. The genetic background of the starting phage K8 in the present invention is clear, and on this basis, we strive to find a new mutant phage that recognizes O-antigen deficiency, so that the follow-up research is more scientific. Using O-antigen deficient host cells as indicator bacteria to isolate phage K8 mutants, the research is more purposeful.

5. The present invention provides a class of hypothetical protein GP075 mutants whose function is related to bacteriophage infection, and this class of mutants is an important structural protein for phages to recognize core oligosaccharides of host cells; the present invention provides multiple broad-spectrum Pseudomonas aeruginosa phages, Infection of lipopolysaccharide O-antigen deficient or core oligosaccharide-only host cells.

6. The significance of the present invention is: in recent years, Pseudomonas aeruginosa has become the main pathogenic strain of hospital infection diseases, but in the process of using antibiotics for treatment, Pseudomonas aeruginosa gradually produces resistance to antibiotics, and evolves. multidrug resistance. In order to better treat infections caused by Pseudomonas aeruginosa, scientists are actively developing new antibiotics while constantly looking for alternatives to antibiotics. As the nemesis of bacteria, bacteriophages can identify and kill bacteria and have a good therapeutic effect on infections caused by bacteria. No virulence factors were found in all proteins with known functions in the whole genome of bacteriophage K8 and its mutants, which laid a good foundation for future bacteriophage therapy and provided a safety guarantee during the use of bacteriophage 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. K8 was unable to infect this host cell, and the function of GP075 has not been studied at this stage. A number of K8 spontaneous mutants were isolated, the most common feature of which is that they can infect lipopolysaccharide O-antigen-deficient host cells, and the whole genome of phage K8-T239A was sequenced. Threonine T was mutated to alanine A, and sequencing of GP075 of other phages revealed different mutations in this gene. Taking this as the starting point in the present invention, the new function of GP075 and the application of inhibiting the growth of Pseudomonas aeruginosa are studied.

Description of drawings

Fig. 1 is an analysis diagram of the basic properties of the interaction between phage K8 mutant strain and host cells in the present invention; wherein, A is the host range diagram of phage 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 identification diagram of mutant gene of phage K8 mutant strain in the present invention;

Fig. 3 is the analysis diagram of particle function protein of K8 phage mutant strain detected by LC-MS in the present invention;

Figure 4 is an application diagram of the bacteriostatic and bactericidal ability of phage K8 mutants in the present invention; wherein, A is the growth curve of phage K8 and mutant K8-T239A inhibiting PAK, and B is the growth curve of phage K8 and mutant K8-T239A inhibiting SK75 Figure, C is the screening map of PAK-tolerant phage K8 and mutant K8-T239A, D is the screening map of SK75-tolerant phage K8-T239A, and E is the spontaneous mutation rate map of host strain resistant to phage K8 and mutant K8-T239A.

Detailed ways

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

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

A pseudomonas aeruginosa phage K8 hypothetical protein GP075, a wild-type GP075 protein, 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 capable of infecting lipopolysaccharide O-antigen deficient host cells, the sequence of which is at amino acids 105, 106 of the hypothetical protein GP075 as described above One amino acid sequence that is completely repeated with 106 to 112 is inserted between; the nucleotide sequence of the mutant K8-D7 is SEQ No. 2, and its amino acid sequence is SEQ No. 11.

A mutein GP075-14 containing the above-mentioned hypothetical protein GP075, the sequence of which is to insert 2 amino acid sequences that are completely repeated with 106 to 112 between amino acids 105 and 106 of the GP075, the mutein GP075-14 Its nucleotide sequence is SEQ No.3, and its amino acid sequence is SEQ No.12.

A mutein GP075-21 containing the hypothetical protein GP075 according to claim 1, the sequence of which is that 3 amino acid sequences that are completely repeated with 106 to 102 are inserted between amino acids 105 and 106 of GP075, the mutein GP075-21 Its nucleotide sequence is SEQ No.4, and its amino acid sequence is SEQ No.13.

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

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

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

A Pseudomonas aeruginosa phage K8 mutant strain K8-P197L capable of infecting lipopolysaccharide O-antigen-deficient host cells, the sequence of which is mutated P197L in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant strain K8-P197L is SEQ No.8, and its amino acid sequence is SEQ No.17.

A Pseudomonas aeruginosa phage K8 mutant strain K8-T239A, the mutant strain K8-T239A can infect lipopolysaccharide O-antigen deficient host cells, and its sequence is mutated T239A in the hypothetical protein GP075 as described above, so The nucleotide sequence of the mutant strain 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 is not mutated.

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

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

The specific isolation and identification steps of the Pseudomonas aeruginosa phage K8 mutant strain of the present invention may 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 the phage K8 mutant strain;

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

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

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

(6) The inhibitory effect of phage K8 mutants 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 for details.

Table 1 Strain and phage used in the present invention

2. Isolation of phage K8 mutants

The mutant subgroups of phage K8, with receptor-deficient Pseudomonas aeruginosa SK2 (wbpV), SK15 (wbpO), SK45 (wbpR) as indicator bacteria, 200 μL (OD ₆₀₀ =0.6) and 100 μL of K8 phage (10 ⁹ ) A subpopulation of mutant phages capable of infecting O-antigen deficient strains was isolated using the double-layer plate method. The double-layer plate was incubated at 37°C for 4 hours. When clear plaques appeared, the plaques that appeared at this time were formed by mutant phages. Pick a single plaque and put it in fresh LB medium. After three purifications, mutant phages with uniform size and the same degree of transparency were obtained. At this time, the phages were K8 mutants.

3. Host range analysis of phage mutants

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

4. Whole genome sequencing of phage K8 mutants

Extract the genomic DNA of phage K8-T239A, analyze the purity and concentration of phage DNA with a nucleic acid analyzer, and meet three indicators: the DNA sample concentration 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 is greater than 1.8. Send the samples that meet the specifications to the sequencing company.

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

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 to analyze the sequencing results. First, use the software Chromas version2.4 to observe the reliability of the sequencing results (no sets of peaks, etc.), and use the TranslationsProtein in the software Primer Premier 5.exe to translate the base sequence into an amino acid sequence for reliable sequencing. Align two sequences in Protein BLAST of the online software NCBI ( https://www.ncbi.nlm.nih.gov/ ) were respectively aligned with the amino acid sequence of protein GP075 for analysis of changes in the putative protein GP075 in single mutant phage.

6. Adsorption rate

The overnight cultured host bacteria were inoculated into 5 mL of liquid LB medium according to 3% of the transfer volume, cultured to logarithmic phase (OD ₆₀₀ =0.6), and 650 μL of host bacteria and 650 μL (MOI=0.001) of bacteriophage were drawn, mixed evenly, statically Set the adsorption for 1 min, immediately draw 100 μL, which is recorded as the total number of phage infection centers, then centrifuge at 13,000 rpm for 30 s, discard the supernatant, add 1200 μL of liquid LB to mix well, and draw 100 μL again, this time is recorded as the phage infection after the first wash The number of centers, according to the above washing method, was washed once every minute, a total of 6 times, three parallels each time, and the double-layer plate method was used to determine the number of remaining infected centers of adsorbed phage after each wash. The formula for calculating the phage adsorption rate is as follows:

A—the adsorption rate of phage (%);

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

C—the titer (pfu/mL) of phage in the control group without added bacteria;

7. Extraction of strain LPS (Lipopolysaccharides)

Referring 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) Culture of strains: Pick up single colonies after purification, inoculate in 5mL LB liquid medium under sterile environment, cultivate overnight at 37°C, 220rpm, and transfer 3% of the transfer volume to 100mL LB liquid the next day In the medium, cultivate to logarithmic phase (OD600=0.6), centrifuge at 4°C, 7000 rpm for 10 min, discard the supernatant, collect the cells, resuspend the cells with 5 mL of 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 bacterial solution, 68 ° C, 120 rpm, water bath shaker, act for 30 min, post ice bath for 30 min, 4 ° C, 7000 rpm, centrifuge for 10 min, collect water phase, repeat the above operation twice.

(3) Dialysis: the collected aqueous phase is drawn into a dialysis bag, stirred with distilled water for dialysis, replaced with distilled water every 4 hours, and dialyzed for 20 hours until the water-saturated phenol is completely removed by dialysis.

(4) Concentrate the water phase: Concentrate the water 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 sedimentation: suck out the concentrated solution, add 2 times the volume of anhydrous ethanol and 1/10 volume of sodium acetate solution, and settle at -20°C overnight.

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

8. LC-MS detection of bacteriophage 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.), the phage granule proteins were separated using SDS-PAGE to obtain the separation After the phage particle protein gel, the protein is cut into small fragments by a 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 proteolytic peptide fragment is retrieved in the corresponding database, Find matching protein fragments.

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

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

LC-MS detection and 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; 85-100 min, 25%-35% B; 100-105 min, 35%-95%; 105-120 min, 95% B; C18 column.

(2) Mass spectrometry conditions: voltage 2.2kv, MS scanning range 350-1550m/z, detector orbitrap; MS2 was 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 on the LC-MS mass spectrometer (LC-MS) used: Obitrap Fusion (Thermofisher, San Jose, USA).

Data filtering principles:

(1) The pep_expect value ≤ 0.01 is more reliable

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

(3) Other data are for reference.

Nine, bacteriophage antibacterial experimental application

Bacteriophage inhibition curve experimental method: the host bacteria cultured overnight were inoculated into a 96-well bacterial culture dish according to 3% of the transfer volume, and the diluted phage (MOI=0.001) was added, and the control group did not add phage (MOI=0), Each group is paralleled three times. Incubate at 37°C at 160 rpm, measure OD ₆₀₀ with a multi-function microplate reader every 0.5h until 5.5h, and measure the growth rate of host bacteria in the presence or absence of phage.

1. Pseudomonas aeruginosa Tolerance Phage Spontaneous Mutation Assay

The frequency of spontaneous mutation of wild-type Pseudomonas aeruginosa strains resistant to phage under the action of phage was determined by the back-turbidity method (Zhang Kebin, Chen Zhijin, Jin Xiaolin, et al. Isolation and identification of Pseudomonas aeruginosa phage and determination of phage-resistant mutation frequency [J] ]. Bulletin of Microbiology, 2002, 29(1): 40-45.). The overnight cultured host bacteria PAK and RO2-15 were serially diluted to 10 ^-10 . 100 μL was sampled from each of 10 ^-6 , 10 ^-7 , and 10 ^-8 dilution tubes, and the number of bacteria/mL was measured by colony counting method. At the same time, 10 μL of phage stock solution (10 ¹⁰ pfu/mL) was added to each dilution tube, mixed well, and shaken at 37°C for culture. After 4 to 6 hours, each tube gradually became clear, indicating that the bacteria had been lysed. Continue to culture for 20 hours. , it can be found that the high-concentration bacterial liquid tube begins to gradually return to turbidity until no new turbidity tube appears the next day. Interpretation results 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 mutation. 10 parallel experiments were performed in each group.

2. Results and Discussion

In the present invention, O-antigen deficient host cells are used to obtain multiple phage K8 mutant strains. The present invention takes K8-T239A as a typical representative, and conducts the following research.

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

In the present invention, O-antigen deficient host cells are used to obtain multiple phage K8 mutant strains. The present invention takes K8-T239A as a typical representative, and conducts 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), Spottingassay were all resistant to K8, among which SK75, SK2, SK15, SK45 were sensitive to phage K8-T239A to form a transparent circle of plaque, and SK98, M21, P2-25 were resistant to phage K8-T239A (Fig. 1A). In the adsorption capacity results of PAK and 7 insertion mutants and phage K8, K8-T239A, it was found that the adsorption capacity of PAK, SK75, SK2, SK15, SK45 to K8 and K8-T239A was significantly different, 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 has both LPS OSA and Coreoligosaccharide, and the mutant strains SK75, SK2, SK15, and SK45 with the deletion genes of wzy, wbpV, wbpO, and wbpR, respectively, are presumed to have LPS OSA deletion. , from the SK98, M21, P2-25 indel genes were ssg, galU, wapH inferred, the LPS of these three bacteria was completely deleted (Figure 1C). Tricine-SDS-PAGE analysis of the lipopolysaccharide components of PAK and 7 insertion mutants showed that LPSCore oligosaccharide existed in the strains PAK, SK75, SK2, SK15, and SK45 that were sensitive to phage K8-T239A, and those that were resistant to phage K8-T239A The lipopolysaccharide component of the SK98, M21, and P2-25 strains was completely absent (Fig. 1C). The results of Tricine-SDS-PAGE are completely consistent with the previous genotype prediction. 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 has only one point mutation in the whole genome sequence. Further analysis found that the point mutation appeared at the 715th position of the gene gp075 base level and changed from A→G (adenine mutation to Guanine), T→A at amino acid level 239 (threonine is mutated to alanine), this protein is annotated as a hypothetical protein; at the same time, the amplicon sequencing of the gene gp075 of other phage mutants isolated, there are 25 strains GP075 has SVN (point mutation, Single nucleotide site variation), the mutation occurs in four different positions on the gene gp075, amino acid level analysis, the 126th position of E126K is 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 mutation, it is speculated that the mutation occurs in other hypothetical proteins or structural proteins; there is another insertion of the 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 start and end of the repeat sequence and the adjacent 4 nucleotide sequences are all CGGT (such as nucleic acid sequence 2), precisely because 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 has a 7-amino acid repeat sequence with the 106th to 112th of GP075, namely GAPWY S V (eg. sequence 11), (Fig. 2).

(3) LC-MS detection of phage mutant granule protein

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 the same. Including 8 functional proteins, namely GP035, GP057, GP062, GP063, GP068, GP074, GP076, GP078, the corresponding protein functions are DNA ligase (DNA ligase), major capsid protein (majorcapsidprotein), putative structural protein ( putative structural protein), putative structural protein, putative tape measure protein, putative baseplate relatedprotein, putative tailfiberprotein, putative tail fiberprotein ); four hypothetical proteins were encoded by GP053, GP056, GP071, and GP110, respectively (Fig. 3). This result indicates that the GP075 protein mutant has a new function and can act as a structural protein without an annotated function.

(4) Antibacterial application of bacteriophage

One of the basic properties of bacteriophage is to infect host cells and lyse the host cells. The co-culture of bacteriophage and host cells can inhibit the growth of host cells. In the growth curve of wild-type Pseudomonas aeruginosa PAK, the control group without phage was cultured to 5.5h, and the OD600 reached 0.7. It was co-cultured with phage K8 and K8-T239A respectively. After 2.0h, PAK almost stopped growing, and phage K8 , K8-T239A has a strong inhibitory effect on the growth of PAK (Figure 4A). The same number of K8 and K8-T239A were co-cultured with different dilutions (10-fold gradient dilution) of PAK, and the turbidity of the bacterial solution at each dilution was observed. The minimum turbidity concentration of PAK can calculate the spontaneous mutation rate of PAK-tolerant phages. , PAK and K8 were co-cultured, only one tube appeared 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 turbid concentration of PAK The mean value was 7.0×10 ⁵ cfu/ml, and the mean frequency of spontaneous mutation of K8 in PAK tolerance was 2.91×10 ^-6 ; K8-T239A co-cultured with PAK, the minimum turbidity concentration was 7.0×10 ⁸ cfu/ml, and the PAK tolerance was calculated The mean frequency of spontaneous mutation of K8-T239A was 2.83×10 ^-9 . The frequency of spontaneous mutation of PAK-tolerant K8-T239A was much lower than that of K8, and there was a significant difference (Fig. 4B, 4E).

SK75 growth curve, the growth of SK75 without phage was used as a control, cultured to 5.5h, the OD600 reached 0.7, after adding phage K8, the growth of SK75 was not inhibited, and the OD600 reached 0.69; adding phage K8-TT239A and SK75 together After culturing for 3.0 h, the growth of SK75 was inhibited, and then almost stopped, so it can be seen that K8-T239A has an inhibitory effect on the growth of SK75 (Fig. 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 1.04×10 cfu/ml, the co-culture solution was completely clear, indicating that the minimum turbidity concentration of SK75 was mainly concentrated in 1.04 ×10 ³ cfu/ml, the mean frequency of spontaneous mutation of SK75 resistant K8-T239A was calculated to be 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 much lower than that of K8. It inhibits the spontaneous mutation of SK75, which is 10 ⁶ higher than the mutation frequency of PAK resistant to 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 have been disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, therefore , the scope of the present invention is not limited to the contents disclosed in the embodiments and drawings.

sequence listing

Sequence 1 Pseudomonas aeruginosa phage K8 gene gp075 nucleic acid sequence

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 2 Pseudomonas aeruginosa phage K8-D7 gene gp075 nucleic acid sequence

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 3 protein GP075-14 nucleic acid sequence

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 4 protein GP075-21 nucleic acid sequence

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGACCAACCTAGTGACATAA

Sequence 5 Pseudomonas aeruginosa phage K8-E126K gene gp075 nucleic acid sequence

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 Pseudomonas aeruginosa phage K8-T239A gene gp075 nucleic acid sequence

ATGGCTGTCAACCAATTTGACAGAGAAGATTATCTGGAGGTGGCCCGGGAACGGGTCACTGAACAGTTTAAAGAGAAGCCGATCTTTGATCGCTTCCTGCAAGTGCTATTGTCTGGTAAGTTTGATATCCAGAATGCACTGGAAGACCTCCAGACTCTCCGGTCTCTGGACACAGCCACCGGGAAGCAACTGGACATTATCGGAGACATTGTAGGGCGACCACGCGGTCTAGTGTACCAAGATATTTTCAACTATTTTGGATTCGCTGGAACGGAGCGTGCAGGTTCTTTCGGAAGCCTGTCGGACCCTACGGTCGGTGCTCCATGGTACTCGGTCGGTGCTCCAACTGGTAACGCCAGAGAGCCGAGCGACGAAGAGTATCGGATGATCCTGAAAGCAAAGATCATCAAGAACAGAACAAACTCAACCCCAGAGCAAGTTATCGAAGCTTATAAATTTGTATTCGGGGTTCCTGAAGTATTCCTAGAGGAGTACGCTCCCGCTGCTGTCCGTATCGGCATCGGTAAGATTCTAACGAACGTAGAGCGTAGTCTTCTATTCGACCTAGGTGGTGCAGGTGCATTGCTTCCTAAGACTATCGGGGTTAACTACACATACACTGAGTTCCAAGCTGGCCGGGTATTTGCTACAGAAGGCTTCCCCGGAGGACAAGGCGTTGGAGACCTAAATGATCCCACTGTTGGTGGAATTCTGGCCAACCTAGTGACATAA

Sequence 10 GP075 amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVTYLDPDLGVFATEGGQGVTEFQAGRVFATETGLV

Sequence 11 GP075-7 amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLGFGGILGLVGTDTNPKTIGVNYTYLPGGGILGLVFATEGFPQ

Sequence 12 GP075-14 amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPWYSVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPWYSVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLDPEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTQVFTGLNDPTEFQAGRVAGALLPKTIGVNYGDGGATELV

Sequence 13 GP075-21 amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPWYSVGAPWYSVGAPWYSVGAPTGNAREPSDEEYRMILKAKIITYKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGTIGKILTNVERLFDLVAGILFGTGLVGDGVNY

Sequence 14 GP075-E126K amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEKYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVTYDPTGLNGGTLVGVNYDPTV

Sequence 15 GP075-S142L amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNLTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVTYTYFQAGRVFATEGFPGGQGVTYTEFQAGRVFATEGFPGGQNY

Sequence 16 GP075-L189R amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDRGGAGALLPKTIGVTYPDLNDPTVGFPGVTEFQAGRVFATETGLVGVNY

Sequence 17 GP075-P197L amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLLKTIGVLNDPTVILGGTLVGGQNY

Sequence 18 GP075-T239A amino acid sequence

MAVNQFDREDYLEVARERVTEQFKEKPIFDRFLQVLLSGKFDIQNALEDLQTLRSLDTATGKQLDIIGDIVGRPRGLVYQDIFNYFGFAGTERAGSFGSLSDPTVGAPWYSVGAPTGNAREPSDEEYRMILKAKIIKNRTNSTPEQVIEAYKFVFGVPEVFLEEYAPAAVRIGIGKILTNVERSLLFDLGGAGALLPKTIGVNYGDPTEFQAGRVFATETGGVNYGDLPTVGLV.

sequence listing

<110> Tianjin University of Science and Technology

<120> A Pseudomonas aeruginosa phage K8 hypothetical protein GP075 and its mutants, mutant proteins and applications

<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 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 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 (1)

1. A pseudomonas aeruginosa bacteriophage K8 mutant K8-T239A is characterized in that: the mutant strain K8-T239A can infect lipopolysaccharide O-antigen defective host cells, the sequence of the mutant strain is that the protein GP075 is supposed to have mutation T239A in pseudomonas aeruginosa bacteriophage K8, the nucleotide sequence of the GP075 mutant protein in the mutant strain K8-T239A is SEQ No.9, and the amino acid sequence of the GP075 mutant protein is SEQ No. 18;

wherein, the nucleotide sequence of the assumed protein GP075 of the pseudomonas aeruginosa bacteriophage K8 is SEQ No.1, and the amino acid sequence thereof is SEQ No. 10.

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