CN108410840A - A kind of Pseudomonas aeruginosa phage endolysin and its coding gene and application - Google Patents

Abstract

The invention discloses a Pseudomonas aeruginosa phage endolysin and its encoding gene and application. The invention provides the following proteins as shown in a) or b): a) a protein composed of the amino acid sequence shown in sequence 1 in the sequence list; b) a protein derived from sequence 1 in the sequence list, in which the amino acid sequence of sequence 1 in the sequence list is replaced and/or deleted and/or added with one or more amino acid residues and has the function of lysing Gram-negative bacteria. The protein provided by the invention can lyse a variety of Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium. The protein acts synergistically with the surfactant EDTA to produce a stronger inhibitory effect on Gram-negative bacteria. It lays a foundation for the development of new antibacterial preparations.

Description

A kind of Pseudomonas aeruginosa phage endolysin and its coding gene and application

technical field

The invention belongs to the field of biotechnology, and relates to a Pseudomonas aeruginosa phage endolysin, its coding gene and its application.

Background technique

In recent years, many bacteria have developed drug resistance, and even multi-drug resistant bacteria have emerged. Multi-drug resistant bacteria pose a serious threat to people's health. Therefore, it is urgent to find a new type of antibacterial agent to deal with the threat of drug-resistant bacteria. As a bacterial virus, bacteriophage has the advantages of high specificity and no side effects on the human body, and has been valued by scientists. Phage is a virus that can infect bacteria, fungi, actinomycetes and spirochetes, and its content in nature is about 10 ³¹ , or it can be said that there are about 10 ⁷ phages/cm ³ in the environment. Phages can be divided into lysogenic and lytic phages. The lysogenic phage coexists with the genome combination of the host bacterium and its own genome; while the lytic phage injects its own genome into the host bacterium, replicates, assembles into progeny phages, and then lyses the host bacterium to release the progeny phages. According to this characteristic of lytic phages, phages can be used as antibacterial agents to treat related bacterial infections. Some countries in Eastern Europe have already used lytic phages to treat bacterial infections in humans. With the emergence of bacterial drug resistance worldwide, more attention has been paid to the antibacterial properties of phages, and related research involves human and veterinary clinics, as well as food safety and environmental purification.

As an independent living organism, phage will produce anti-phage strains when used as an antibacterial agent, and its biological safety has been questioned. Bacteriophage endolysins (bacteriophage endolysins) are a kind of hydrolase expressed by dsDNA phage in the late stage of bacterial infection. Researchers believe that phage endolysin has the following advantages: 1. Strong specificity, it will not interfere with normal flora while killing pathogenic bacteria. 2. It is not easy for bacteria to develop drug resistance. Since endolysin is produced during the co-evolution process of phage and bacteria, it targets the highly conserved peptidoglycan on the bacterial cell wall, so the probability of drug resistance is very small. 3 can kill the colonization of pathogenic bacteria on the mucosal surface. As a protein macromolecule, endolysin may produce corresponding antibodies to neutralize its bactericidal ability in the body, but some experiments have found that the antibodies have no neutralizing effect on it. Phage endolysin is less likely to produce drug resistance than phage. Idelevich et al. found that the chimeric enzyme PRF-119 is resistant to phage-resistant Staphylococcus aureus mutants. At present, there is no relevant report of endolysin resistance. Experiments on the safety and pharmacokinetic properties of endolysin in clinical application are being prepared. For example, endolysin P128 has entered phase II clinical trials to test its bactericidal efficacy against Staphylococcus aureus carried in the human nasal cavity. Moreover, the research on the physicochemical properties of endolysin as a protein is very mature, and it can be modified, so phage endolysin has the potential of new antibacterial agents. In recent years, some animal models established by researchers have also proved the antibacterial activity of endolysin.

Contents of the invention

The object of the present invention is to provide a kind of Pseudomonas aeruginosa bacteriophage endolysin and its coding gene and application. The endolysin can degrade the protoplasts of various Gram-negative (G-) bacteria, and has a synergistic effect with EDTA to enhance the inhibition of G- bacteria.

Pseudomonas aeruginosa phage endolysin provided by the invention, name is Lysin-G78, derived from Pseudomonas aeruginosa phage (Pseudomonas aeruginosa) vB_PaeM_G1, is the protein of following (a) or (b):

(a) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing;

(b) The amino acid sequence of Sequence 1 in the sequence listing is substituted and/or deleted and/or added by one or several amino acid residues, and the protein derived from (a) has the function of lysing G-bacteria.

Wherein, the sequence 1 in the sequence listing consists of 187 amino acids.

The invention patent application of 201710089442.1 "A Pseudomonas aeruginosa phage and its application" records a phage vB_PaeM_QKL1, and the phage involved in the present invention can be selected as the phage involved in the invention.

In order to facilitate the purification of Lysin-G78 protein, the amino-terminal or carboxy-terminal of the protein composed of the amino acid residue sequence of Sequence 1 in the sequence listing can be attached with the tags shown in the table below.

Table: Sequence of Labels

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH c-myc 10 EQKLISEEDL

The protein in (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression.

The protein-encoding gene in (b) above can delete the codons of one or several amino acid residues in the DNA sequence shown in Sequence 2 in the sequence listing, and/or carry out missense mutations of one or several base pairs .

The nucleic acid molecule encoding the Lysin-G78 protein also belongs to the protection scope of the present invention.

The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA, etc.; the nucleic acid molecule can also be RNA, such as mRNA, hnRNA or tRNA, etc.

In one embodiment of the present invention, the nucleic acid molecule is specifically the gene encoding the Lysin-G78 protein (named Lysin-G78), and the Lysin-G78 gene is any one of the following DNA molecules:

1) The DNA molecule shown in sequence 2 in the sequence listing;

2) A DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the sequence defined in 1) and encoding the protein Lysin-G78.

Wherein, sequence 2 consists of 564 nucleotides, and the whole sequence 2 is ORF, which encodes the protein shown in sequence 1 in the sequence listing.

Recombinant vectors, expression cassettes or recombinant bacteria containing the nucleic acid molecules also belong to the protection scope of the present invention.

The recombinant vector can be a recombinant expression vector or a recombinant cloning vector.

In one embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by inserting the gene between the multiple cloning sites of pET-28a(+).

The expression cassette consists of a promoter capable of initiating expression of the gene, the gene, and a transcription termination sequence.

In one embodiment of the present invention, the recombinant bacterium is Escherichia coli containing the recombinant vector; specifically, the Escherichia coli is BL21(DE3).

The application of the protein in inhibiting G-bacteria also belongs to the protection scope of the present invention.

The application of the nucleic acid in inhibiting G-bacteria also belongs to the protection scope of the present invention.

The application of the recombinant vector, expression cassette or recombinant bacterium of the nucleic acid molecule in inhibiting G-bacteria also belongs to the protection scope of the present invention.

The application of the protein in the following (c1) or (c2) or (c1), (c2) combination also belongs to the protection scope of the present invention:

(c1) Preparation of lysed Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium;

(c2) Preparation of products for the treatment of diseases caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium infections.

The application of the nucleic acid molecule in the following (c1) or (c2) or (c1), (c2) combination also belongs to the protection scope of the present invention:

(c1) Preparation of lysed Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium;

(c2) Preparation of products for the treatment of diseases caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium infections.

The application of the recombinant vector, expression cassette or recombinant bacteria of the nucleic acid molecule in the following (c1) or (c2) or (c1), (c2) combination also belongs to the protection scope of the present invention:

(c1) Preparation of lysed Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium;

(c2) Preparation of products for the treatment of diseases caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium infections.

The method for preparing Pseudomonas aeruginosa phage endolysin Lysin-G78 provided by the present invention, concrete steps are as follows:

S1 Design specific primers according to the sequence 2 in the sequence table to amplify the artificial Pseudomonas aeruginosa phage endolysin gene from the Pseudomonas aeruginosa phage genomic DNA;

S2 Construction of recombinant expression vector of artificial Pseudomonas aeruginosa phage endolysin;

S3 transforms the recombinant expression vector into Escherichia coli competent cells, and screens the engineering bacteria expressing the artificial Pseudomonas aeruginosa phage endolysin;

S4 uses isopropyl-β-D-thiogalactopyranoside to induce the expression of Pseudomonas aeruginosa phage endolysin engineering bacteria to obtain expression products;

In S5, the recombinant gene expression product is purified and separated by nickel column affinity chromatography to obtain a recombinant artificial Pseudomonas aeruginosa phage endolysin.

Specifically, the primers described in step S1 are:

Upstream primer: 5'-GC GGATCC ATGATCACCGACAGAGAGTATCAG-3', where the underlined part is the restriction site of BamHI

Downstream primer: 5'-GC CTCGAG TCAGCCACTAGCTTCAGCATA-3', wherein the underlined part is the restriction site of Xho I.

Advantage and positive effect of the present invention are:

1 The present invention isolates a strain of Pseudomonas aeruginosa phage vB_PaeM_G1, and clones therefrom the coding gene of endolysin Lysin-G78. Using engineering strains to produce endolysin Lysin-G78, the protein finally lyses the host cell by hydrolyzing the glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid on the bacterial cell wall peptidoglycan, and the protein is water-soluble Good, high activity, wide antibacterial spectrum and other advantages.

2 The Pseudomonas aeruginosa phage artificial endolysin provided by the present invention can inhibit the growth of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium. The monocystic phage endolysin has a synergistic effect with the surfactant EDTA, which can improve the bactericidal activity against G- bacteria and prepare a new type of bactericide.

Description of drawings:

Figure 1 is the plaque morphology of Pseudomonas aeruginosa phage vB_PaeM_G1;

Fig. 2 is the transmission electron micrograph of Pseudomonas aeruginosa phage vB_PaeM_G1;

Fig. 3 is the PCR identification of expression strain BL21-G78 monoclonal colony;

Figure 4 is the SDS-PAGE identification result of Lysin-G78 endolysin;

Figure 5 is the cleavage activity and cleavage spectrum of endolysin Lysin-G78;

Figure 6 shows the synergistic effect of endolysin Lysin-G78 and EDTA.

Detailed ways

Below by specific embodiment the present invention is described in further detail, and the embodiment that gives is only in order to clarify the present invention, is not in order to limit the scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: Isolation and identification of phage

1. Isolation of phage

Take 1L of sewage before treatment in the hospital, add CaCl ₂ to 1mmol/L, centrifuge at 5000rpm at 4°C for 10min to remove the precipitated particles in the sewage, take the supernatant and filter it with a 0.22μm filter membrane to sterilize; take the filtrate 20mL and 20mL 2× LB medium was mixed, and 400 μL of Pseudomonas aeruginosa in the logarithmic growth phase was inoculated at an inoculum volume of 1%, and enriched and cultured at 37° C. for 12 hours. Take 5ml of the above-mentioned bacterial solution and centrifuge at 5000rpm for 10min at 4°C, and filter the supernatant through a 0.22μm filter membrane to obtain the phage stock solution. The obtained phage stock solution was diluted 10 times (10 ^-1 -10 ^-8 ), and 300 μL of the diluted solution was mixed with Pseudomonas aeruginosa in logarithmic growth phase 1:1, incubated at 37°C for 15 minutes, and mixed with 4mL of 55°C The liquid LB medium was mixed, evenly poured onto a solid LB agar plate, cooled for 15 minutes, and incubated overnight at 37°C. The next day, a single plaque was obtained. Use a pipette tip to pick a larger, independent plaque on the plaque formation plate and put it into 1mL SM solution (gelatin 0.1g, MgSO4 7H2O 2g, NaCl 5.8g, add 50mL 1MTris-HCl (pH 7.5) , add water to 1000mL) in an EP tube, place at room temperature for 1h and then overnight at 4°C, take 0.1mL after appropriate dilution (10 ^-1 -10 ^-8 , a total of 8 gradients) the next day, and culture to logarithmic growth phase Pseudomonas aeruginosa was mixed at a ratio of 1:1 to make a double-layer plate for purification, and phage plaques of uniform size were obtained the next day. After obtaining evenly sized plaques, double-layer plate experiments were performed on the phages again, and the morphology of the plaques was observed after culturing at 37°C for 24 hours and 48 hours, respectively, as shown in Figure 1 . It was found that there was a translucent halo outside the transparent area of the phage plaque, which indicated that there was an extracellular polymer degrading enzyme in the phage tail spine, which could degrade the extracellular polymer of the biofilm. As time increases, the phage diffuses out of the clear zone of the plaque to lyse the surrounding bacteria, so the halo increases in size over time. Use a sterilized gun tip to pick the above-mentioned purified phage plaques and add them to the Pseudomonas aeruginosa cultured to the logarithmic growth phase. Cultivate at 37°C and 140rpm for about 6 hours. The culture medium becomes relatively clear. Centrifuge at 8000rpm for 5min, take the supernatant and filter it with a 0.22μm filter membrane to remove a small amount of bacteria and bacterial fragments to obtain a high-titer phage stock solution.

2. Identification of phage

The specific method is: immerse the 350-mesh copper mesh in the phage purification solution, take out the copper mesh after 10 minutes, and absorb the excess liquid with filter paper; put the copper mesh on the 5% (w/v) uranyl acetate droplet, and dye Take it out and put it on filter paper for testing; after all the samples are processed, use TEM with an accelerating voltage of 80kV to observe the phage morphology. The electron micrograph shows that it belongs to Myoviridae (Myoviridae), the diameter of the head is about 50nm, and the length of the tail is about 65nm. The morphology of the phage is shown in Figure 2.

Embodiment 2: Extraction of phage genome

1. Concentration of phage particles

The overnight culture of Pseudomonas aeruginosa was transferred to 100mL liquid LB medium, the inoculum size was 1%, and the culture was expanded to the logarithmic phase (OD600 about 0.4), and 5mL Pseudomonas aeruginosa phage culture solution was added, 37 The phage lysates were obtained after 4-6 hours of shaking culture at ℃. Add DNase I and RNase A to the lysate to a final concentration of 5 μg/mL, mix well and let stand at 37°C for 1 h. Then add NaCl to a final concentration of 0.1mol/L, mix and dissolve, put in ice bath for 1h, and centrifuge at 12000rpm for 20min. After the supernatant was transferred to another centrifuge tube, PEG8000 was added to a final concentration of 10% (w/v), fully shaken to dissolve, then left at 4°C overnight, centrifuged at 12,000 rpm for 20 min, and the supernatant was discarded. Resuspend the precipitate with 500 μL TM (0.05mol/L Tris-HCl pH7.5, 0.2% MgSO4·7H2O) solution, extract once with an equal volume of chloroform, centrifuge at 12000rpm for 10min to remove PEG8000 in the resuspension, and finally A crude extract of phage particles was obtained.

2. Preparation of phage genomic DNA

S1 Add DNase I and RNase A to the crude extract of phage particles at a final concentration of 1 μg/mL, and let stand at 37°C for 1 hour to degrade residual host bacterial DNA or RNA.

S2 then added 1mol/L EDTA (pH8.0) to a final concentration of 50mmol/L to terminate the DNase I and RNase A activities;

S3 Add proteinase K to a final concentration of 50 μg/mL, add SDS to a final concentration of 0.5%, mix well, digest protein at 56°C for 1 hour;

S4 Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and mix well, centrifuge at 12000 rpm for 10 min, and collect the supernatant;

Step 4 of S5 is repeated 3 times;

S6 Add an equal volume of chloroform, mix well, 12000rpm, 10min, collect the supernatant;

S7 Add 1/10 volume of 3mol/L NaAc and 2 times the volume of pre-cooled 95% ethanol, mix well, 12000rpm, 10min, precipitate DNA; add 70% ethanol (500μL) to the precipitate, and turn the capped centrifuge tube upside down Several times, centrifuge at 12000rpm for 5min to recover DNA.

S8 Remove the supernatant, remove the alcohol droplets on the tube wall, dry the open centrifuge tube at room temperature for 10 min, and then resuspend the DNA with double distilled water.

Example 3: Cloning of endolysin gene Lysin-G78, construction of expression vector

1. Obtaining the target fragment

S1 designs a pair of specific primers according to the sequence encoded by the Lysin-G78 (sequence 2) gene, and the primer sequences are as follows:

Upstream primer: 5'-GC GGATCC ATGATCACCGACAGAGAGTATCAG-3', where the underlined part is the restriction site of BamHI

Downstream primer: 5'-GC CTCGAG TCAGCCACTAGCTTCAGCATA-3', where the underlined part is the restriction site of Xho I

The reflection system is:

PCR reaction conditions: pre-denaturation at 95°C for 5 min, 30 cycles of amplification (denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1.5 min); extension at 72°C for 10 min, storage at 4°C. PCR amplification products were subjected to 1% agarose gel electrophoresis to observe whether there were specific bands.

Using the phage genome DNA as a template, amplify the Lysin-G78 gene with the above primers, electrophoresis on 1% agarose, and identify the size of the amplified fragment.

S2 The PCR product was directly digested with QuickCut restriction endonucleases (Takara) BamH I and Xho I, bathed in water at 37°C for 5 min, electrophoresed on 1% agarose, and the gel recovery kit was used to purify and recover the fragments, and the fragments were compared with the previous The purified pET-28a vector digested with BamHI and XhoI was ligated overnight at 16°C with T4 ligase, and transformed into Escherichia coli DH5α competent cells the next day. The transformation mixture was added to LB medium and incubated at 37°C for 45min. The mixture was then spread on an LB plate containing kanamycin (50 μg/ml), and incubated overnight at 37° C.;

Identification of S3-positive clones: Pick positive clones and inoculate them in LB liquid medium containing kanamycin (50 μg/mL), culture overnight at 37°C, extract plasmids with the kit, and send them to Shanghai Sangon for sequencing. The plasmid with correct sequencing was named pET-28a-G78.

S4 transforms the plasmid pET-28a-G78 with correct sequencing into the competent expression strain Escherichia coli BL21 (DE3). Then spread the mixture on LB plates containing kanamycin (50 μg/ml) and incubate overnight at 37° C.; use the above primers for colony PCR identification. The identification result is shown in Figure 3, the amplified fragment is between 750bp and 500bp, and the size is consistent with the target fragment. The expression strain containing the positive recombinant plasmid was named BL21-G78.

Example 4: Induced expression and purification of endolysin gene Lysin-G78

Inoculate a single colony of expressing bacteria BL21-G78 containing the recombinant plasmid into LB culture medium containing kanamycin (50 μg/mL), shake overnight at 37°C; the next day, transfer to 100 mL LB culture at a ratio of 1:100 In the culture medium, shake culture at 37°C until the OD600 value is about 0.6, add IPTG (isopropylthiogalactopyranoside) to a final concentration of 0.5mmol/L, and induce at 16°C for 16h. Collect the bacteria, sonicate the cells (130W, super 3s and stop for 10s, sonicate for 5min), centrifuge at 10000rpm for 10min at 4°C, collect the supernatant, and filter the supernatant through a 0.22μm filter membrane, analyze the protein in the supernatant by SDS-PAGE Express the situation. The filtered supernatant was purified with a His affinity chromatography nickel column (GE Healthcare, Sweden), specifically according to the kit instructions, and the obtained protein was named Lysin-G78.

The results of SDS-PAGE analysis are shown in Figure 4. After the engineering bacteria BL21-G78 containing the recombinant plasmid was induced by IPTG, there was an induced protein band at the position of about 21kD in the supernatant, which was consistent with the expected 20.8KD, thus indicating that the recombinant Bacteria BL21-G78 was constructed correctly, and the expressed endolysin Lysin-G78 was a soluble protein

Example 5: Endolysin Lysin-G78 lyses outer membrane permeabilized bacterial cells

S1 permeabilized bacterial cells

Permeabilized cells refer to cells that change the permeability of cell walls and cell membranes without causing cell lysis or destroying the internal organic structure of cells, so that small molecules and some larger molecules can freely enter and exit cells.

Permeabilized bacterial cells of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia, Salmonella typhimurium, Staphylococcus aureus and Staphylococcus epidermidis were prepared as follows.

The strains cultivated overnight were transferred to 250mL LB medium, cultivated to OD600=0.6, cultured at 4°C, 4000×g, centrifuged for 15min, and the bacterial cells were collected. Use chloroform-saturated 0.05mol/L Tris buffer to resuspend the bacteria, and shake gently at room temperature for 45min. Centrifuge at 4000×g for 15 minutes at 4°C, discard the supernatant and collect the precipitate. Then use 10mmol/L phosphate buffer to wash and resuspend, and finally resuspend the permeabilized bacterial cells to OD600=0.6-1.0.

Lysis of S2 Permeabilized Bacterial Cells

The seven permeabilized bacterial cells prepared in S1 were lysed as follows.

30 μl endolysin (final concentration 2 μg/mL) with a final concentration of 2 μg/mL was mixed with 170 μl permeabilized bacterial cells (OD=0.8) and added to a 96-well plate, at room temperature, using a full-wavelength microplate reader Measure the absorbance OD600 of each sample, and use 10mmol/L phosphate buffer as a control. The absorbance OD600 value was significantly lower than the negative control value, indicating that the bacteria were lysed.

Results: The endolysin Lysin-G78 can efficiently degrade the permeabilized bacteria of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium at a concentration of 2 μg/ml cell. As shown in Figure 5, judging from the OD600 value when endolysin Lysin-G78 interacted with protoplasts for 2 hours, the OD600 of G-bacteria treated with endolysin Lysin-G78 was significantly reduced, while endolysin Lysin-G78 was effective against G+ bacteria. Such as Staphylococcus aureus permeabilized bacterial cells and Staphylococcus epidermidis permeabilized bacterial cells without lysis.

The synergistic effect of embodiment 6 endolysin Lysin-G78 and surfactant EDTA

Transfer the overnight activated bacterial solution to 100ml LB medium according to the inoculum size of 1%, and cultivate it to OD600=0.6; add 100ul bacterial solution to 10ml 50°C semi-solid LB (8% agar), and then add In the bacterial culture dish, let it stand for 15 minutes in the super workbench, punch a hole (about 7mm in diameter) with a 200ul sterile pipette tip, and set aside.

0.9% normal saline was used as negative control group, EDTA, endolysin Lysin-G78, endolysin Lysin-G78 and EDTA were used together as experimental group. Add 10ul of the sample to be tested to the well of the petri dish, incubate overnight at 37°C, and observe the diameter of the inhibition zone.

The results are shown in Figure 6. In the experiment, when the final concentration of EDTA was 2.5uM and the final concentration of endolysin Lysin-G78 was 2 μg/ml, there was a zone of inhibition, and the combination of the two had a stronger antibacterial effect (zone of inhibition diameter increases). It shows that the surfactant EDTA has a synergistic effect with the endolysin Lysin-G78, which can further improve the antibacterial effect of Lysin-G78, and provide effective reference data for the application of the endolysin Lysin-G78.

SEQUENCE LISTING

<110> Dalian University of Technology

<120> A Pseudomonas aeruginosa bacteriophage endolysin and its coding gene and application

<130> ZR181046LQ

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 187

<212> PRT

<213> Pseudomonas aeruginosa

<400> 1

Met Ile Thr Asp Arg Glu Tyr Gln Gln Ala Ala Glu Met Leu Gly Val

1 5 10 15

Asp Val Pro Ala Ile Lys Ala Val Thr Lys Val Glu Ala Pro Val Gly

20 25 30

Gly Phe Gln Pro Thr Gly Glu Pro Thr Ile Leu Tyr Glu Arg His Gln

35 40 45

Met Tyr Arg Gln Leu Gln Ala Lys Gly Leu Pro Thr Glu Gly His Pro

50 55 60

Pro Asp Leu Val Asn Lys Val Ala Gly Gly Tyr Gly Lys Tyr Ser Glu

65 70 75 80

Gln His Ala Lys Leu Ala Arg Ala Val Lys Ile Asp Arg Asp Ser Ala

85 90 95

Leu Glu Ser Cys Ser Trp Gly Met Phe Gln Ile Met Gly Tyr His Trp

100 105 110

Lys Leu Met Gly Tyr Pro Thr Leu Gln Ala Phe Val Asn Ala Met Tyr

115 120 125

Ala Ser Glu Gly Ala Gln Met Asp Ala Phe Cys Arg Phe Ile Lys Ala

130 135 140

Gln Pro Thr Thr His Ala Ala Leu Lys Ala His Asp Trp Ala Lys Phe

145 150 155 160

Ala Arg Leu Tyr Asn Gly Pro Gly Tyr Ala Lys Asn Lys Tyr Asp Val

165 170 175

Lys Leu Glu Lys Ala Tyr Ala Glu Ala Ser Gly

180 185

<210> 2

<211> 564

<212>DNA

<213> Pseudomonas aeruginosa

<400> 2

atgatcaccg acagagagta tcagcaagct gctgagatgt tgggggtaga tgtcccagcg 60

atcaaggcag tgaccaaggt ggaggccccg gtagggggct tccagcctac aggagagcca 120

acgatcctct acgagcgtca ccagatgtac cgacagctcc aggccaaagg gctcccaacg 180

gaaggtcatc ccccagacct ggtaaataag gtagctggtg ggtatggaaa atacagcgag 240

caacacgcta aactggcccg cgccgtaaag atcgacaggg acagcgccct ggagtcctgc 300

tcctggggga tgttccagat catgggctac cactggaagc tgatggggta ccctaccctt 360

caagctttcg taaacgccat gtacgccagc gaaggagccc agatggacgc cttctgccgg 420

ttcatcaagg cacaacccac cacgcatgct gccttgaaag cccatgattg ggccaagttt 480

gccagactgt acaacggtcc aggctacgcc aagaacaagt atgacgtgaa attggagaaa 540

gcatatgctg aagctagtgg ctga 564

Claims (14)

1. a Pseudomonas aeruginosa phage endolysin, is characterized in that: be the protein of following (a) or (b): (a) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing; (b) The amino acid sequence of Sequence 1 in the sequence listing is subjected to substitution and/or deletion and/or addition of one or several amino acid residues, and the protein derived from (a) has the function of lysing Gram-negative bacteria. 2. A nucleic acid molecule encoding the protein of claim 1. 3. The nucleic acid molecule according to claim 2, characterized in that: the nucleic acid molecule is a gene encoding the protein of claim 1, and the gene is a DNA molecule as shown in any of the following: 1) The DNA molecule shown in sequence 2 in the sequence listing; 2) A DNA molecule that has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the sequence defined in 1) and encodes the protein described in claim 1. 4. According to claim 2, characterized in that said nucleic acid molecule is DNA or RNA. 5. A recombinant vector, expression cassette or recombinant bacterium containing the nucleic acid molecule of claim 2 or 3 or 4. 6. According to claim 5, characterized in that the recombinant vector can be a recombinant expression vector or a recombinant cloning vector. 7. the preparation method containing the Pseudomonas aeruginosa phage endolysin described in claim 1, is characterized in that, comprises the steps: S1 Design specific primers according to the sequence 2 in the sequence table to amplify the artificial Pseudomonas aeruginosa phage endolysin gene from the Pseudomonas aeruginosa phage genomic DNA; S2 Construction of recombinant expression vector of artificial Pseudomonas aeruginosa phage endolysin; S3 Transform the recombinant expression vector into Escherichia coli competent cells, and screen to obtain engineering bacteria expressing artificial Pseudomonas aeruginosa phage endolysin; S4 uses isopropyl-β-D-thiogalactopyranoside to induce the expression of Pseudomonas aeruginosa phage endolysin engineering bacteria to obtain expression products; In S5, the recombinant gene expression product is purified and separated by nickel column affinity chromatography to obtain a recombinant artificial Pseudomonas aeruginosa phage endolysin. 8. according to the preparation method of the described Pseudomonas aeruginosa phage endolysin of claim 7, it is characterized in that, the primer described in step S1 is: Upstream primer: 5'-GCGGATCCATGATCACCGACAGAGAGTATCAG-3' Downstream primer: 5'-GCCTCGAGTCAGCCACTAGCTTCAGCATA-3'. 9. The application of the protein according to claim 1 in inhibiting Gram-negative bacteria. 10. The use of the nucleic acid molecule according to claim 2 or 3 in inhibiting Gram-negative bacteria. 11. The application of the recombinant vector, expression cassette or recombinant bacterium of the nucleic acid molecule described in claim 5 in inhibiting Gram-negative bacteria. 12. The application of the protein according to claim 1 in the following (c1) or (c2) or (c1), (c2) combination: (c1) Preparation of products for lysing Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium; (c2) Preparation of products for treating diseases caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium infection. 13. The application of the nucleic acid molecule according to claim 3 or 4 in the following (c1) or (c2) or (c1), (c2) combination: (c1) Preparation of products for lysing Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium; (c2) Preparation of products for treating diseases caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium infection. 14. The application of the recombinant vector, expression cassette or recombinant bacterium of the nucleic acid molecule described in claim 5 in the following (c1) or (c2) or (c1), (c2) combination: (c1) Preparation of products for lysing Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium; (c2) Preparation of products for treating diseases caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium infection.