CN118240809A - Preparation and application of Acinetobacter baumannii phage lyase LysZHSHW - Google Patents

Abstract

The present invention belongs to the technical field of biological preparations, and discloses the preparation and application of Acinetobacter baumannii phage lytic enzyme LysZHSHW. The present invention provides the application of Acinetobacter baumannii phage lytic enzyme LysZHSHW, a vector containing a LysZHSHW expression element, an expression cassette containing a LysZHSHW expression element, or a host cell containing a LysZHSHW expression element in the preparation of an antibacterial agent, and the amino acid sequence of the Acinetobacter baumannii phage lytic enzyme LysZHSHW is shown in SEQ ID NO.1. The present invention also provides a method for preparing the Acinetobacter baumannii phage lytic enzyme LysZHSHW. The lytic enzyme LysZHSHW can lyse Acinetobacter baumannii, and also has an inhibitory effect on other bacteria including Escherichia coli and Pseudomonas aeruginosa, and further evaluates its application and safety in vivo, providing a new antibacterial agent for combating Acinetobacter baumannii infection.

Description

Preparation and application of Acinetobacter baumannii phage lytic enzyme LysZHSHW

Technical Field

The invention belongs to the technical field of biological preparations, and specifically relates to the preparation and application of Acinetobacter baumannii phage lytic enzyme LysZHSHW.

Background technique

As the most typical and common clinical pathogen in the genus Acinetobacter, Acinetobacter baumannii (Ab) is a common Gram-negative bacterium in nature. Initially, Acinetobacter baumannii was considered a low-level pathogen, but it is now considered an opportunistic pathogen for humans and animals because Acinetobacter baumannii has a high frequency of self-mutation and a strong ability to acquire and integrate exogenous DNA, making it resistant to a variety of antibiotics and external environmental factors, promoting its popularity in hospital environments. In addition, in recent years, there have been reports of Acinetobacter baumannii causing infections in the pet industry and animal husbandry. Abroad, Acinetobacter baumannii carrying the blaNDM-1 carbapenem resistance gene was isolated from the urine of pet dogs with urinary tract infections; in China, there have been reports of harm caused by Acinetobacter baumannii in poultry, cattle and pig farming. In 2019, a brown bear died of infection with Acinetobacter baumannii in a zoo in Shanghai. Although extensively drug-resistant Acinetobacter baumannii has not yet been detected in animals, the prevalence of carbapenem-resistant or multidrug-resistant Acinetobacter baumannii in veterinary clinics and aquaculture has made the treatment effect of antibiotics very unsatisfactory, and there is also a risk of important drug-resistant genes being transmitted to humans. In the face of the potential harm that Acinetobacter baumannii brings to hospitals, aquaculture environments, and veterinary clinics, especially the emergence of multidrug-resistant Acinetobacter baumannii, new prevention and control strategies are urgently needed.

Bacteriophage (phage) is a general term for viruses that can specifically kill bacteria and other microorganisms. According to the characteristics of the life cycle, phages can be divided into lytic, mild and pseudo-lysogenic phages. Lytic phages are often used in phage therapy because of their rapid bactericidal ability. Phage therapy refers to the use of phages to control or remove bacterial infections. Compared with antibiotics, phage therapy has the advantages of not destroying the balance of the microecology, no adverse reactions, being able to penetrate blood vessels to reach tissues, even through the blood-brain barrier, and having little impact on the environment. However, bacteria can produce a series of resistance mechanisms against each stage of the phage life cycle, and the emergence of phage-resistant bacteria reduces the effectiveness of phage therapy or even makes it ineffective. In order to overcome the shortcomings of phage therapy, many new strategies have been proposed, such as phage cocktails, phage-antibiotic combined applications, and phage product applications. Among them, endolysin, which can destroy the peptidoglycan in the bacterial cell wall, has attracted widespread attention from the scientific community due to its specificity, mode of action, transformable potential and lack of resistance mechanism, and many successful applications have been developed. As for the Acinetobacter baumannii phage lytic enzyme LysZHSHW, there are relatively few studies on it, and there is still a lack of relevant data on whether the lytic enzyme has an inhibitory effect on Acinetobacter baumannii or other bacteria.

Summary of the invention

In view of the deficiencies in the prior art, the present application provides a preparation and application of Acinetobacter baumannii phage lytic enzyme LysZHSHW.

The primary purpose of the present invention is to provide the use of Acinetobacter baumannii phage lytic enzyme LysZHSHW, a vector containing a LysZHSHW expression element, an expression cassette containing a LysZHSHW expression element, or a host cell containing an expression LysZHSHW element in the preparation of an antibacterial agent, wherein the amino acid sequence of the LysZHSHW is shown in SEQ ID NO.1.

Preferably, the nucleotide sequence of the gene encoding the Acinetobacter baumannii phage lytic enzyme LysZHSHW is shown as SEQ ID NO.2.

Preferably, the antibacterial agent has an antibacterial spectrum of Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa.

Preferably, the present invention also provides a method for preparing the above-mentioned Acinetobacter baumannii phage lytic enzyme LysZHSHW, which specifically comprises the following steps: the present invention uses the expression plasmid pET28a as the skeleton, uses PCR and enzyme cutting and ligation methods to construct a pET28a-Lys recombinant plasmid containing the lytic enzyme LysZHSHW encoding gene, transforms it into competent cells, cultures those verified as positive, induces expression, collects the bacterial solution, extracts, and purifies.

Preferably, the primers used in the PCR are Lys-F and Lys-R, the nucleotide sequence of the Lys-F is as follows: CGCGGATCCATGATTCTGACTAAAGACG, and the nucleotide sequence of the Lys-R is as follows: CCGGAATTCCTATAAGCTCCGTAGAGCA.

Preferably, the enzyme digestion uses double digestion with BamH I and EcoR I.

Preferably, the competent cells are E. coli BL21 (DE3).

After verifying the expression of the lytic enzyme LysZHSHW by the Western blot method, the present invention characterizes the lytic enzyme and evaluates its in vivo/in vitro antibacterial application potential.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The present invention provides the bacteriostatic effect of Acinetobacter baumannii phage lytic enzyme LysZHSHW, which can inhibit the growth of Acinetobacter baumannii, has the effect of killing Acinetobacter baumannii, and has a certain lytic effect on Escherichia coli and Pseudomonas aeruginosa, and is non-toxic to human cells. Through the greater wax moth and mouse thigh muscle models, its application and safety in vivo are further evaluated, providing a new antibacterial agent for combating Acinetobacter baumannii infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the bioinformatics analysis result of Acinetobacter baumannii phage lytic enzyme LysZHSHW, wherein FIG. 1A is the identification of the amphipathic helical structure of ALysZHSHW, and FIG. 1B is the tertiary structure analysis of LysZHSHW.

FIG2 is the PCR verification result of the recombinant plasmid, wherein 1 and 3 are plasmid pET28a fragments; 2 and 4 are LysZHSHW encoding gene fragments; and M is DL1000 DNA marker.

Figure 3 is the SDS-PAGE and Western blot verification results of prokaryotic expressed LysZHSHW, wherein 1, 2, and 3 are the bacterial lysate, precipitate, and supernatant of E. coli BL21 (DE3) carrying pET28a-Lys, respectively; 4, 5, and 6 are the bacterial lysate, precipitate, and supernatant of E. coli BL21 (DE3) carrying pET28a, respectively; and M is a protein molecular weight standard.

FIG4 shows the stability results of LysZHSHW, wherein FIG4 shows the temperature stability of LysZHSHW; and FIG4 shows the pH stability of LysZHSHW.

FIG5 is a transmission electron microscopy observation result of the cleavage effect of LysZHSHW.

FIG6 shows the in vitro bactericidal effect of LysZHSHW.

FIG7 is the result of the fragmentation spectrum determination of LysZHSHW, wherein ab and b represent significant differences, and ab and ab represent no significant differences.

FIG8 is the cytotoxicity assay result of LysZHSHW, wherein ab and b represent significant differences, and ab and ab represent no significant differences.

FIG. 9 is the result of measuring the survival rate of G. mellonella.

FIG. 10 is the observation result of pathological section of mouse thigh muscle.

Detailed ways

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Unless otherwise specified, the test methods used in the examples of the present invention are all conventional methods; the strain materials, reagents, etc. used, unless otherwise specified, are all reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

1 Materials and Methods

1.1 Test materials

The bacteria and plasmids used in the experiment are shown in Table 1. Acinetobacter_phage_vB_AbaP_ZHSHW phage isolation method Reference "Zhu Yinuo. Isolation and identification of two strains of Acinetobacter baumannii phages and analysis of biological characteristics and genomics [D]. Beijing University of Chemical Technology, 2022. DOI: 10.26939/d.cnki.gbhgu.2022.002092", the gene sequence information has been uploaded to GenBank, NCBI accession number: 2930334. LysZHSHW was amplified by the primers in Table 2, and the primers were synthesized by Guangzhou Qingke Technology Co., Ltd.

Table 1

Table 2 Primer information

Note: The bold and underlined sequences are BamHI and EcoRI restriction sites, respectively.

High-fidelity enzyme Taq Mix was purchased from Beijing PolyMei Biotechnology Co., Ltd.; DNA gel recovery kit and Plasmid DNA Kit were purchased from OmegaBio-Tek Biotechnology Co., Ltd., USA; BamH I, EcoR I and 10×FastDigest buffer were purchased from Beijing New EnglandBiolabs Co., Ltd.; Soulution I was purchased from Takara Beijing Bao Ri Yi Biotechnology Co., Ltd.; PBS pre-mixed powder was purchased from Beijing Jinsha Biotechnology Co., Ltd.; IPTG, EDTA and cyclophosphamide were purchased from BioFroxx, Germany; PAGE gel rapid preparation kit, instant protein loading buffer, multi-color pre-stained protein molecular weight standard, Coomassie brilliant blue staining solution, WB primary antibody diluent, and universal antibody diluent were purchased from Shanghai Yamei Biomedical Technology Co., Ltd.; methanol (analytical grade) was purchased from Guangzhou Chemical Reagent Factory; PVDF membrane and dialysis bag (8000d) were purchased from Mil lipore; anti-His tag monoclonal antibody (mouse) and goat anti-mouse IgG-FITC labeled protein were purchased from Wuhan Abbkine Biotechnology Co., Ltd.; ECL luminescence kit was purchased from Hangzhou Fude Biotechnology Co., Ltd.; His tag protein pre-loaded nickel column was purchased from Jiaxing Qianchun Biotechnology Co., Ltd.; BCA protein quantitative analysis kit was purchased from Thermo Fisher Scientific; HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) was purchased from Beijing Meiyimei Biotechnology Co., Ltd.; Britton-Robinson Buffer (BR buffer) was purchased from Guangzhou Yitao Biotechnology Co., Ltd.; DMEM (containing various amino acids and glucose culture medium) was purchased from Giboc, USA; MTT solution was purchased from Shanghai Lianmai Bioengineering Co., Ltd. The experimental animal mice were ICR mice, purchased from Guangdong Medical Experimental Animal Center, and the animal experiment ethics number was 2023c003. The difference analysis was performed using SPSS software (version 16.0).

1.2 Bioinformatics analysis of LysZHSHW

According to the sequence encoding LysZHSHW in the complete genome sequence of Acinetobacter_phage_vB_AbaP_ZHSHW published by our laboratory on NCBI, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2, and the amino acid sequence of LysZHSHW is shown in SEQ ID NO.1. The domain of the lyase was predicted (InterPro (https://www.ebi.ac.uk/interpro/)); theoretical molecular weight, theoretical isoelectric point, number of positively and negatively charged amino acids and other physicochemical properties (ExPASy (https://web.expasy.org/protparam/)); transmembrane domain (HMMTOP (http://www.enzim.hu/hmmtop/html/submit.html)); secondary structure (Phyre (http://www.sbg.bio.ic.ac.uk/phyre2/html/)) and tertiary structure (SWISS-MODEL (https://swissmodel.expasy.org/)).

2. Preparation of LysZHSHW

2.1 Construction of recombinant plasmid

Using the DNA of bacteriophage (Acinetobacter_phage_vB_AbaP_ZHSHW) as a template, primers Lys-F and Lys-R were used to amplify the target gene LysZHSHW fragment, and BamH I and EcoR I restriction sites were added at both ends. The PCR reaction program was: 95℃ pre-denaturation for 3min; 94℃ denaturation for 25sec, 60.2℃ annealing for 25sec, 72℃ extension for 15sec, a total of 35 cycles; 72℃ extension for 5min. The PCR product was subjected to 1.5% agarose gel electrophoresis, and the LysZHSHW fragment was recovered using a gel recovery kit. The pET28a vector fragment double-digested with BamH I and EcoR I was linearized using a gel recovery kit. Solution I was used to connect the LysZHSHW fragment and the pET28a vector fragment overnight at 16°C. The ligation product was transformed into E. coli DH5α competent bacteria and cultured overnight at 37°C. The primers pET28a-F and pET28a-R were used to verify the positive clones. The positive clones verified by PCR were sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing.

2.2 Expression, purification and verification of LysZHSHW

The correct recombinant plasmid pET28a-Lys was extracted and verified using a plasmid extraction kit, and the recombinant plasmid was transferred to E.coli BL21 (DE3) competent bacteria and cultured at 37°C overnight; the positive clones were verified using primers pET28a-F and pET28a-R, and the positive clones verified by PCR were sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The E.coli BL21 (DE3) carrying pET28a-Lys was cultured to D600 nm = 0.6-1.0, and IPTG with a final mass concentration of 1 μmol/L was added, and the culture was induced at 16°C for 16 hours. The induced bacterial solution was centrifuged and washed three times with PBS; the precipitate was resuspended with Buffer A (20 μmol/L potassium phosphate buffer, 40 μmol/L imidazole, 500 μmol/L sodium chloride; pH = 7.4); and the supercharged crusher was used to crush until the liquid was transparent. The samples to be tested are: bacterial lysate, precipitate and supernatant of E.coli BL21 (DE3) carrying pET28a-Lys. The samples to be tested are mixed with protein loading buffer and boiled, added to PAGE gel, and the gel is Coomassie stained and destained after electrophoresis, and photographed and recorded in the fully automatic chemiluminescence image analysis system. The PAGE gel is cut, transferred and blocked with a PVDF membrane. A mouse anti-His tag monoclonal antibody diluted 1:1000 is used as the primary antibody; a goat anti-mouse IgG-FITC labeled protein diluted 1:2000 is used as the secondary antibody. After the development is completed, it is photographed and recorded in the fully automatic chemiluminescence image analysis system. The bacterial lysate of E.coli BL21 (DE3) carrying pET28a-Lys verified correctly by Western Blot is purified using a nickel column pre-loaded with His tag protein. The protein sample solution was passed through the BIO-RAD chromatography system, and Buffer B (20 μmol/L potassium phosphate buffer, 500 μmol/L imidazole, 500 μmol/L sodium chloride; pH = 7.4) was used to competitively elute LysZHSHW carrying the 6×His tag from the nickel column, and the protein sample solution after chromatography was collected. The protein purification effect was verified by SDS-PAGE and Western Blot; the verified correct LysZHSHW was dialyzed overnight using an 8000d dialysis bag; the mass concentration of LysZHSHW after dialysis was determined using a BCA protein quantitative analysis kit.

Result analysis

LysZHSHW has a molecular weight of 24.6 kDa, a theoretical isoelectric point of 9.46, 19 negatively charged residues (Asp+Glu), and a total of 27 positively charged residues (Arg+Lys); the C-terminus of the enzyme is 79-128 aa, which is the catalytic domain of the glycoside hydrolase family 19 (Glycoside hydrolase, family 19, catalytic, IPR000726), and the chitinase produced is involved in the hydrolysis of β-1,4-linked polysaccharides. According to topological prediction, the C-terminus of LysZHSHW contains an amphipathic helical structure located at 121-141 aa, indicating that the enzyme has potential transmembrane ability (Figure 1A). The secondary structure prediction results show that LysZHSHW contains multiple α-helices (54%), multiple random coils (8%), and a β-turn (2%). Among them, the α-helix accounts for the largest proportion, indicating that most of the polypeptide chains of the protein are in an α-helical state. Through tertiary structure prediction and homology modeling using PDB:4ok7.1.A as a template, it was found that the transmembrane region of LysZHSHW is distributed on the outside, with a simple structure and only one transmembrane helix; its spatial conformation is spherical (Figure 1B).

The LysZHSHW encoding gene with BamH I and EcoR I restriction sites was cloned into the pET28a plasmid and verified by PCR using primers pET28a-F and pET28a-R. Detection by 1.5% agarose gel electrophoresis showed that a specific band was amplified at 913 bp, which was consistent with the theoretical size (see Figure 2).

E.coli BL21(DE3) carrying pET28a-Lys was induced by IPTG for expression, and after the bacteria were broken, the protein expression was preliminarily determined by SDS-PAGE. The electrophoresis results showed that a specific band was detected at 24.6kDa in the bacterial lysate and supernatant samples, which was consistent with the theoretical size. Further detection by Western Blot showed that the band was single, which was consistent with the theoretical size (Figure 3). No target protein expression was detected in the samples prepared by E.coli BL21(DE3) carrying pET28a, indicating that the pET28a-Lys recombinant plasmid was well expressed in vitro. LysZHSHW was then purified and the mass concentration was determined. The standard curve established was y=1163.6x-162.75 (R ² =0.997), and the mass concentration of LysZHSHW was calculated to be 4086mg/L.

Antibacterial activity and application of 3-lyase LysZHSHW

3.1 Stability of LysZHSHW

The enzyme activity was determined by referring to the high-throughput analytical method for determining the activity of cell wall hydrolase (Briers Y, Lavigne R, Volckaert G, et al. A standardized approach for accurate quantification of murein hydrolase activity in high-throughput assays [J]. J Biochem Biophys Methods. 2007, 70 (3): 531-533.), wherein the reaction substrate was selected to be the outer membrane body. The general steps are as follows: the logarithmic phase host bacteria Ab_8_4 was centrifuged to obtain a precipitate, a 50 μmol/L Tris-HCl solution (pH = 7.7) saturated with chloroform was added, and the precipitate was placed at 37°C, incubated at 120 rpm for 45 min, and centrifuged. The precipitate was washed three times with PBS, and the precipitate was resuspended with 50 μmol/L KH ₂ PO ₄ /K ₂ HPO ₄ buffer (pH = 7.2), and the D _600nm was adjusted to 1.0-1.6. The prepared outer membrane body was placed at 4°C for use. 270 μL of the outer membrane-free body was added to a 96-well plate, and 30 μL of different mass concentrations of LysZHSHW (400, 200, 150, 112.5, 84.3, 63.28, 47.4, 35.6, 26.7, 13.35, 6.68, 3.33 mg/L) were added respectively. D _600nm was measured every 3 min, and shaken for 10 s before measurement. Each group had three parallels. According to the enzyme activity calculator (http://www.biw.kuleuven.be/logt/ActivityCalculator.htm), the average change of D _600nm per minute under different mass concentrations of LysZHSHW was calculated, and the enzyme activity was calculated according to the formula.

(1) Temperature stability 270 μL of the above-mentioned outer membrane-removed cells were added to a 96-well plate, and 30 μL of LysZHSHW (final mass concentration of 40 mg/L) was added. The plates were incubated at 4, 25, 37, 45, 55, and 65°C, and D _600nm was measured every 3 min. PBS buffer was added to the control group. The relative enzyme activity at each temperature was calculated.

(2) pH stability LysZHSHW was incubated in BR buffer at pH = 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 for 1 h. 270 μL of the above-mentioned outer membrane-removed bodies was added to a 96-well plate, and 30 μL of LysZHSHW incubated in BR buffer at different pH values (final mass concentration of 40 mg/L) was added respectively. D _600nm was measured every 3 min, and PBS buffer was added to the control group. The relative enzyme activity at each temperature was calculated.

Result analysis

The enzyme activity calculator was used to obtain the average value of the change in D _600nm per minute at different mass concentrations of the enzyme. The standard curve established was y=0.0021x (R ² =0.899), and the enzyme activity of LysZHSHW was calculated to be 630 U/μg.

The enzyme activity of LysZHSHW under different conditions was determined by stability tests. In terms of temperature stability, we found that LysZHSHW was most active at 25°C; at 37°C and 45°C, the activity of the enzyme weakened; when the temperature was 4°C, 55°C and 65°C, LysZHSHW was basically inactivated (Figure 4A). In terms of pH stability, it was found that LysZHSHW was most active at pH 8.0 and 9.0; when the pH was 6.0 and 7.0, the activity of the enzyme weakened; when the pH was less than 5.0, or greater than 9.0, LysZHSHW was basically inactivated (Figure 4B). The above results show that LysZHSHW is most active at room temperature and weakly alkaline conditions.

3.2 LysZHSHW cleavage

Ab_8_4 was cultured to the logarithmic phase and centrifuged. The precipitate was washed three times with PBS, and PBS, 3mgLysZHSHW, 0.5μmol/L EDTA and 3mg LysZHSHW+0.5μmol/L EDTA were added to the precipitate, respectively, and the volume was made up to 1mL with HEPES, and centrifuged after incubation for 30min. 2.5% glutaraldehyde electron microscopy fixative was added, fixed at -20℃ for 2h, and stored at 4℃. The sample was sent to the Analysis and Testing Center of South China Agricultural University for sample preparation and transmission electron microscopy analysis.

Result analysis

The lysis effect of LysZHSHW was directly observed by projection electron microscopy. It was found that in the blank control group and EDTA group, the cell wall structure of Ab_8_4 was intact and the edges were clear; after adding LysZHSHW, the cell wall structure of Ab_8_4 was intact, and bacteria with incomplete cell wall structure were occasionally found (3/42); after adding LysZHSHW+EDTA, the cell wall structure of Ab_8_4 collapsed and shattered, and the contents flowed out (Figure 5). The results show that LysZHSHW has a certain transmembrane function due to its amphipathic helical structure, but it is not obvious. When LysZHSHW is used in combination with EDTA, the rupture of the bacterial cell wall can be directly observed.

3.3 In vitro application of LysZHSHW

(1) Killing curve Ab_8_4 was cultured to the logarithmic phase and centrifuged. The precipitate was washed three times with PBS, resuspended with HEPES solution, and plated for counting. LysZHSHW was diluted to 100, 250, 500, 750, and 1000 mg/L with HEPES solution, and 0.5 μmol/L EDTA was added. The diluted LysZHSHW and PBS were mixed with the above bacterial solution in a ratio of 1:1, incubated at 37°C, and samples were taken for plate counting at 0.5, 1.0, 1.5, 2.0, and 2.5 h, respectively, with three replicates per group.

(2) Lysis spectrum Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus and Enterococcus faecalis were prepared by removing the outer membrane. 180 μL of the outer membrane-removed body was added to a 96-well plate, and 20 μL of LysZHSHW (final mass concentration 200 mg/L) or PBS was added, respectively, and D _600nm was measured after incubation for 1 hour.

(3) Cytotoxicity test The MTT method ^[18] was used to detect the effects of 1500, 500, and 150 mg/L LysZHSHW and PBS on the survival rate of A549 human lung adenocarcinoma cells. D _490nm was measured after oscillation decolorization. The calculation formula is:

Result analysis

The bactericidal ability of LysZHSHW was determined by in vitro bactericidal tests. The results showed that the bactericidal effect was most significant when the mass concentration of LysZHSHW was 1000 or 750 mg/L. Among them, after 0.5 h, the bacterial concentration dropped rapidly by about 2.4 to 2.8 orders of magnitude; after 2.5 h, the bacterial concentration dropped by 4.0 orders of magnitude. When the concentration of LysZHSHW was 500 or 250 mg/L, the bacterial concentration dropped by 2.5 or 1.5 orders of magnitude after 2.5 h, respectively, and had a certain bactericidal effect (Figure 6).

LysZHSHW (final concentration of 200 mg/L) was used to react with the outer membrane bodies of different bacteria to observe the cleavage spectrum of LysZHSHW. The results showed that within 1 hour, LysZHSHW reduced the D _600nm of Ab_8_4 by 0.8 (P<0.01); reduced the D 600nm of E. coli LGC1 and 16FS-27-68 by about 0.2 (P<0.01); reduced the D 600nm of P. aeruginosa Z2P136 by about 0.1 (P<0.05); and had no significant effect on other bacteria (Figure 7). This shows that LysZHSHW has a high specificity and has a certain cleavage effect on Escherichia coli and Pseudomonas aeruginosa, but has no significant effect on Gram-positive bacteria.

The toxicity of LysZHSHW was determined on A549 human lung adenocarcinoma cells. The results showed that under the action of 1500, 500 and 150 mg/L LysZHSHW, the relative cell survival rate of A549 human lung adenocarcinoma cells was 100.9% to 108.8%; under the action of PBS, the relative cell survival rate of A549 cells was 96.2% to 97.9% (Figure 8). This indicates that LysZHSHW has no cytotoxicity to A549 human lung adenocarcinoma cells and can be further used for in vivo experiments.

3.4 Therapeutic effect of LysZHSHW in the wax moth model

The weight of the wax moth larvae used in this experiment was 300-400 mg. Unhealthy larvae with weak vitality and black spots were discarded, and healthy larvae with symmetrical body and white color were retained. Ten healthy larvae were randomly selected and placed in the same culture dish as a group. Ab_8_4 was cultured to the logarithmic phase and centrifuged. The precipitate was washed three times with PBS and resuspended with PBS. According to the results of the preliminary experiment, LD90 (2×10 ⁷ Cfu/mL) was used as the challenge dose. LysZHSHW was diluted to 500, 350, and 150 mg/L with PBS, and 0.5μmol/L EDTA was added. PBS was used as a blank control. 5μL of bacterial solution or PBS was injected into the left foot of the wax moth with a microsyringe. After 1 hour, 5μL of different mass concentrations of LysZHSHW or phage ZHSHW (2×10 ⁷ Pfu/mL) or PBS was injected into the right foot of the wax moth with a microsyringe. The wax moth was placed in a dark room at 30°C for cultivation, and the status and survival rate of the wax moth were recorded every 12 hours, and the health index was scored (reference: Loh JM, Adenwalla N, Wiles S, et al. Galleria mellonella larvae as an infection model for group A streptococcus [J]. Virulence. 2013, 4 (5): 419-428). The treatment plan is shown in Table 3.

table 3

Result analysis

The therapeutic effect of LysZHSHW in vivo was evaluated by using the G. mellonella model. Preliminary experimental results showed that when the concentration of Ab_8_4 was 10 ⁵ CFU/larvae, the mortality rate of G. mellonella larvae was 90% (LD90), so this concentration was selected for subsequent experiments. The results of the treatment experiment showed that after injection of phage ZHSHW, the survival rates at 24 and 48 hpi were 66.67% and 46.67%, and the average health index scores were 4.00 and 3.20, respectively; after injection of 2.5 μg LysZHSHW+EDTA, the survival rates at 24 and 48 hpi were 92.86% and 85.71%, and the average health index scores were 5.47 and 5.07, respectively; after injection of 1.75 μg LysZHSHW+EDTA, the survival rates at 24 and 48 hpi were 64.29% and 28.57%, and the average health index scores were 3.97 and 2.20, respectively; after injection of Ab_8_4+0.75 μg LysZHSHW+EDTA, the survival rates at 24 and 48 hpi were 46.67% and 20.00%, and the average health index scores were 2.13 and 0.80, respectively (Figure 9) (Table 4). The above results show that the therapeutic effect of LysZHSHW on G. mellonella is concentration-dependent, and LysZHSHW can effectively improve the survival rate of G. mellonella when the mass concentration is 500 mg/L.

Table 4 Health index of greater wax moth (n=15)

The data in the table are mean ± standard error

3.5 Therapeutic effect of LysZHSHW in mouse thigh model

The ICR mice used in this experiment were all 15-20 g in weight, 35-40 days old, and free of specific pathogens. Three female mice and three male mice were randomly selected as a group. After completing the quarantine period, on the 4th day and the 1st day before the mouse infection, 150 g/L and 100 g/L cyclophosphamide solution (CTX) or PBS were injected intraperitoneally per kg of the mouse body weight. Ab_8_4 was cultured to the logarithmic phase and centrifuged. The precipitate was washed three times with PBS, and the bacteria were concentrated to 10 ¹⁰ CFU/mL with PBS. 1.5 g/L LysZHSHW, 10 ⁹ Pfu/mL phage ZHSHW and 0.9% NaCl solution were prepared respectively, and 100 μL of bacterial solution or 0.9% NaCl was injected into the thigh of the mouse. 2h later, 100 μL of different mass concentrations of LysZHSHW, phage ZHSHW and 0.9% NaCl solution were injected at the same site. After 1h or 12h of treatment, the mice were euthanized, the thigh muscles were removed aseptically, homogenized, diluted with PBS to an appropriate multiple, plated and counted, and the data were recorded. The above experiment was repeated, and the muscle tissues of mice injected with LysZHSHW, phage ZHSHW and 0.9% NaCl solution were stained with HE for pathological sections and observed under an optical microscope.

Result analysis

The application of LysZHSHW in vivo was further evaluated by the mouse thigh model. The results showed that at 1 h after the challenge, the bacterial load in the thighs of immunodeficient mice injected with cyclophosphamide (CTX) increased significantly by 2 orders of magnitude compared with normal mice (P<0.05), indicating that CTX helps Ab_8_4 colonize in mouse muscle tissue. At 1 h and 24 h after injection of LysZHSHW or phage, the bacterial load in the thighs of mice decreased significantly by 2.3 to 3.3 orders of magnitude compared with immunodeficient mice (P<0.05); at 24 h, the bacterial load in the thighs of mice injected with phages was less than that of mice injected with LysZHSHW (P<0.05) (Table 5). In addition, pathological sections of the thigh muscles of mice were observed, and the results showed that the muscle tissues of the normal mice group and the blank control group showed no obvious abnormalities at both 1h and 24h. Inflammatory cell infiltration was found between the muscle fibers of mice at 1h and 24h after infection with Ab_8_4, mainly neutrophils, plasma cells and lymphocytes. After 1h or 24h of treatment with phage or LysZHSHW, no obvious abnormalities were found in the muscle tissues of mice (Figure 10). The above results show that phage ZHSHW or LysZHSHW can effectively kill bacteria in the infection site in a short time and reduce the inflammatory response between muscle fibers.

Table 5 Bacterial load in mouse thigh muscle (n=6) log10 (Cfu/g)

The data in the table are mean ± standard error

The above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims (7)

1. Use of Acinetobacter baumannii phage lytic enzyme LysZHSHW, a vector containing a LysZHSHW expression element, an expression cassette containing a LysZHSHW expression element, or a host cell containing a LysZHSHW expression element in the preparation of an antibacterial agent, characterized in that the amino acid sequence of the Acinetobacter baumannii phage lytic enzyme LysZHSHW is shown in SEQ ID NO.1. 2. The use according to claim 1, characterized in that the nucleotide sequence of the gene encoding the Acinetobacter baumannii phage lytic enzyme LysZHSHW is shown in SEQ ID NO.2. 3. The use according to claim 1, characterized in that the antibacterial agent has an antibacterial spectrum of Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa. 4. The application according to claim 1, characterized in that the preparation method of the Acinetobacter baumannii phage lytic enzyme LysZHSHW mainly comprises the following steps: using the expression plasmid pET28a as the skeleton, using PCR and enzyme ligation method to construct the pET28a-Lys recombinant plasmid containing the lytic enzyme LysZHSHW encoding gene, transforming it into competent cells, culturing those verified to be positive, inducing expression, collecting bacterial liquid, extracting and purifying. 5. The use according to claim 4, characterized in that the primers used in the PCR are Lys-F and Lys-R, the nucleotide sequence of the Lys-F is CGCGGATCCATGATTCTGACTAAAGACG, and the nucleotide sequence of the Lys-R is CCGGAATTCCTATAAGCTCCGTAGAGCA. 6. The use according to claim 4, characterized in that the enzyme digestion adopts double digestion of BamH I and EcoR I. 7. The use according to claim 4, characterized in that the competent cells are E. coli BL21 (DE3).