CN114344451A

CN114344451A - Application of Reg4 antibacterial peptide in treatment of pseudomonas aeruginosa infectious pneumonia

Info

Publication number: CN114344451A
Application number: CN202210085094.1A
Authority: CN
Inventors: 万晓玉; 肖永陶; 王伟鹏
Original assignee: Shanghai Institute Of Pediatric Research; Shanghai Pulmonary Hospital
Current assignee: Shanghai Institute Of Pediatric Research; Shanghai Pulmonary Hospital
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-04-15
Anticipated expiration: 2042-01-25
Also published as: CN114344451B

Abstract

The invention provides application of Reg4 antibacterial peptide in treatment of pseudomonas aeruginosa infectious pneumonia.

Description

Application of Reg4 antibacterial peptide in treatment of pseudomonas aeruginosa infectious pneumonia

Technical Field

The invention relates to the field of new medicine application, in particular to application of Reg4 antibacterial peptide in treatment of pseudomonas aeruginosa infectious pneumonia.

Background

Pseudomonas aeruginosa (PAO 1), also known as Pseudomonas aeruginosa, belongs to the genus Pseudomonas, is a unidirectional-movement gram-negative opportunistic pathogen that is distributed in various environments and organisms, such as soil, water, plants, and animals.

Clinically, PAO1 is one of the major pathogens of nosocomial infections and respiratory infections and can cause acute and chronic infections of the patient's respiratory, urinary and burn sites, leading to severe morbidity and mortality. Particularly in patients with underlying diseases such as cancer, cystic fibrosis, ventilator-associated pneumonia, or other immune deficiencies, can lead to a significant incidence of infection and even mortality.

Pseudomonas aeruginosa is one of the leading causes of morbidity and mortality in cystic fibrosis patients. Studies have shown that PAO1 is the second leading cause of hospital-acquired pneumonia (HAP), accounting for 14% -16%.

The current primary treatment regimen for PAO1 infection is antibiotic and supportive care. In recent years, however, resistance to multiple antibiotics has been shown due to excessive abuse of antibiotics, which severely limits the therapeutic options available for treating patients with PAO1 infection. Once patients are infected with pan-resistant PA01 strain, the therapeutic effect is very limited even if different antibiotics are changed. Carbapenem antibiotics, including imipenem and meropenem, are the last resort to the treatment of gram-negative bacterial infections. However, the situation is not optimistic, and according to the monitoring statistics of the Chinese bacterial drug resistance monitoring network: in 2019, 27.5% of the pseudomonas aeruginosa strains clinically isolated from the third-class hospital in china developed resistance to imipenem antibiotics. The development period of the novel antibiotics is long, and the novel antibiotics cannot effectively solve the drug resistance problem of PAO 1. Therefore, there is an urgent need to develop new effective non-antibiotic treatment strategies for PAO1 infection to control and treat refractory infections caused by multi-drug resistant bacteria.

Disclosure of Invention

The invention aims to overcome the defects and provides an ideal substitute of antibiotics, namely, Reg4 antibacterial peptide.

The invention provides an application of an active ingredient, which is characterized in that:

the active ingredients are Reg4 protein;

the Reg4 protein is shown in SEQ ID NO. 1:

the application comprises the preparation of the medicine for treating or relieving the symptoms of PAO1 infection.

Further, the invention provides an application of the active ingredient, which is characterized in that:

the above uses also include at least one of the following uses:

use a. for the preparation of a medicament for the treatment and alleviation of pulmonary inflammatory response induced by PAO 1;

use B for the preparation of a medicament for the treatment and alleviation of lung injury caused by PAO 1.

the application also comprises the medicine for preparing the medicine for treating and relieving the weight loss caused by the PAO1 infection.

the application also comprises the preparation of the medicine capable of combining with the PAO 1.

the application comprises the preparation of the medicine for inhibiting the growth of PAO1 bacteria.

the use also includes the use for preparing a medicament for reducing PAO1 adhesion and invading alveolar epithelial cells.

the application also comprises the preparation of a medicament for reducing the pulmonary colonization of the PAO 1.

the application also comprises the application of the medicine in preparing the medicine capable of inhibiting migration of PAO 1.

the application also comprises the preparation of the medicine for relieving the spleen invasion caused by the PAO 1.

In addition, the invention provides a PAO1 chronic infection model experiment model, which is characterized by being modeled by the following steps:

s1, preparing agar bead suspension containing PAO1 bacteria;

s2, injecting the agar beads into a specific part of the target model.

The invention has the following functions and effects:

the regenerating family (Reg) protein 4(Reg4) is a secretory protein expressed by organisms, and the research of the invention shows that Reg4 has antibacterial activity to PAO 1.

In the research of the invention, the Reg4 protein and the PAO1 are incubated together, and the Reg4 protein is found to be capable of obviously inhibiting the growth of the PAO 1.

In the research of the invention, through successfully establishing a mouse PAO1 chronic infection model, the Reg4 is found to reduce the colonization of PA01 in the lung and improve the pathological damage and inflammatory reaction of the lung of a mouse infected with PAO 1.

Therefore, the Reg4 protein is considered to have strong antibacterial activity, small molecular weight, protease degradation resistance, wide antibacterial spectrum, an antibacterial mechanism different from that of the traditional antibiotics, and good antibacterial activity on super-drug-resistant strains generated by the antibiotics, and meanwhile, the protein superior to the Reg4 protein belongs to one of human antibacterial peptides, does not cause organism rejection reaction, and has no risks such as drug residue.

Therefore, the Reg4 protein can be used as a novel antibacterial peptide to replace the traditional antibiotics, improve the chronic lung infection of PAO1, become the best medicine for treating pseudomonas aeruginosa infection in the future, and provide a new treatment strategy for clinical treatment of PAO1 infection.

Drawings

FIG. 1 Reg4 protein inhibits growth of PAO1

In this, figure 1A shows the growth kinetics of PAO1 bacteria (×, PA01+ PBS vs. PAO1+10 μ g/ml, p < 0.0001).

FIG. 1B shows the co-incubation of Reg4 protein with PAO1 bacteria at 4h OD₆₀₀And (6) measuring the result.

FIG. 1C shows the results of plate coating of bacteria incubated with Reg4 protein and PAO1 bacteria at 24 h.

FIG. 1D shows the results of measurement of the number of bacteria incubated for 24h with Reg4 protein and PAO1 bacteria.

FIG. 2.PAO1 flowchart of mice infected with PAO1 and weight changes of mice

Wherein, FIG. 2A is a flowchart of PA01 lung chronic infection mouse.

FIG. 2B shows the change in Reg expression following PA01 infection.

FIG. 2C shows the weight change of PA01 lung-infected mice.

FIG. 3 pathological changes in the lungs of mice infected with PAO1

Wherein, FIG. 3A is a mouse lung HE staining optical lens (magnification:. times.200 in each group).

FIG. 3B is a Masson staining light mirror (magnification:. times.200 for each group) for mouse lung.

FIG. 4 detection of the expression level of related inflammatory factors in mouse lung and alveolar lavage fluid

In FIG. 4A, the content of Reg4 in mouse alveolar lavage fluid was measured by ELISA.

FIG. 4A shows the detection of IL-22 content in mouse alveolar lavage fluid by ELISA.

FIG. 4C is the expression level of IL-1. beta. in mouse colonic mucosal tissue.

FIG. 4D is the expression level of IL-10 in mouse colonic mucosal tissue.

FIG. 4E is the expression level of CCL-28 in mouse colonic mucosal tissue.

FIG. 4F is the level of CXCL-2 expression in mouse colonic mucosal tissue.

FIG. 5 bacterial content of lung, spleen and alveolar lavage fluid in mice

Among them, FIG. 5A is the effect of recombinant murine Reg4 protein on the levels of PAO1 bacteria in the lungs, spleen and alveoli.

FIG. 5B is a comparison of the PAO1 bacteria content in the lungs;

FIG. 5C is a comparison of the bacterial content of PAO1 in the spleen;

FIG. 5D is a comparison of the content of PAO1 bacteria in alveolar lavage fluid;

FIG. 6 Effect of Reg4 protein on PAO1 adhesion and invasion of MLE 12 cells

Among them, FIG. 6A is the effect of recombinant murine Reg4 protein on PAO1 adhesion to MLE 12 cells.

FIG. 6B is a comparison of the content of PAO1 bacteria adhering to MLE 12 cells.

FIG. 6C is a graph of the effect of recombinant murine Reg4 protein on PAO1 invasion of MLE 12 cells.

FIG. 6D is a comparison of the content of PAO1 bacteria invading MLE 12 cells.

FIG. 7 Effect of Reg4 protein on PAO1 adhesion and invasion of MH-S cells

Among them, FIG. 7A is the effect of recombinant murine Reg4 protein on adhesion of PAO1 to MH-S cells.

FIG. 7B is a comparison of the bacterial content of PAO1 adhering to MH-S cells.

FIG. 7C is a graph of the effect of recombinant murine Reg4 protein on PAO1 invasion of MH-S cells.

FIG. 8 binding of Reg4 to PAO1

In FIG. 8A, the binding of PAO1 to Reg4 protein was detected by ELISA.

FIG. 8B shows the migration of PAO1 in a semi-solid agarose plate.

FIG. 8C shows the measurement result of halo diameter formed by migration of PAO1 in a semi-solid agarose plate.

Detailed Description

Example 1 Reg4 inhibition of PAO1 growth in vitro

1.1 Main Material

Pseudomonas aeruginosa PAO1 (ATCC-15692): bio-82076, available from Biotech, Inc. of Baiohbowei, Beijing.

1.2 methods

1.2.1 culture of Pseudomonas aeruginosa

0.5ml of TSA liquid culture medium was aspirated with a sterile pipette to dissolve all lyophilized powder in a lyophilization tube. The dissolved bacterial suspension was transferred to a test tube containing 5ml of liquid medium and mixed well, and 100. mu.l of the suspension was transferred to a solid medium. And (4) performing static culture on the liquid test tube and the inclined plane test tube. Adding 100 μ l PA01 bacterial liquid into 1000ml TSA culture medium containing 50 μ g/ml kanamycin, culturing overnight at 37 ℃, after the culture reaches the logarithmic growth phase, at 4000rpm for 20min, centrifugally collecting thalli, resuspending the thalli by PBS buffer solution, determining the total bacterial colony number of the bacterial liquid by using a plate counting method, and preserving the thalli at-80 ℃ for later use.

1.2.2 Reg4 protein concentration adjustment

The drug concentration was adjusted to 1mg/ml with sterile PBS buffer. All protein solutions were filter sterilized using a 0.22 μm filter before use and stored in sterile 1.5ml EP tubes.

1.2.3 Co-incubation of Reg4 protein with PAO1

Mu.l of resuscitated PA01 bacterial suspension was inoculated into a total of 200. mu.l containing different concentrations (0, 2 or 10. mu.g/ml) of Reg4 protein. Per 2 hour OD passage₆₀₀The measurement monitors the growth of the bacteria.

PAO1 was diluted to a final concentration of 2X 10 with sterile PBS buffer⁵CFU/ml. Take 4 pieces of 15ml centrifuge tube, arrange them into a row, add 1980. mu.l TSA medium and 20. mu.l diluted PAO1 bacterial solution to tube 1 (as a control group), add 1978. mu.l TSA medium, 20. mu.l bacterial solution and 2. mu.l recombinant murine Reg4 protein to tube 2 (as a 1. mu.g/ml group), add 1960. mu.l TSA medium, 20. mu.l bacterial solution and 20. mu.l recombinant murine Reg4 protein to tube 3 (as a 10. mu.g/ml group), and mix them well. The lid was closed and incubated in a 37 ℃ incubator for 16 hours.

1.2.4 Total bacterial colony count assay

Continuously diluting the incubated bacterial liquid with sterilized water to a final concentration of 1 × 10⁷CFU/ml. 100 mul of the incubated bacterial liquid was evenly smeared on kanamycin agar plates. The plate was placed flat on a clean bench for 30 minutes to allow the bacterial solution to penetrate into the surface layer of the medium. And (3) inversely placing the plate in a 37 ℃ incubator for culturing for 24h, calculating the colony number of the plate after 24h, and multiplying the colony number by the dilution multiple to obtain the colony number of each group.

1.3 results

1.3.1 Total bacterial colony count assay

Bacterial plate counts were performed according to GB 4789.2-2016. The total number of PAO1 colonies was 8.3X 10⁹ CFU/ml。

1.2.2 recombinant Reg4 protein inhibits the growth of PAO1 bacteria

Respectively incubating PAO1 with recombinant murine Reg4 protein with different concentrations for OD₆₀₀Measurement (FIGS. 1A-B). The results of the bacterial growth kinetics curve show that the Reg4 protein concentration reaches 10 mu g/ml, and the growth of PAO1 can be effectively inhibited.Total bacterial colonies were determined by incubating PAO1 with 0. mu.g/ml, 1. mu.g/ml, and 10. mu.g/ml recombinant murine Reg4 protein, respectively, for 16h (FIGS. 1A-B). The results showed that the total number of bacteria in the PAO1+ 0. mu.g/ml Reg4 group was 8.37X 10⁸CFU/ml, total bacterial count was reduced to 6.73X 10 after administration of 1. mu.g/ml recombinant murine Reg4 protein⁸CFU/ml, the total number of bacteria is reduced by about 5 times when 10 mu g/ml of recombinant mouse is given with Reg4 protein, and the total number is 1.63 multiplied by 10⁹CFU/ml (p ═ 0.0023) (fig. 1C-D). The result shows that the Reg4 recombinant protein can effectively inhibit the growth of PAO 1.

Example 2 in vivo bacteriostasis test

2.1 Main Material

(1) C57BL/6 mice: shanghai Jihui laboratory animal feeding Co., Ltd

(2) Gavage needle No. 8: GWZ-8-45, Shanghai Jing Sangbiotech GmbH

2.2 methods

2.2.1 Reg4 protein concentration adjustment

The drug concentration was adjusted to 1mg/ml with sterile PBS buffer. All proteins were filter sterilized using a 0.22 μm filter before use and stored in sterile 1.5ml EP tubes.

2.2.2 formulation of PAO1 agar bead suspension

(1) 100ul of cryopreserved cell suspension was aspirated, applied to a TSA plate, and cultured overnight (37 ℃ C., 200rpm, about 16 hours)

(2) A single colony was inoculated into 5ml of TSB medium (15ml centrifuge tube) and cultured overnight (37 ℃ C., 200rpm, ca. 16h)

(3) The bacteria were diluted in PBS (1:50) in overnight cultures and the OD was determined₆₀₀The value is obtained.

(4) Dilution of bacterial overnight cultures: 2OD bacteria were added to a tube containing 20ml of fresh TSB and incubated at 37 ℃ and 200rpm for about 3-4 hours until 10-15OD (bacteria reached logarithmic phase).

(5) TSA plates (TSB + 1.5% agar) were formulated, autoclaved, and equilibrated in a 50 ℃ water bath. In a water bath at 50 ℃ 150ml of pre-self-adhesive heavy mineral oil was added.

(6) When P.aeruginosa reached log phase, bacterial cells were collected at 2700 Xg at 4 ℃ by centrifugation for 15min and the supernatant discarded.

(7) The bacterial pellet was resuspended in 1ml sterile PBS and the bacterial pellet resuspended.

(8) 1ml of the bacterial suspension was mixed with 9ml of TSA liquid preheated at 50 ℃. 10ml of the TSA-Pseudomonas aeruginosa mixture was added to the heavy mineral oil (preheated at 50 ℃) and stirred immediately at room temperature for 6 minutes. The agitation must create a visible vortex in the oil.

(9) The mixture was cooled to 4 ℃ and stirred at minimum speed for 35 minutes.

(10) The agar-bead-oil mixture was placed in ice for an additional 20 minutes.

(11) The agar beads were transferred to 50ml Falcon tubes and centrifuged at 2, 700 Xg for 15min at 4 ℃. Six washes with sterile PBS removed mineral oil. After three washes, the beads can be pelleted by gravity without a centrifuge. After the last wash, the agar beads were resuspended in 20-30ml PBS.

(12) An aliquot of the beads (about 0.5ml) was taken and homogenized aseptically.

(13) 100ul of homogenized beads were taken and diluted in 900ul of sterile PBS. Continuous 1:10 dilution to 10^-6。

(14) Serial dilution of plates on TSA plates, including dilution to 10^-6Double undiluted sample and incubated at 37 ℃.

(15) The bead diameters were measured in several regions using an inverted optical microscope. The bead diameter must be between 100 and 200 meters.

(16) The beads were stored at 4 ℃ overnight.

2.2.3 mice Chronic infection test

Wild Type (WT) C57BL/6 mice (female: male: 1) at 8 weeks of age were used as infection subjects and randomly divided into 6 groups, which were grouped as follows: including Con group (n 10), PAO1+ PBS group and PAO1+ Reg4 group (n 10), the experimental scheme is shown in fig. 2A.

The bacterial count in the agar bead suspension was determined by plate coating and diluted to 4X 10 in PBS⁷CFU/ml agar bead suspension. Anaesthetizing mice with 1% sodium pentobarbital solution, taking supine position, disinfecting the skin, vertically incising the skin to expose the trachea, and using sterile and flexibleThe 22G iv catheter was cannulated and attention was given to removing the stylet as it was moved down into the trachea. The catheter is inserted into the trachea (not too deep) and stopped before reaching the carina (bifurcation). Immediately 50ul of the agar bead suspension was taken with a 1ml syringe and connected to a catheter. The plunger of the syringe is gently pushed to allow the beads to implant into the lung. Mice were stood for 1 minute after dosing to ensure that the bacterial fluid entered both lungs. The incision is sutured. The animal was placed on a heating pad until fully awake. Mice in the PAO1+ Reg4 group were given 100. mu.l of Reg4 protein (10. mu.g/ml) intraperitoneally daily, starting on day 1 after infection, and the Con group and the PAO1+ PBS group were given a metered amount of sterile PBS solution intraperitoneally.

Body weights were recorded daily and taken on day 28 of infection. The eyeballs were bled after mice were anesthetized. Intact lungs were dissected out, left lungs fixed in 4% paraformaldehyde and right lungs used for lung homogenate counting of bacterial load (CFU). Spleen was fixed in 4% paraformaldehyde for some mice and used for tissue homogenization to count bacterial load.

Collecting alveolar lavage fluid from some mice: mice were anesthetized, the airway and lung tissue were fully exposed by surgery, a "v" incision was made along the airway, a catheter was inserted into the incision, fixed, and 700 μ L sterile PBS was aspirated by syringe, repeatedly perfused, aspirated, and collected as alveolar lavage fluid.

2.2.4 measurement of bacterial load on organs

The lungs and spleen of the mice were removed with sterile scissors and forceps, and the mice were homogenized in sterile PBS by weight. The homogenate was then serially diluted in a gradient, and 100. mu.l of each homogenate was pipetted and spread on a TSA plate containing kanamycin, cultured overnight, observed for colony status, and counted. 100 μ L of alveolar lavage fluid was applied to TSA plates containing kanamycin, cultured overnight, and the colony status was observed and counted.

2.2.6 Lung histopathology score

Fixing mouse lung in 4% paraformaldehyde, dehydrating, embedding, slicing, HE staining, Masson staining, and observing lung result integrity and inflammatory cell infiltration under microscope.

2.2.7 detection of inflammatory reaction in the Lung

The mouse alveolar lavage fluid was assayed for levels of Reg4 and I L-22 according to the protocol provided by the reagent supplier. RNA of a mouse lung tissue is extracted by using an RNA extraction kit, and the expression level of lung-related inflammatory genes of the mouse, including IL-1 beta, IL-10, CXCL-2, CCL-8 and CCL-2, is detected by quantitative PER.

2.3 results

2.3.1 Reg4 protein can improve weight loss of PAO1 infected mice

The content of Reg4 in alveolar lavage fluid of each group of mice is detected by using an ELISA kit, and the result is shown in figure 2B, and the content of Reg4 in the alveolar lavage fluid infected by PAO1 is obviously increased compared with that of the mice in the PBS group. Weight loss in mice in each group as shown in FIG. 2C, the weight of Con mice, which were not given PAO1 infection, increased slowly, while the weight loss in mice in each group, which were infected with PAO1, was varied. On day 2 post-infection, mice in PAO1+ PBS and PA01+ Reg4 groups weighed 92.6% and 93% of initial body weight, respectively, significantly lower than Con group (p both < 0.01). On day 3 post-infection, mice in PAO1+ Reg4 and PA01 groups began to gain weight. Mice in the PAO1+ Reg4 group weighed significantly better than mice in the PAO1 group (p <0.05) from day 22 to day 28 of infection. The result shows that the Reg4 protein treatment can improve the body weight condition of a mouse infected by PA 01.

2.3.2 Reg4 protein can relieve lung injury caused by PAO1

Pathological examination results are shown in fig. 3A-C, and compared with the ileum and colon tissues of the Con group mice, lung space edema can be seen by the lung HE staining of the PAO1+ PBS group mice, a large amount of neutrophil infiltration can be seen between alveoli, lung fibrosis can be seen by the Masson staining, and a large amount of fibrous deposition can be seen in the lung space. And the lung injury of mice in the PAO1+ Reg4 group is improved, inflammatory cell infiltration in the lung is reduced, and lung fibrosis is reduced. Quantitative analysis shows that the lung tissues of the PAO1+ Reg4 mice have lower neutrophil score and Ashcroft score than the PAO1 mice. The results show that the Reg4 protein can effectively relieve chronic lung injury caused by PAO 1.

2.3.3 Reg4 protein can improve lung inflammatory reaction induced by PAO1

The content of IL-22 in alveolar lavage fluid of each group of mice was measured by ELISA kit. The results are shown in FIG. 4A, the IL-22 content of PAO 1-infected alveolar lavage fluid is obviously increased compared with that of uninfected mice, and the IL-22 content in serum can be obviously reduced by the intervention of the administered Reg4 protein. The expression level of the lung-related inflammatory genes in mice was measured using quantitative PCR, and the results are shown in FIGS. 4B-F. The expression level of IL-1 beta, IL-10, CXCL-2 and CCL-28 in lung tissues of the PA-infected mice is obviously higher than that of mice in an uninfected group, and the expression level of IL-1 beta, IL-10, CXCL-2, CCL-8 and CCL-2 in the lung tissues of the mice can be obviously reduced by the intervention of the administered Reg4 protein. The results show that the Reg4 protein can improve lung inflammatory reaction induced by PAO 1.

2.3.4 Reg4 protein can reduce the colonization of PAO1 in lung

The number of PAO1 bacteria in lung and alveolar lavage fluid was measured in each group of mice, and the results are shown in FIGS. 5A-C. PAO1 was not detected in both lungs and alveolar lavage of Con group mice, whereas PAO1 detected large amounts of PAO1 in both lungs and alveolar lavage. The number of PAO1 in the lungs and alveolar lavage fluid of the group PAO1+ Reg4 was significantly reduced after administration of Reg4 protein treatment (p < 0.05).

2.3.5 Reg4 protein was able to reduce the invasion of PAO1 to mouse spleen the number of PAO1 bacteria in the spleen of each group of mice was examined and the results are shown in FIGS. 5A and D. PAO1 was not detected in the lung external organs of Con group, and PAO1 was found to colonize the spleen of mice infected with PAO1 in large numbers. The number of PAO1 in the spleen was significantly reduced in PAO1+ Reg4 group mice compared to PA01 group mice. The result shows that the Reg4 protein can effectively inhibit PAO1 from invading other extrapulmonary organ tissues.

Example 3 Reg4 protein reduces PAO1 adhesion and invasion of alveolar epithelial cells

3.1 Main Material

(1) Mouse alveolar epithelial cells (MLE-12) C432, Shanghai Yubo Biotech Co., Ltd

(2) Mouse alveolar macrophages (MH-S), C4704, Shanghai Yubo Biotech Co., Ltd

(2) Standard 24-well cell culture plate: 3524, Shanghai Baisai Biotechnology Ltd

(3) Gentamicin sulfate: PXTG80002, Shanghai Yuanzao Biotechnology Ltd

3.2 methods

(1) MLE-12 was subcultured routinely in cell culture medium containing 10% fetal bovine serum in DMEM, 100U/mL streptomycin and 100U/mL penicillin, and cultured in a CO2 incubator (5% CO2, 95% air) at 37 ℃ with medium change every 48 h. After the cells are grown and fused to 70% -80%, the cells are digested and passaged by 0.25% pancreatin-EDTA, and the experiment is generally carried out about 5 times or so, and the concentration is adjusted to be about 5 multiplied by 10⁵cells/ml for use.

(2) Effect of Reg4 on PAO1 adhesion to MLE-12 cells

The cell suspension (1 mL/well) was added to a 24-well cell culture plate and incubated at 37 ℃ in a 5% CO2 incubator until the cells reached a monolayer (approximately 5X 10 cells/well)⁵cells/well), washed 3 times with PBS, and co-cultured with MLE-12 cells for 2h with PAO1 at an infection ratio of 100:1 (centrifugation at 500 Xg for 5min to promote binding of bacteria to epithelial cells). 10min after the addition of bacteria, 10. mu.g/ml of Reg4 protein was added to the Reg4 group and an equal amount of PBS solution was added to the PBS group. After the incubation was completed, the cells were washed 3 times with PBS, and the bacteria not attached to the cells were washed away. Then 100 μ L of 0.5% Triton X-100 was added for 5min, and the cells were lysed by repeated pipetting. The lysate is serially diluted by 10 times of PBS buffer solution, an appropriate amount of the dilution solution is taken and coated on a Luria-Bertani (LB) plate, and the colony count is carried out after 24 hours of culture at 37 ℃, and 3 repeat wells are arranged.

(3) Effect of Reg4 on PAO1 invasion of MLE-12 cells

After 2h incubation of PAO1 with cells, the cells were washed 3 times with PBS to remove non-adhering bacteria, and then DMEM medium containing gentamicin (100. mu.g/mL) was added and incubated in a 5% CO2 cell incubator at 37 ℃ for 2h to kill non-invading PAO1 adhering to the cell surface. Washing cells with PBS 3 times, adding 100 μ L of 0.5% Triton X-100 per well, reacting for 5min, and repeatedly blowing to crack and shed cells. The lysate is serially diluted by 10 times, and an appropriate amount of the diluent is inoculated on an LB plate for viable bacteria plate counting, and 3 repeat wells are arranged.

(4) Culturing MH-S in conventional subculture modeThe cells were cultured in a cell culture medium of 10% fetal bovine serum in RPMI-1640medium, 100U/mL streptomycin and 100U/mL penicillin at 37 ℃ in a CO2 incubator (5% CO2, 95% air) with the medium changed every 48 hours. Adjusting the concentration to about 5X 10⁵cells/mL are ready for use.

(5) Effect of Reg4 on PAO1 adhesion to MH-S cells

The cell suspension (1 mL/well) was added to a 24-well cell culture plate, incubated at 37 ℃ in a 5% CO2 incubator, and PAO1 was CO-cultured with MLE-12 cells at an infection ratio of 100:1 for 2h (500 Xg centrifugation for 5min to promote binding of the bacteria to epithelial cells). 10min after the addition of bacteria, 10. mu.g/ml of Reg4 protein was added to the Reg4 group and an equal amount of PBS solution was added to the PBS group. After the incubation was completed, the cells were washed 3 times with PBS, and the bacteria not attached to the cells were washed away. Then 100 μ L of 0.5% Triton X-100 was added for 5min, and the cells were lysed by repeated pipetting. The lysate is serially diluted by 10 times of PBS buffer solution, an appropriate amount of the diluent is taken and coated on an LB plate, and the plate is cultured at 37 ℃ for 24h for colony counting, and 3 repeat wells are arranged.

(6) Effect of Reg4 on PAO1 invasion of MH-S cells

After 2h incubation of PAO1 with cells, the cells were washed 3 times with PBS to remove non-adhering bacteria, and then RPMI-1640medium containing gentamicin (100. mu.g/mL) was added and incubated in 5% CO2 cell incubator at 37 ℃ for 2h to kill non-invasive PAO1 adhering to the cell surface. Washing cells with PBS 3 times, adding 100 μ L of 0.5% Triton X-100 per well, reacting for 5min, and repeatedly blowing to crack and shed cells. The lysate is serially diluted by 10 times, and an appropriate amount of the diluent is inoculated on an LB plate for viable bacteria plate counting, and 3 repeat wells are arranged.

3.3 results

3.3.1 Reg4 protein reduces PAO1 adhesion and invasion of MLE-12 cells

Reg4 was co-incubated with MLE-12 cells and the bacterial content of adherent and invading cells was measured by plate counting and the results are shown in FIGS. 6A-D. In the Reg4 protein-treated group, the number of PAO1 adhering to and invading MLE-12 cells was less than that in the PBS-treated group, indicating that the Reg4 protein can reduce the PAO1 adhering to and invading MLE-12 cells.

3.3.2 Reg4 protein reduces PAO1 adhesion and invasion of MH-S cells

Reg4 was co-incubated with MH-S cells and the bacterial content of adherent and invading cells was measured by plate counting and the results are shown in FIGS. 7A-C. In the Reg4 protein treated group, the number of PA01 adhering to and invading MH-S cells was less than that in the PBS treated group, indicating that the Reg4 protein can reduce PAO1 adhering to and invading MH-S cells.

Example 4 binding of Reg4 protein to PAO1 inhibits migration

4.1 Main Material

(1) 96-well EIA/RIA plates: 9018 Shanghai Yujin Biotechnology Ltd

(2) Reg4 antibody: bs-10036R, Shanghai Kogya Biotech Co., Ltd

(3) TMB color development liquid: p0209-100ml, Shanghai Biyuntian Biotech Co., Ltd

4.2 methods

4.2.1 ELISA assay to detect binding to Reg4 protein

ELISA plates were incubated with 100ul of PA01 bacterial suspension (bacterial count 5X 10^ a)⁷CFU/ml) ug/ml Reg4 protein or BSA were coated overnight (4 ℃, 16 h). The plates were washed 3 times with PBS, different concentrations of Reg4 protein and mutant Reg4 protein (0, 2, 4, 6, 8 and 10ug/ml) were formulated with PBS containing 1% BSA, and the protein containing solutions were incubated with the ELISA plates for 2h (37 ℃). The plate was washed 3 times with PBS and incubated for 2h with the addition of the antibody Reg4 (1: 1000). The plate was washed 3 times with PBS, and a secondary antibody (1:5000) was added and incubated at 37 ℃ for 2 h. PBS was washed 3 times, TMB was added for color development, and OD was measured₄₅₀The value is obtained.

4.2.2 Effect of Reg4 protein on migration of PAO1

PA01 in logarithmic growth phase was incubated with Reg4 protein for 1 h. The size of halo diameter was measured by inoculating 10u l cultures onto semi-solid agar plates containing 0.3% agar, incubating at 37 ℃ for 10h and allowing the bacteria to move to form halos.

4.3 results

4.3.1 mutual binding of Reg4 protein and PAO1

The binding of PAO1 flagella to Reg4 was detected using ELISA assay and the results are shown in FIG. 8A. PAO1+ BSA group OD₄₅₀No significant change in value was observed, whereas the PAO1+ Reg4 group OD₄₅₀Value is followed byThe increase of the dosage of the Reg4 protein suggests that the Reg4 protein and the PAO1 are combined with each other.

4.3.2 Reg4 protein inhibits migration of PAO1

The results are shown in FIGS. 8B-C, comparing the migration of PAO1 on semi-solid agar plates. The PBS group PAO1 migrated on the semi-solid agar plate to form halos with diameters of (7.75. + -. 0.27) cm, whereas the bacterial halos of the Reg4+ PAO1 group decreased in diameter to (6.21. + -. 0.46) cm after administration of the Reg4 protein, suggesting that the Reg4 protein inhibits migration of PAO 1.

Sequence listing

<110> pulmonale Hospital of Shanghai city

Shanghai Institute of Pediatrics

Application of <120> Reg4 antibacterial peptide in treatment of pseudomonas aeruginosa infectious pneumonia

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 157

<212> PRT

<213> Artificial

<400> 1

Met Ala Ser Lys Gly Val Arg Leu Leu Leu Leu Leu Ser Trp Val Ala

1 5 10 15

Gly Pro Glu Val Leu Ser Asp Ile Leu Arg Pro Ser Cys Ala Pro Gly

20 25 30

Trp Phe Tyr Tyr Arg Ser His Cys Tyr Gly Tyr Phe Arg Lys Leu Arg

35 40 45

Asn Trp Ser His Ala Glu Leu Glu Cys Gln Ser Tyr Gly Asn Gly Ser

50 55 60

His Leu Ala Ser Val Leu Asn Gln Lys Glu Ala Ser Val Ile Ser Lys

65 70 75 80

Tyr Ile Thr Gly Tyr Gln Arg Asn Leu Pro Val Trp Ile Gly Leu His

85 90 95

Asp Pro Gln Lys Lys Gln Leu Trp Gln Trp Thr Asp Gly Ser Thr Asn

100 105 110

Leu Tyr Arg Arg Trp Asn Pro Arg Thr Lys Ser Glu Ala Arg His Cys

115 120 125

Ala Glu Met Asn Pro Lys Asp Lys Phe Leu Thr Trp Asn Lys Asn Gly

130 135 140

Cys Ala Asn Arg Gln His Phe Leu Cys Lys Tyr Lys Thr

145 150 155

Claims

1. Use of an active ingredient characterized by:

the active ingredients are Reg4 protein;

the application comprises the preparation of the medicine for treating or relieving the PAO1 infection symptoms.

2. Use of an active ingredient according to claim 1, characterized in that:

the use also includes at least one of the following uses:

3. Use of an active ingredient according to claim 1, characterized in that:

4. Use of an active ingredient according to claim 1, characterized in that:

the use also includes the use for preparing a medicament capable of binding to PAO 1.

5. Use of an active ingredient according to claim 1, characterized in that:

6. Use of an active ingredient according to claim 1, characterized in that:

the use also includes the use for the manufacture of a medicament for reducing PAO1 adhesion and invasion of alveolar epithelial cells.

7. Use of an active ingredient according to claim 1, characterized in that:

8. Use of an active ingredient according to claim 1, characterized in that:

the use also includes the use for preparing a medicament capable of inhibiting migration of PAO 1.

9. Use of an active ingredient according to claim 1, characterized in that:

the application also comprises the application of the PAO1 in preparing the medicine for relieving the spleen invasion.

10. An experimental model of a PAO1 chronic infection model, which is characterized by comprising the following steps:

s1, preparing agar bead suspension containing PAO1 bacteria;

s2, injecting the agar beads into a specific part of the target model.