CN116870134A - Application of theta-defensin in preparation of medicine for resisting feline calicivirus infection - Google Patents

Abstract

The invention discloses application of theta-defensin in preparation of medicines for resisting feline calicivirus infection. The invention discovers for the first time that the theta-defensin not only can inhibit the replication of feline calicivirus (including malignant FCV) genes and proteins and inhibit the inflammation of the oral cavity and the respiratory tract of a cat caused by FCV infection, but also has quick response, can basically eliminate the inflammation reaction of the oral cavity and the respiratory tract of the cat caused by FCV after continuous use for 5 days, and can be repeatedly used after recurrence; meanwhile, the medicine has natural sources, does not hurt the liver and kidney, has no side effect, has good medication safety, does not cause drug resistance, and provides an effective treatment way for cat calicivirus infection.

Description

Application of theta-defensin in preparation of medicine for resisting feline calicivirus infection

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of theta-defensin in preparation of medicines for resisting feline calicivirus infection.

Background

Cat stomatitis refers to mucosal inflammation of any part in the oral cavity of a cat, and clinically refers to extensive, chronic and progressive oral mucosal inflammation. 0.7-10% of all cats are affected by the disease, and severe stomatitis can cause eating difficulty, mental state change and weight loss of the suffering cats, and seriously affect the quality of life.

The etiology of cat stomatitis is unknown, the influencing factors are numerous and cannot be locked, and the pathogenic pathogens are numerous, which may be inflammation caused by excessive immune response of the body's immune system to one or more currently unknown mediators.

The ideal therapeutic goal of cat stomatitis is to completely eliminate inflammation in abnormal physiological states in the oral cavity, but no 100% successful therapeutic regimen exists at present due to unknown etiology. The mainstay of therapies includes full mouth tooth extraction (including canine teeth, incisors) with half mouth tooth extraction (all molar teeth, premolars), carbon dioxide laser burning of inflamed tissues, long term use of hormones, cyclosporins, interferons and antibiotics to control inflammation. However, antibiotics are easy to generate drug resistance, hormone medicines can generate serious side effects while inhibiting inflammation, the full mouth tooth extraction cannot be thoroughly cured, the recurrence after stopping the medicine is very frequent, and in most cases, the medicine must be taken for life.

Therefore, defining the etiology of cat stomatitis and targeted treatment is an effective way to radically treat cat stomatitis. However, it has been studied for many years that the suspicion of cat stomatitis-related factors, cat cup virus (Feline calicivirus, FCV), is greatest. Calcivirus is a single-stranded positive strand RNA virus of the genus Calcivirus of the family Calciviraceae, which mainly causes oral and respiratory infections in cats. Feline calicivirus is unstable during RNA-dependent RNA polymerase replication, is prone to mutagenic strains, and highly pathogenic FCV can even cause fatal systemic disease in cats.

The incidence of feline calicivirus varies from region to region worldwide and from cat population to cat population of different species, typically between 5-10%, but may be as high as 80% or more in some high risk venues (e.g., pet stores, animal hospitals, animal-handling centers, etc.; in addition, the cat with the toxin can become an asymptomatic pathogen carrier after being treated, but can expel toxin for a long time after clinical rehabilitation, and is a dangerous infectious source.

However, there is no effective antiviral therapeutic against FCV at present, and since caliciviruses are RNA viruses, the virus mutation is frequent, and some prophylactic vaccine effects are general, there is a great need to develop therapeutic agents against FCV.

Disclosure of Invention

The invention aims to provide the application of the theta-defensin in preparing medicines for resisting the infection of the feline calicivirus, and provides an effective treatment way for the infection of the feline calicivirus.

In order to achieve the above object, the present invention has the following technical scheme:

the application of the theta-defensin in preparing medicines for resisting feline calicivirus infection;

and the use of a theta-defensin in the preparation of a feline calicivirus inhibitor.

The invention discovers for the first time that the theta-defensin not only can inhibit the replication of feline calicivirus (including malignant FCV) genes and proteins and inhibit the inflammation of the oral cavity and the respiratory tract of a cat caused by FCV infection, but also has quick response, can basically eliminate the inflammatory reaction of the oral cavity and the respiratory tract of the cat caused by FCV after continuous use for 5 days, and can be repeatedly used after recurrence; meanwhile, the medicine has natural sources, does not hurt the liver and kidney, has no side effect, has good medication safety, does not cause drug resistance, and provides an effective treatment way for cat calicivirus infection.

Based on the above, the invention also provides application of the theta-defensin in preparing a medicine for preventing and treating cat stomatitis; and the application of the theta-defensin in preparing medicines for preventing and treating the respiratory diseases of cats. Clearly, both the cat stomatitis and the cat respiratory disease are caused by FCV infection.

Preferably, the amino acid sequence of the θ -defensin is as follows: GICRCICGKGICRCICGR. The amino acid sequences are joined end to form a cyclic peptide.

The invention also provides an oral spray for treating cat stomatitis, and the effective component of the treatment spray at least comprises theta-defensin. The spray can not only directly reach the affected part (oral cavity or upper respiratory tract), but also is convenient to use.

Preferably, in the oral spray for treating cat stomatitis, the amino acid sequence of the θ -defensin is: GICRCICGKGICRCICGR, and the concentration of θ -defensin is not lower than 3.125 μg/ml.

Research shows that θ -defensin starts to inhibit FCV virus gene replication at a concentration of 3.125 mug/ml, the inhibition rate reaches 50% at a concentration of 6.25 mug/ml, and the inhibition rate on FCV virus gene replication and protein expression can reach 100% basically at a concentration of 50 mug/ml; no substantial FCV virus-induced cytopathy was observed after use at a concentration of 25 μg/ml.

Compared with the prior art, the invention has the beneficial effects that:

the invention discovers for the first time that the theta-defensin not only can inhibit the replication of feline calicivirus (including malignant FCV) genes and proteins and inhibit the inflammation of the oral cavity and the respiratory tract of a cat caused by FCV infection, but also has quick response, can basically eliminate the inflammation reaction of the oral cavity and the respiratory tract of the cat caused by FCV after continuous use for 5 days, and can be repeatedly used after recurrence; meanwhile, the medicine has natural sources, does not hurt the liver and kidney, has no side effect, has good medication safety, does not cause drug resistance, and provides an effective treatment way for cat calicivirus infection.

Drawings

FIG. 1 is a graph showing the reversal of cytopathic effects of FCV infection by varying concentrations of θ -defensin;

wherein Ctrl represents Control group, i.e. CRFK cells are not treated with FCV virus and RC 101; WT means CRFK cells were treated with FCV virus only; 12.5 μg/ml indicates that CRFK cells were treated with FCV virus and RC101 at a concentration of 12.5 μg/ml; 25 μg/ml indicated that CRFK cells were treated with FCV virus and RC101 at a concentration of 25 μg/ml, as follows;

FIG. 2 is a graph showing the effect of different concentrations of θ -defensin treatment on FCV viral gene replication;

wherein, -virus/0 means not treated with FCV virus and RC101, +/(3.125. Mu.g/ml, 6.25. Mu.g/ml, 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml and 200. Mu.g/ml) means treated with FCV virus and RC101 at the corresponding concentrations; the Relative FCV 5'UTR mRNA (fold change) represents the Relative copy amount (fold difference) of FCV 5' UTR mRNA;

FIG. 3 shows the effect of different concentrations of θ -defensin treatment on the expression of the VP-1 protein of FCV virus (10-fold fluorescence microscopy);

FIG. 4 is a graph showing the effect of different concentrations of θ -defensin treatment on the expression of the VP-1 protein of FCV virus (20-fold fluorescence microscopy);

FIG. 5 is a graph showing the effect of θ -defensin on cellular activity;

fig. 6-8 show the clinical treatment effect of the oral spray for treating cat stomatitis of the present invention on cat pet stomatitis.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the attached drawings and the detailed description.

Example 1 Effect of theta-defensins on cytopathic effects of FCV infection

CRFK cells were seeded in 96-well plates with cell culture medium (high sugar DMEM+10% FBS) and placed at 37℃in 5% CO ₂ Culturing in an incubator for 16 hours; subsequently, RC101 was diluted to 25. Mu.g/ml and 50. Mu.g/ml with cell culture medium in gradient; diluting the virus by using a cell culture solution to make the virus amount be 0.2MOI; theta-defensin (hereinafter abbreviated as RC101, purchased from Kirsrui, amino acid sequence shown in SEQ ID No.1: GICRCICGKGICRCICGR) and virus liquid are mixed according to a volume ratio of 1:1, the final concentration of RC101 is 12.5 mug/ml and 25 mug/ml, the virus amount is 0.1MOI; replacing cell culture solution in original 96-well plate with RC101 and virus mixture, and continuing at 37deg.C and 5% CO ₂ The cells were incubated in the incubator for 24-36 hours (control group (without FCV virus and RC101 treatment) and WT group (with FCV virus only)), and cytopathic effect (CPE) was observed under a microscope, and the observation results are shown in FIG. 1.

As can be seen from FIG. 1, the cytopathic effects of the WT group are very severe compared to the control group; whereas the group of RC101, 12.5. Mu.g/ml and the group of RC101, 25. Mu.g/ml showed reduced cytopathic effect compared to the WT group, and had a dose-dependence, the higher the concentration of RC101 treatment, the lighter the cytopathic effect, and no cytopathic effect was substantially observed at 25. Mu.g/ml; RC101 was shown to be able to inhibit FCV virus infection of cells.

Example 2 fluorescent quantitative PCR method for analysis of the effects of θ -defensins on FCV viral replication

CRFK cells were seeded in 96-well plates with cell culture medium (high sugar DMEM+10% FBS) and placed at 37℃in 5% CO ₂ Culturing in an incubator for 16 hours; subsequently, RC101 was gradient diluted to 3.125. Mu.g/ml, 6.25. Mu.g/ml, 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml and 200. Mu.g/ml with cell culture broth; diluting the virus by using a cell culture solution to make the virus amount be 0.2MOI; RC101 and virus liquid are mixed according to the volume ratio of 1:1, the final concentration of RC101 is 1.5625. Mu.g/ml, 3.125. Mu.g/ml, 6.25. Mu.g/ml, 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, and the virus amount is 0.1MOI; replacing cell culture solution in original 96-well plate with RC101 and virus mixture, and continuing at 37deg.C and 5% CO ₂ Collecting cell culture supernatant after culturing in an incubator for 12 hours, extracting RNA by using a TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 kit, and obtaining cDNA by reverse transcription, wherein FCV RNA is frequently mutated, then three pairs of primers (shown in table 1) are designed for a 5'UTR region, and fluorescence quantitative PCR detection is respectively carried out by adopting the three pairs of primers, so that the content of FCV virus gene FCV 5' UTR in the cell culture supernatant is analyzed, and the optimal primer is selected as No. 2; the analysis results of the primer # 2 are shown in FIG. 2.

TABLE 1

As can be seen from FIG. 2, RC101 inhibited FCV viral gene replication at a concentration of 3.125. Mu.g/ml, with the gene copy number of FCV 5' UTR in FCV viral mRNA in cell culture supernatant gradually decreasing as the concentration of RC101 treatment increases; 50% inhibition was achieved by 6.25. Mu.g/ml, with 50. Mu.g/ml inhibition being substantially complete; RC101 was shown to be able to inhibit FCV viral replication.

Example 3 Indirect immunofluorescence assay to analyze the effects of θ -defensins on FCV viral replication

CRFK cells were seeded in 96-well plates with cell culture medium (high sugar DMEM+10% FBS) and placed at 37℃,5％CO ₂ Culturing in an incubator for 16 hours; subsequently, RC101 was gradient diluted to 3.125. Mu.g/ml, 6.25. Mu.g/ml, 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml and 200. Mu.g/ml with cell culture broth; diluting the virus by using a cell culture solution to make the virus amount be 0.2MOI; RC101 and virus liquid are mixed according to the volume ratio of 1:1, the final concentration of RC101 is 1.5625. Mu.g/ml, 3.125. Mu.g/ml, 6.25. Mu.g/ml, 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, and the virus amount is 0.1MOI; replacing cell culture solution in original 96-well plate with RC101 and virus mixture, and continuing at 37deg.C and 5% CO ₂ After 6h incubation in incubator, cells were plated with 4% paraformaldehyde for 10min at 25℃followed by 0.1% Triton X-100; blocking with 2% BSA was performed for 1h at room temperature, and primary antibody was incubated with FCV VP1 mab (1:1000) at room temperature for 1h; the secondary antibody uses Goat anti-Mouse, alexa Fluor ^TM 594 (1:1000), incubating for 1h at room temperature, and finally adding DAPI (1:1000), and incubating for 1h at room temperature; the observation under a fluorescence microscope is shown in fig. 3 and 4.

As can be seen from FIGS. 3 and 4, VP1 protein of FCV virus has not been detected under RC101 treatment at a concentration of 50. Mu.g/ml, and it is also shown that RC101 is capable of inhibiting replication of FCV virus.

Example 4 cytotoxicity assay of theta-defensins

CRFK cells were seeded in 96-well plates with cell culture medium (high sugar DMEM+10% FBS) and placed at 37℃in 5% CO ₂ Culturing in an incubator for 16 hours; subsequently, RC101 was gradient diluted to 0.78125. Mu.g/ml, 1.5625. Mu.g/ml, 3.125. Mu.g/ml, 6.25. Mu.g/ml, 12.5. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, 200. Mu.g/ml with cell culture solution instead of the cell culture solution in the original 96-well plate, and continued at 37℃with 5% CO ₂ Incubate for 12h (while setting a blank with no RC101 added); then WST-8 reagent (Cell Counting Kit-8) is added, and the mixture is continuously heated to 37 ℃ and 5% CO ₂ Incubating for 3h; finally, detecting the OD value at 450 nm; the detection results are shown in FIG. 5.

As can be seen from FIG. 5, the increase in RC101 concentration versus cell OD ₄₅₀ Has no influence, shows that RC101 does not inhibit cell activity basically (no obvious cytotoxicity), and has good biological safety.

EXAMPLE 5 preparation of oral spray for treatment of cat stomatitis Using θ -defensin

The oral spray for treating cat stomatitis of the embodiment is prepared by adopting theta-defensin, and the preparation method comprises the following steps: taking 5mg of RC101, dissolving by adopting 20mL of phosphate buffer solution with the concentration of 10mM, and then adding 5mL of distilled water to fix the volume to 30mL, thus obtaining the oral spray (hereinafter referred to as RC101 spray) for treating cat stomatitis.

Clinical trials were conducted on the RC101 spray prepared as described above to verify the therapeutic effect of θ -defensin on cat stomatitis. The clinical test is carried out by a pet animal hospital of the department of academy of agricultural science, guangdong, and the test method is as follows:

3 cats with cat stomatitis are collected, after general anesthesia, the oral cavity is cleaned, then RC101 spray (the effective concentration of theta-defensin is 0.167 mg/ml) is sprayed into the oral cavity of the cat, the spraying amount is 5 ml/day, the spraying is continued for 5 days, and the cat stomatitis symptoms before and after spraying are shown in figure 6.

As can be seen from fig. 6, after 5 days of continuous spraying, the oral inflammation of the cat pet has completely disappeared, and the effect is quick. After the inflammation disappears for 4 weeks, the stomatitis of three cats recurs again, and the RC101 spray is continuously used by adopting the same method, the inflammation still disappears after 5 days of administration, and the symptoms of the stomatitis of the cats before and after administration are shown in figure 7; after the inflammation disappears for six months, the stomatitis of three cats recurs again, the RC101 spray is continuously used by adopting the same method, the inflammation disappears after continuous use for 10 days, and the symptoms of the stomatitis of the cats before and after the use are shown in figure 8; after the inflammation disappears for 4 weeks, the three cats relapse the stomatitis again, and the RC101 spray is continuously used by adopting the same method; during the administration period, the cat pets have normal appetite, and the appetite is increased along with the disappearance of inflammation.

Claims (10)

1. The application of theta-defensin in preparing medicines for resisting feline calicivirus infection is provided.

2. The use according to claim 1, wherein the θ -defensin has the amino acid sequence: GICRCICGKGICRCICGR.

3. Use of θ -defensin in the preparation of feline calicivirus inhibitor.

4. The use according to claim 3, wherein the θ -defensin has the amino acid sequence: GICRCICGKGICRCICGR.

5. Application of theta-defensin in preparing medicine for preventing and treating cat stomatitis.

6. The use according to claim 5, wherein the θ -defensin has the amino acid sequence: GICRCICGKGICRCICGR.

7. The application of theta-defensin in preparing medicines for preventing and treating cat respiratory diseases.

8. The use of claim 7, wherein the θ -defensin has the amino acid sequence: GICRCICGKGICRCICGR.

9. An oral spray for treating cat stomatitis, wherein the active ingredient comprises at least θ -defensin.

10. The oral spray for treating cat stomatitis according to claim 9, wherein the θ -defensin has an amino acid sequence of: GICRCICGKGICRCICGR;

and the concentration of the theta-defensin is not less than 3.125 mug/ml.