Application of KN-93 in preparation of medicine for inhibiting porcine deltacoronavirus

Abstract

The invention discloses application of KN-93 in preparation of a medicine for inhibiting porcine deltacoronavirus, and relates to the technical field of cytobiology and virology. According to the invention, technologies of cytobiology, virology and the like are comprehensively utilized, and the calmodulin-dependent protein kinase II inhibitor KN-93 is found to have the effect of inhibiting porcine delta coronavirus in-vitro infection. The method comprises the following steps: firstly, carrying out cytotoxicity detection on KN-93 to determine the highest safe concentration of the drug, and then identifying the in-vitro PDCoV infection inhibition effect of 10 [mu] mol / L KN-93 through fluorescent quantitative PCR (RT-qPCR), TCID50 and IFA to find that the KN-93 can significantly inhibit porcine delta coronavirus. Therefore, the KN-93 can be used for preparing the medicine for inhibiting the porcine deltacoronavirus.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology and virology, and in particular to the application of KN-93 in the preparation of drugs for inhibiting porcine delta coronavirus. Background Art

[0002] Porcine deltacorovirus (PDCoV) is a new member of the genus Deltacoronavirus, belonging to the family Coronaviridae. It is a single-stranded positive-sense RNA virus with an envelope and a diameter of about 60-180nm. Under an electron microscope, the PDCoV virus particles are spherical with typical crown-like protrusions of coronaviruses on their surface. The genome length is about 25.4kb (excluding the Poly A tail), making it the smallest coronavirus known to date.

[0003] Currently, PDCoV is prevalent worldwide, seriously restricting the development of the pig farming industry. PDCoV infection occurs widely in pigs, and the virus spreads quickly and widely. It is highly pathogenic and can cause vomiting, diarrhea, dehydration and other symptoms in pigs. The mortality rate of infected suckling piglets can reach 30% to 40%, causing serious economic losses to the pig farming industry.

[0004] Since key scientific issues such as the infection and pathogenic mechanism of the virus have not yet been resolved, the development of vaccines and antiviral drugs has been seriously hindered, resulting in huge challenges in the prevention and control of the virus. PDCoV has a wide spectrum of infection. In addition to infecting pigs, it can also infect cattle, mice, chickens and other animals. It has the risk of cross-species transmission, posing a huge challenge to global public health security. Therefore, it is necessary to develop new anti-PDCoV drugs to inhibit PDCoV infection. Summary of the invention

[0005] The purpose of the present invention is to provide the use of KN-93 in the preparation of drugs for inhibiting porcine delta coronavirus to solve the problems existing in the above-mentioned prior art. The present invention has found that the calmodulin-dependent protein kinase II inhibitor KN-93 has the effect of inhibiting PDCoV infection in vitro, so it can be used to prepare drugs for inhibiting porcine delta coronavirus.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of KN-93 in preparing a drug for inhibiting porcine deltacoronavirus.

[0008] The present invention also provides a drug for inhibiting porcine deltacoronavirus, wherein the active ingredient includes KN-93.

[0009] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0010] Furthermore, the auxiliary materials include solubilizers, colorants, binders, disintegrants, fillers, stabilizers and / or preservatives.

[0011] Furthermore, the dosage form of the drug is granules, tablets, powders, capsules, emulsions or injections.

[0012] The present invention also provides the use of KN-93 in the preparation of an environmental disinfectant for inhibiting swine delta coronavirus.

[0013] The present invention also provides an environmental disinfectant for inhibiting swine deltacoronavirus, wherein the active ingredient includes KN-93.

[0014] The present invention discloses the following technical effects:

[0015] The present invention uses a combination of cell biology and virology techniques to discover that the calmodulin-dependent protein kinase II inhibitor KN-93 has the effect of inhibiting PDCoV infection in vitro. First, KN-93 was tested for cytotoxicity to determine the highest safe concentration of the drug, and then fluorescent quantitative PCR (RT-qPCR), TCID 50 The identification of the inhibitory effect of 10 μmol / L KN-93 on PDCoV infection in vitro by IFA showed that it could significantly inhibit PDCoV. Therefore, KN-93 can be used to prepare drugs against PDCoV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 is a bar graph of the cytotoxicity of KN-93 to LLC-PK1 cells;

[0018] Figure 2 This is a bar graph of the RT-qPCR method to detect the effect of KN-93 in inhibiting PDCoV infection; A is the result of 12 hours of cell culture; B is the result of 18 hours of cell culture;

[0019] Figure 3 TCID 50 Methods Bar graph of KN-93 inhibitory effect on PDCoV infection;

[0020] Figure 4 This is an immunofluorescence image to detect the effect of KN-93 in inhibiting PDCoV infection. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Terminology Note:

[0027] KN-93 is a calmodulin-dependent protein kinase II inhibitor, with a CAS number of 139298-40-1 and a molecular formula of C 26 H 29 ClN2O4S, the structural formula is as follows:

[0028]

[0029] The MEM medium used in the following examples contained 5% (v / v) heat-inactivated fetal bovine serum, 1 wt % penicillin and 1 wt % streptomycin.

[0030] Example 1

[0031] 1. Materials

[0032] PDCoV HNZK-02 strain (GenBank accession number MH708123) and LLC-PK1 cells were provided by the Animal Molecular Pathogenesis Laboratory of Henan Agricultural University; MEM culture medium and fetal bovine serum were purchased from Gibco, USA; β-actin antibody was purchased from Wuhan Mitaka Biotechnology Co., Ltd.; PDCoV N protein monoclonal antibody has been disclosed in Chinese patent CN202110376120.1 and was prepared and preserved by the Animal Molecular Pathogenesis Laboratory of Henan Agricultural University.

[0033] 2. Methods and Results

[0034] 1. Identification of KN-93 cytotoxicity to LLC-PK1 cells

[0035] LLC-PK1 cells were plated in 96-well cell plates, and when the cells were confluent, the cells were washed three times with D-Hanks balanced salt solution, and different concentrations of KN-93 were added and cultured at 37°C for 48 h. Cytotoxicity was identified according to the instructions of the CCK8 kit, and statistical analysis was performed using GraphPad software.

[0036] The results are as follows Figure 1 As shown, compared with the cell activity of the untreated group (0 μmo1 / L) (cell activity is 100%), the addition of 20 μmol / L KN-93 had a significant effect on cell activity, while the addition of 5 μmol / L or 10 μmol / L KN-93 had no significant effect on cell activity (ns), that is, there was no cytotoxicity, so 10 μmol / L KN-93 was used for subsequent experiments.

[0037] 2. RT-qPCR method to detect the inhibitory effect of KN-93 on PDCoV infection

[0038] LLC-PK1 cells were plated in 12-well plates and cultured in a 37°C, 5% CO2 incubator. The cells were washed three times with D-Hanks balanced salt solution, and the PDCoV HNZK-02 strain (GenBank accession number MH708123) with an MOI of 2 was infected with LLC-PK1 cells at 37°C for 1 h. The cells were washed three times with D-Hanks balanced salt solution, and MEM medium containing trypsin and 10 μmol / L KN-93 was added and cultured for 12 and 18 h. The cells were collected, total RNA was extracted, and reverse transcribed into cDNA, and cDNA was used as a template for RT-qPCR. The primer sequences, reaction system, and reaction procedures are shown in Tables 1, 2, and 3.

[0039] The results are as follows Figure 2 Compared with the untreated group, the PDCoVRNA content in the KN-93 treated group was significantly reduced.

[0040] Table 1 RT-qPCR primer sequences

[0041]

[0042] Table 2 RT-qPCR reaction system

[0043]

[0044] Table 3 RT-qPCR reaction procedure

[0045]

[0046] 3.TCID 50 Methods to detect the inhibitory effect of KN-93 on PDCoV infection

[0047] LLC-PK1 cells were plated onto 12-well plates and cultured in a 37°C, 5% CO2 incubator. The cells were washed three times with D-Hanks balanced salt solution, and the PDCoV HNZK-02 strain with an MOI of 2 was used to infect LLC-PK1 cells at 37°C for 1 hour. The cells were washed three times with D-Hanks balanced salt solution, and MEM medium containing trypsin and 10 μmol / L KN-93 was added and cultured for 12 and 18 hours. The cell supernatant was collected and diluted 10-fold, and the LLC-PK1 cells that had been plated on 96-well cell culture plates were inoculated. Eight replicates were made for each dilution, and the cells were cultured in a 37°C, 5% CO2 incubator for 3 days. The number of cytopathic wells no longer increased. The TCID of the virus was calculated using the Reed-Muench method. 50 .

[0048] The results are as follows Figure 3 As shown, the PDCoV virus titer in the KN-93-treated group was significantly reduced compared with the untreated group.

[0049] 4. Immunofluorescence detection of KN-93 inhibitory effect on PDCoV infection

[0050] LLC-PK1 cells were plated in 12-well plates and cultured in a 37°C, 5% CO2 incubator. The cells were washed three times with D-Hanks balanced salt solution, and the PDCoV HNZK-02 strain with MOI = 2 was infected with LLC-PK1 cells at 37°C for 1 hour. The cells were washed three times with D-Hanks balanced salt solution, and MEM medium containing trypsin and 10μmol / L KN-93 was added and cultured for 18 hours. Anhydrous ethanol was added to fix the cells, and the cells were washed twice with PBS. IFA blocking solution was added to each well and blocked at room temperature for 2 hours. After blocking, PDCoVN protein monoclonal antibody (1:3000 dilution) was added and incubated at 4°C overnight. The cells were washed three times with PBS, and FITC-labeled goat anti-mouse secondary antibody (1:3000 dilution) was added, and incubated in a 37°C constant temperature incubator in the dark for 1 hour. The cells were washed three times with PBS, and DAPI was added to stain the cell nuclei at room temperature for 15 minutes. The cells were placed on a horizontal shaker and washed with PBS at 130 rpm for 5 min, washed three times, and observed and photographed with a fluorescence microscope.

[0051] The results are as follows Figure 4 As shown, the fluorescence intensity of the KN-93 treated group was significantly decreased compared with the untreated group.

[0052] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of KN-93 in the preparation of drugs for inhibiting porcine deltacoronavirus.

2. A drug for inhibiting porcine delta coronavirus, characterized in that: Active ingredients include KN-93.

3. The drug according to claim 2, characterized in that The drug also includes pharmaceutically acceptable excipients.

4. The drug according to claim 3, characterized in that The auxiliary materials include solubilizers, colorants, binders, disintegrants, fillers, stabilizers and / or preservatives.

5. The drug according to claim 4, characterized in that The dosage form of the medicine is granules, tablets, powders, capsules, emulsions or injections.

6. Application of KN-93 in the preparation of environmental disinfectants for inhibiting swine deltacoronavirus.

7. An environmental disinfectant for inhibiting porcine delta coronavirus, characterized in that Active ingredients include KN-93.

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