Method for screening antiviral drugs

Abstract

The invention belongs to the field of biological medicine, and relates to immunology and virology. Specifically, the invention relates to a method for screening antiviral drugs, in particular to a method for screening anti-influenza virus drugs. Specifically, the invention relates to a method for screening antiviral drugs, which comprises the following steps: (1) culturing host cells; (2) infecting host cells with the virus, and culturing under conditions suitable for virus and cell growth; (3) adding a covering solution and a candidate drug with a proper concentration, and continuing to culture, the covering solution comprising microcrystalline cellulose, a cell culture medium and TPCK pancreatin; (4) detecting virus antigens through ELISA (enzyme-linked immuno sorbent assay), and reading original data by using an ELISPOT instrument; (5) calculating IC50 according to the original data; and (6) selecting the candidate drugs according to the IC50 value. The method is rapid, simple, accurate and reliable, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to immunology and virology. Specifically, the present invention relates to a method for screening antiviral drugs, and more specifically, the present invention relates to a method for screening anti-influenza virus drugs. Background Art

[0002] In virological research, known influenza viruses are inoculated into a monolayer of dog kidney cells (hereinafter referred to as MDCK cells) and covered with a layer of low-melting-point agar containing nutrients. The plaques formed after culture can be used to quantify influenza viruses. This method is called the virus plaque assay, which provides a means of virus quantification.

[0003] The principle of the virus plaque assay is: first prepare a sheet of MDCK cells on a cell plate, then infect the cells with influenza virus, and then cover a single layer of low-melting-point agar containing nutrients. As time goes on, the virus continues to replicate and then spreads to the surrounding cells. In this way, using the principle of crystal violet staining, only normal surviving cells can be stained with crystal violet; that is, cells destroyed by virus infection will not be stained, while surrounding cells not infected by the virus will be stained, thus showing virus plaques. It is often used for screening anti-influenza virus drugs and studying whether drugs lead to the emergence of drug-resistant strains.

[0004] The traditional plaque assay method for influenza virus is used to determine the plaque forming unit (PFU) of influenza virus, which includes the following steps: 1) first combining the virus with cells and incubating at 35°C for 1 hour; 2) adding low melting point agar containing nutrients, solidifying, and incubating at 35°C for 72 hours; 3) then adding crystal violet, staining, and incubating at 35°C for 24 hours; 4) calculating the plaque forming unit (PFU) of influenza virus, that is, calculating the total amount of virus with infectious ability;

[0005] When it comes to the effective concentration (EC50) of a compound 50 When the value is 0.05, the method further comprises the following steps: 5) combining 100 PFU influenza virus with different concentrations of compounds and adding them to the MDCK cells in the film; 6) incubating at 35°C for 1 hour; 7) adding low melting point agar containing nutrients, solidifying, and incubating at 35°C for 72 hours; 8) adding crystal violet, staining, and incubating at 35°C for 24 hours; 9) after the appearance of plaques, visually observing the cytopathic effect (CPE) and counting, and calculating the EC 50 value.

[0006] The disadvantages of the traditional virus plaque assay are: the experimental cycle is long, and the fastest measurement takes 8 days; since the solidification time of low-melting point agar is difficult to control, cells are easily killed by uneven heating or the low-melting point agar solidifies first; the staining method (crystal violet) is used to obtain the number of virus plaques, which easily causes incomplete plaque formation or cell detachment during decolorization; the virus spots formed need to be counted by observing the CPE of the virus, and the results are inaccurate.

[0007] There is still a need to develop new plaque assay methods for viruses, especially influenza viruses. Summary of the invention

[0008] After in-depth research and creative work, the inventors have developed a new virus plaque test method, using microcrystalline cellulose instead of low melting point agar, avoiding the phenomenon of cell shedding or self-solidification of low melting point agar due to the use of low melting point agar, and reducing experimental errors. In addition, the inventors have also improved the detection method during identification to ELISA detection. After staining with Trμe Blμe, the clearly visible blue spot value can be directly calculated on the ELiSPOT instrument, making the experimental results accurate and reliable. The present invention can quickly detect the binding of influenza virus and compounds, significantly shorten the binding time of the virus and the compound (for example, as low as 22 hours), and effectively reduce the time of the entire experimental process (for example, as low as 3 days). The following invention is thus provided:

[0009] One aspect of the present invention relates to a method for screening antiviral drugs, comprising the following steps:

[0010] (1) Cultivating host cells;

[0011] (2) infecting host cells with the virus and culturing them under conditions suitable for virus and cell growth;

[0012] (3) adding a covering solution and an appropriate concentration of the candidate drug, and continuing the culture, wherein the covering solution comprises microcrystalline cellulose, a cell culture medium, and TPCK pancreatin;

[0013] (4) Detect viral antigens by ELISA and obtain raw data by Enzyme Linked Immunospot Assay (ELiSPOT) technology (ELiSPOT instrument reading);

[0014] (5) Calculate IC based on raw data 50 ;and

[0015] (6) According to IC 50 Numerical values ​​are used to select candidate drugs.

[0016] In some embodiments of the present invention, the method for screening antiviral drugs, wherein, in step (1), the host cells are cultured 8-36, 12-24, 8-24 or 8-12 hours before the virus infects the host cells in step (2);

[0017] Preferably, the culture temperature is 36°C-37°C;

[0018] Preferably, the cultured cells are washed with a virus maintenance solution once or more (eg, 2, 3, 4 or 5 times).

[0019] In some embodiments of the present invention, the method for screening antiviral drugs, wherein, in step (2), the virus is prepared into a suitable concentration using a virus maintenance solution, such as 80-120 PFU per 100 μL or 100 PFU per 100 μL;

[0020] Preferably, the culture temperature is 35°C-36°C;

[0021] Preferably, the culture time is 1-5 hours, 1-3 hours or 1-2 hours.

[0022] In some embodiments of the present invention, the method for screening antiviral drugs, wherein in step (3), the microcrystalline cellulose is LOOP:15-375;

[0023] Preferably, the concentration of microcrystalline cellulose is 1%-5%, 2%-4%, 2%-3% or 2%-2.5%;

[0024] Preferably, the concentration of TPCK pancreatin is 1-5 μg / ml, 1-3 μg / ml or 1-2 μg / ml;

[0025] Preferably, the cell culture medium is 2X MEM;

[0026] Preferably, the culture temperature is 35°C-36°C;

[0027] Preferably, the culture time is 8-36, 12-24, 22-24 or 12-22 hours;

[0028] Preferably, cells to which the virus is added but not the candidate drug are added serve as a positive control;

[0029] Preferably, cells without the addition of virus and without the addition of candidate drug serve as negative controls.

[0030] In some embodiments of the present invention, in the method for screening antiviral drugs, in step (4), the viral antigen is a viral nucleoprotein, such as an influenza virus nucleoprotein.

[0031] In some embodiments of the present invention, the method for screening antiviral drugs, wherein in step (4), the detection is performed by ELISA method and the raw data is read by ELiSPOT instrument. For example, the reading of 363 represents 363 PFU / 100 μl.

[0032] In some embodiments of the present invention, the method for screening antiviral drugs, wherein in step (5), Graphpad Prism 7 is used to prepare a curve and calculate IC 50 ;

[0033] Preferably, after the concentration of the candidate drug is converted to a logarithmic value, a nonlinear regression curve is fitted between the converted logarithmic concentration value and the percentage of inhibition of the number of viral plaques, and the half inhibition concentration IC is obtained by the above regression curve. 50 ;

[0034] Preferably, the calculation formula for the percentage inhibition of the number of virus plaques is as follows:

[0035] The percentage of inhibition of the number of virus plaques = 100% - (the readings of different dilutions of the candidate drug - the average value of the cell control readings) / (the average value of the virus control readings - the average value of the cell control readings) * 100%.

[0036] In some embodiments of the present invention, the method for screening antiviral drugs, wherein the virus is an influenza virus, preferably an influenza A virus such as an H1N1 or H3N2 subtype, or an influenza B virus; the antiviral drug is an anti-influenza virus drug; and the viral antigen is an influenza virus antigen;

[0037] Preferably, the host cell is a dog kidney cell.

[0038] In some embodiments of the present invention, the method for screening antiviral drugs comprises the following steps:

[0039] A. Dilute MDCK cells with cell culture medium at 3 x 10 4 MDCK cells / 100 μL cell culture medium / well were seeded into a 96-well cell culture plate. After overnight culture at 37°C, the cells were washed twice with virus maintenance solution (200 μL / well).

[0040] B. Dilute the influenza virus with virus maintenance solution to a virus working solution containing 100 PFU per 100 μL culture medium. Take 100 μL of the virus working solution and add it to each cell culture well. After culturing at 35°C for 1 hour, remove the virus working solution (discard the virus remaining in the supernatant; the cells in the well are attached and the virus has entered the cells).

[0041] C. Take 200 μL of microcrystalline cellulose (LOOP:15-375) containing 2 μg / ml TPCK pancreatin and 2X MEM medium to prepare compound working solutions of different concentrations (containing 100 μL of 2.4% microcrystalline cellulose, 100 μL of 2X MEM and 2 μg / ml TPCK pancreatin), add to each cell culture well and culture at 35°C for 22 hours.

[0042] D. Discard the overlay solution containing different concentrations of compounds, detect viral antigens by ELISA and calculate the IC of the compounds 50 value.

[0043] In some embodiments of the present invention, in the method for screening antiviral drugs, the ELISA method is an indirect ELISA method.

[0044] In some embodiments of the present invention, the method for screening antiviral drugs, wherein the ELISpot technique comprises the following steps:

[0045] ① After discarding the covering solution containing different concentrations of compounds, add 4% paraformaldehyde (200 μL / well) and place the cell culture plate in a 4°C refrigerator for 40 min;

[0046] ②Discard 4% paraformaldehyde, wash the cells twice with phosphate buffered saline (PBS, 100 μL / well), add permeabilization solution (100 μL / well) and leave at room temperature for 30 min;

[0047] ③Discard the permeabilization solution, wash the cells twice with PBS (100 μL / well), add 50 μL / well of mouse anti-influenza virus nucleoprotein (NP) monoclonal antibody (primary antibody, dilution multiple 1:2000) diluted with ELISA buffer, and leave at room temperature for 1 hour;

[0048] ④Discard the primary antibody, wash the cells three times with phosphate-buffered saline (PBST, 200 μL / well), add 50 μL / well of peroxidase-labeled mouse IgG antibody (secondary antibody, dilution multiple 1:1000) diluted with ELISA buffer, and place at room temperature away from light for 1 hour;

[0049] ⑤Discard the secondary antibody, wash the cells 3 times with PBST, add 0.3% H 2 O 2 Trμe Blμe substrate (50μl / well) was placed at room temperature in the dark for 30min;

[0050] ⑥ Discard the peroxidase substrate (containing 0.3% H 2 O 2 Trμe Blμe substrate), double distilled water (ddH 2O, 200 μL / well) once, and discard ddHO 2 O, air-dried naturally, and the raw data were read using an ELiSPOT instrument.

[0051] Advantageous Effects of the Invention

[0052] The present invention achieves one or more of the following technical effects (1) to (4):

[0053] (1) Microcrystalline cellulose is more convenient and simple to replace low melting point agar;

[0054] (2) After permeabilization, Trμe Blμe peroxidase substrate was added for staining, and the formed blue spots were clearly visible;

[0055] (3) ELISA testing is more accurate and the calculated results are more reliable;

[0056] (4) Significantly shortened the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 :The experimental results of two compounds. The value in each well represents the number of cells infected by the virus.

[0058] Figure 2 : The percentage inhibition of viral plaque number is plotted as the effect curve of the compound dosage, wherein the left curve relates to the compound of formula I, and the right curve relates to the compound of formula II. DETAILED DESCRIPTION

[0059] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0060] Reagents are shown in Table A.

[0061] Table A

[0062]

[0063]

[0064] Consumables are shown in Table B.

[0065] Table B

[0066] name brand Part Number 96-well cell plate Gibco A11428-03 Sterile sink Caster 4870 10μl sterile pipette tip Rainin 17002928 200μl sterile pipette tip Rainin 17002927 1000μl sterile pipette tip Rainin 17002902 15ml centrifuge tube Corning 430790 50ml centrifuge tube Corning 430921 2ml cryotube Greiner 126279 T75 cell flask Corning 430720

[0067] The instruments and equipment are shown in Table C.

[0068] Table C

[0069] name brand model Biological Safety Cabinets Thermo Level 2 1389 Electronic balance OH AμS STX123ZH Water Purifier ELGA A10 Magnetic stirrer Zhenrong 90-1 High Pressure Steam Sterilizer SANYO MLS-3030CH <![CDATA[CO 2 Incubator]]> SANYO MCo-15A 4℃ refrigerator Hair SC-316 -20℃ Refrigerator Siemens KK25E76TI -80℃ Ultra Low Temperature Refrigerator Hair DW-86L486 ELiSPOT instrument AID ELRIFL04 ELiSPOT Reader AID Version 7.0 Water bath Changfeng 600 Constant temperature incubator Shanghai Yiheng GHP9270 Inverted microscope LOYMPμS CKX41 Centrifuge Thermo IS-R Cell Counter Coμntstar IC1000

[0070] Cell culture medium: 427.5ml DMEM+5ml PS+5ml GlμtaMax+12.5ml HEPES+50ml FBS.

[0071] Virus maintenance solution: 495ml DMEM + 5ml PS.

[0072] Covering solution: 100 ml 2X MEM + 200 μl Trypsin (2 mg / ml) + 100 ml microcrystalline cellulose (LOOP: 15-375).

[0073] Permeabilization solution: Working concentration: 0.2% X-100: 1ml 10% X-100+49mlPBS.

[0074] ELISA buffer: 50μl 0.1% Tween-80+1665μl 1% BSA (30%)+50ml PBS.

[0075] Example 1

[0076] 1. Dilute the cells with cell culture medium one day before influenza virus (H1N1 subtype) infection to 3x 10 4 The cells were seeded into a 96-well cell culture plate at a density of 10 MDCK cells / 100 μL cell culture medium / well and the layout shown in Table 1. After overnight culture at 37°C, the cells were washed twice with virus maintenance solution (200 μL / well).

[0077] Table 1: Sample layout of a 96-well cell culture plate (each cell in the table represents a well of a cell culture plate)

[0078] PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS cell cell cell cell cell cell cell cell cell cell PBS PBS cell cell cell cell cell cell cell cell cell cell PBS PBS cell cell cell cell cell cell cell cell cell cell PBS PBS cell cell cell cell cell cell cell cell cell cell PBS PBS cell cell cell cell cell cell cell cell cell cell PBS PBS cell cell cell cell cell cell cell cell cell cell PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS

[0079] 2. Dilute the influenza virus with virus maintenance solution to a virus working solution that can produce approximately 100 PFU influenza virus plaques per 100 μL culture solution. Add 100 μL of the virus working solution to each cell culture well and culture at 35°C for 1 hour before removing the virus working solution.

[0080] Table 2: Layout of virus infection on cell culture plates (each cell in the table represents a well of a cell culture plate)

[0081]

[0082] 3. Take 200 μL of working solution (containing 100 μL of 2.4% microcrystalline cellulose, 100 μL of 2X MEM and 1‰ TPCK trypsin) of the test compound (for example, the compounds shown in Formula I and Formula II below) diluted at different concentrations, add it to each cell culture well according to the cell culture plate layout shown in Table 3, and culture at 35°C for 22 hours.

[0083]

[0084] Table 3: Layout of cell culture plates for virus plaque assay (grey filling represents the addition of compounds of formula I, underlined represents the addition of compounds of formula II; each cell in the table represents a well of a cell culture plate)

[0085]

[0086]

[0087] Plaque color development and determination of virus plaque number:

[0088] 4. Discard the covering solution containing the test compound, add 4% paraformaldehyde (200 μl / well) and place the cell culture plate in a 4°C refrigerator. Discard the 4% paraformaldehyde after 40 minutes.

[0089] 5. Wash the cells twice with PBS (100 μl / well), add permeabilization solution (100 μL / well) and place at room temperature for 30 min;

[0090] 6. Discard the permeabilization solution, wash the cells twice with PBS (100 μl / well), add the primary antibody diluted with ELISA buffer (dilution factor 1:2000, 50 μl / well), and leave at room temperature for 1 hour;

[0091] 7. Discard the primary antibody, wash the cells 3 times with PBST (200 μL / well), add the secondary antibody diluted with ELISA buffer (dilution ratio 1:1000, 50 μl / well), and place at room temperature away from light for 1 hour;

[0092] 8. Discard the secondary antibody, wash the cells 3 times with PBST, add 0.3% H 2 O 2 Trμe Blμe substrate (50μl / well) was placed at room temperature in the dark for 30 min; the results were as follows Figure 1 shown.

[0093] 9. Discard the Trμe Blμe substrate and use ddH 2 Wash once with 5% HO (200 μl / well) and discard ddHO. 2 O, air-dry naturally, and read the original data (reading value) using the ELiSPOT instrument. The results are as follows Figure 1 And as shown in Table 4.

[0094] 10. The dose-effect curve was drawn using the percentage inhibition of the number of virus plaques in the control samples (virus control as positive control, i.e., virus was added but no compound was added; cell control as negative control, i.e., no virus was added but no compound was added). The calculation formula is as follows:

[0095] Virus plaque number inhibition percentage = 100% - (the readings of different dilutions of the compound - the average value of the cell control readings) / (the average value of the virus control readings - the average value of the cell control readings) * 100%

[0096] The results of the virus plaque number inhibition percentage obtained are shown in Table 5.

[0097] 11. Use Graphpad Prism 7 to make curves (such as Figure 2 As shown) and calculate IC 50 Specifically, after the compound concentration is converted into a logarithmic value, a nonlinear regression curve fitting is performed on the converted logarithmic concentration value and the percentage of inhibition of the number of viral plaques (the fitting formula is a four-parameter fitting), and the half inhibition concentration IC is obtained through the above regression curve. 50 . IC of Formula I 50 The value is 1.469 nM, and the IC of formula II 50 The value is 9755nM.

[0098] Table 4: Virus plaque numbers (directly read out by ELiSPOT)

[0099] Compound 1 Duplicate hole 1 Duplicate hole 2 Duplicate hole 3 Compound 2 Duplicate hole 1 Duplicate hole 2 Duplicate hole 3 Cell Control 0 0 0 Cell Control 0 0 0 Virus control 156 171 191 Virus control 203 191 185 158nM 0 0 0 100μM 0 0 0 50nM 0 0 0 31.6μM 92 79 70 15.8nM 19 18 24 10μM 99 127 87 5nM 54 65 76 3.16μM 145 152 128 1.58nM 87 119 88 1μM 142 126 137 0.5nM 132 95 155 0.316μM 174 131 141 0.158nM 150 123 140 0.1μM 154 139 149 0.05nM 141 164 173 0.0316μM 138 117 164

[0100] Table 5: Virus plaque number inhibition percentage (%)

[0101]

[0102]

[0103] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for screening antiviral drugs, comprising the following steps: (1) Cultivating host cells; (2) infecting host cells with the virus and culturing them under conditions suitable for virus and cell growth; (3) adding a covering solution and an appropriate concentration of the candidate drug, and continuing the culture, wherein the covering solution comprises microcrystalline cellulose, a cell culture medium, and TPCK pancreatin; (4) Detect viral antigens by ELISA and obtain raw data by enzyme-linked immunospot technology; (5) Calculate IC based on raw data 50 ;and (6) According to IC 50 Numerical values ​​are used to select candidate drugs.

2. The method for screening antiviral drugs according to claim 1, wherein: In step (1), the host cells are cultured 8-36, 12-24, 8-24 or 8-12 hours before the virus infects the host cells in step (2); Preferably, the culture temperature is 36°C-37°C; Preferably, the cultured cells are washed with a virus maintenance solution once or more (eg, 2, 3, 4 or 5 times).

3. The method for screening antiviral drugs according to any one of claims 1 to 2, wherein: In step (2), the virus is prepared into a suitable concentration using a virus maintenance solution, such as 80-120 PFU per 100 μL or 100 PFU per 100 μL; Preferably, the culture temperature is 35°C-36°C; Preferably, the culture time is 1-5 hours, 1-3 hours or 1-2 hours.

4. The method for screening antiviral drugs according to any one of claims 1 to 3, wherein: In step (3), the microcrystalline cellulose is LOOP:15-375; Preferably, the concentration of microcrystalline cellulose is 1%-5%, 2%-4%, 2%-3% or 2%-2.5%; Preferably, the concentration of TPCK pancreatin is 1-5 μg / ml, 1-3 μg / ml or 1-2 μg / ml; Preferably, the cell culture medium is 2X MEM; Preferably, the culture temperature is 35°C-36°C; Preferably, the culture time is 8-36, 12-24, 22-24 or 12-22 hours.

5. The method for screening antiviral drugs according to any one of claims 1 to 4, wherein: In step (3), Cells to which the virus was added but not the candidate drug were used as positive controls; Preferably, cells without the addition of virus and without the addition of candidate drug serve as negative controls.

6. The method for screening antiviral drugs according to any one of claims 1 to 5, wherein: In step (4), the viral antigen is a viral nucleoprotein, such as an influenza virus nucleoprotein.

7. The method for screening antiviral drugs according to any one of claims 1 to 6, wherein: In step (5), Graphpad Prism 7 was used to generate the curve and calculate the IC 50 ; Preferably, after the concentration of the candidate drug is converted to a logarithmic value, a nonlinear regression curve is fitted between the converted logarithmic concentration value and the percentage of inhibition of the number of viral plaques, and the half inhibition concentration IC is obtained by the above regression curve. 50 .

8. The method for screening antiviral drugs according to claim 7, wherein: The calculation formula for the percentage inhibition of virus plaque number is as follows: The percentage of inhibition of the number of virus plaques = 100% - (the readings of different dilutions of the candidate drug - the average value of the cell control readings) / (the average value of the virus control readings - the average value of the cell control readings) * 100%.

9. The method for screening antiviral drugs according to any one of claims 1 to 8, wherein: The virus is an influenza virus, preferably an influenza A virus such as H1N1 or H3N2 subtype, or an influenza B virus; The antiviral drug is an anti-influenza virus drug; The viral antigen is an influenza virus antigen.

10. The method for screening antiviral drugs according to any one of claims 1 to 9, wherein: The host cell is a dog kidney cell.

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