CN112592953A - High-throughput screening method for human adenovirus proliferation inhibition drug and application thereof - Google Patents

High-throughput screening method for human adenovirus proliferation inhibition drug and application thereof Download PDF

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CN112592953A
CN112592953A CN202011514304.1A CN202011514304A CN112592953A CN 112592953 A CN112592953 A CN 112592953A CN 202011514304 A CN202011514304 A CN 202011514304A CN 112592953 A CN112592953 A CN 112592953A
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谷峰
商璐
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Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
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Abstract

The invention discloses a high-throughput screening method of a medicament for inhibiting human adenovirus proliferation and application thereof. The human adenovirus marked with GFP in the patent can express green fluorescent protein after infecting HEK-293 cells, so that the infected cells are green under a fluorescent microscope. The medicinal activity of anti-human adenovirus is realized by detecting the change of the adenovirus content in the supernatant of the culture medium after adding the anti-adenovirus medicament and the human adenovirus to HEK-293 cells by using flow cytometry. Specifically, a proper amount of virus supernatant is added into a certain amount of HEK-293 cells again, and the ratio of cells infected by adenovirus (cells with green fluorescence) in the cells is determined by flow cytometry, so as to evaluate whether the added medicine has obvious inhibition effect on the proliferation of adenovirus. The method can be used for screening whether the candidate drug molecules have the effect of inhibiting the human adenovirus or not in vitro quickly, simply, conveniently, cheaply, effectively and at high flux. The patent can accelerate the research and development of novel anti-human adenovirus drugs.

Description

High-throughput screening method for human adenovirus proliferation inhibition drug and application thereof
Technical Field
The invention relates to a high-flux screening method for inhibiting human adenovirus proliferation by a medicament and application thereof, which can screen whether the medicament has the effect of inhibiting human adenovirus proliferation or not in vitro quickly, simply, conveniently, effectively and at high flux and is beneficial to the research and development of novel anti-adenovirus medicaments.
Background
Human adenoviruses are linear, double-stranded, icosahedral DNA viruses, and up to now 70 kinds of human adenoviruses have been identified and classified into 7 kinds (a-G) according to their genetic relationship. Different adenovirus species have specific organ preference, and the C, E, B type virus infects respiratory tract; some class B viruses infect the urethra; the infection target organs of a and F are the gastrointestinal tract; class D primarily infects the eye, which may be associated with different receptors for different adenoviruses. Class C ( serology 1, 2, 5) causes mainly mild infections in young children; class B ( serology 3, 7, 14, 21) and E mainly cause adult infections. Most adenoviral infections are probably subclinical, but the infection can manifest as relatively mild upper respiratory disease, as well as more severe bronchiolitis and pneumonia, as well as diarrhea and conjunctivitis. Currently, there are no approved antiviral drugs for specific treatment of adenoviral infections. Although Cidofovir (Cidofovir, C) and brincidifovir are used for adenovirus therapy. Cidofovir has severe nephrotoxicity. Brincidovir (B) is a phospholipid conjugate of cidofovir and is mainly used for treating diseases caused by adenovirus infection in clinic. Brincidovir has fewer toxic and side effects, is better than cidofovir, but intestinal toxicity may occur in some patients. Therefore, the development of new, highly effective, low toxic and side effects anti-adenovirus drugs is still a great clinical need for the treatment of adenovirus diseases. At present, tests for determining the inhibitory effect of antiviral drugs on adenovirus mainly comprise immunofluorescence quantification for determining virus content, immunofluorescence and virus TCID50Titer, viral plaque reduction assay, IC50(half inhibitory concentration). When the method is used for high-throughput screening of the anti-adenovirus drugs, the time consumption is long, the workload is large, and the cost is high. How to screen out candidate compounds with the anti-adenovirus effect in vitro with low cost, high efficiency, rapidness and high throughput is the key for developing anti-adenovirus drugs. And a rapid, simple, convenient, effective and high-flux screening platform is established to screen whether the medicament has the effect of inhibiting the proliferation of the adenovirus, which is beneficial to screening new anti-adenovirus medicaments and is new anti-adenovirus medicamentsProvides an effective screening platform. The invention utilizes anti-human adenovirus drugs Cidofovir and Brincidovir as positive drugs to evaluate the effect of the screened drugs.
Disclosure of Invention
The invention aims to establish a high-throughput screening method for determining the inhibition of adenovirus proliferation by a drug, solves the problems of large workload, long time consumption and high cost when the conventional method is used for detecting the inhibition of the drug to adenovirus at high throughput, is beneficial to screening of new anti-adenovirus drugs, and provides an effective screening platform for the research and development of the new anti-adenovirus drugs.
Specifically, the plasmid pAd-YFP is constructed by utilizing pAdTrack-CMV and pAdEasy-1 plasmids, and the plasmid pAd-YFP is transfected into 293FT cells to obtain the GFP-integrated human adenovirus, wherein the specific method refers to https:// media. Briefly, the method comprises the steps of: the plasmid pAdTrack-CMV was linearized with PmeI restriction endonuclease. The linearized pAdTrack-CMV and the adenovirus backbone vector pAdEasy-1 plasmid were co-electroporated into E.coli BJ5183 cells. And (3) carrying out monoclonal screening by using kanamycin, carrying out enzyme digestion identification on the obtained plasmid, transforming the correctly identified plasmid into DH10B competence, picking out a monoclonal, and extracting to obtain the plasmid pAd-YFP. The plasmid pAd-YFP is linearized by using a restriction enzyme PacI, and transfected into 293FT cells by using lipofectamine, and the obtained virus is infected into the cells to obtain adenovirus with higher concentration for subsequent experimental study. After adenovirus infection, the infected cells were green under a fluorescent microscope, which indicated that the obtained adenovirus was a recombinant adenovirus.
The adenovirus can express green fluorescent protein after infecting cells, and the infected adenovirus cells are green and can be used for flow screening. The high-throughput screening method for inhibiting the proliferation of the adenovirus by the medicine comprises the steps of adding the virus supernatant of the 4 th day after the virus infection into the paved HEK-293 cells again, detecting the proportion of green fluorescent cells by flow cytometry at the 2 nd day after the virus addition, and establishing a medicine-adding-free group (NC) which is not infected, a medicine-adding-free group (PC) which is only infected and not added and a medicine-adding group (different doses).
Furthermore, the invention provides a high-throughput screening method for simply and conveniently determining the inhibition of adenovirus proliferation by an anti-adenovirus drug, which mainly comprises the steps of detecting the titer of adenovirus, carrying out MTT test on HEK-293 cells by a positive drug (Brincidovir, Cidofovir) and establishing the high-throughput screening method for the inhibition of adenovirus proliferation by the drug. The establishment of the high-throughput screening method for inhibiting the proliferation of the adenovirus by the drug mainly researches the influence of different adding time points, different dosages, different sampling points and cell densities of the drug on the proliferation of the adenovirus.
The method comprises the following steps:
1. preparation of GFP-integrated adenovirus.
2. The adenovirus titer is detected by using the TCID50 method.
3. MTT assay of HEK-293 cells with positive drugs (Brincidovir, Cidofovir) was performed using the MTT kit according to the instructions.
4. High-throughput screening method for inhibiting adenovirus proliferation by drug
1) Effect of different addition time points of drugs on adenovirus proliferation
Mainly designs the drug Brincidovir 1000nM at 8 h, 6 h, 4 h, 2h and 0 h before adding the virus and at 2h, 4 h and 6 h after adding the virus, and measures the number of green cells in the cells at 4d after the virus infection and the number of green cells in the cells after the HEK-293 cells are infected again by the culture medium supernatant at 4 d.
2) Effect of different doses of drugs on adenovirus proliferation
The drug concentration of brincidiofovir was set at 5000 nM; 1000 nM; 500 nM; 100 nM; the drug concentration of Cidofovir is set to 200 mu M; 100 mu M; 50 mu M; 25 μ M. After HEK-293 cells were infected again with virus supernatants taken 4d after drug and virus addition and 4d, the proportion of green cells in the cells was analyzed by flow analysis, respectively. Initially selecting the concentration of the positive drug for inhibiting the proliferation of the adenovirus.
3) Time points for virus supernatant collection and cell density determination for the second plating.
Adding the culture supernatants of 2d and 4d after adding medicine and virus into HEK-293 cells laid 1d in advance with 24-well plate at cell density of 5 × 104Cell/well and 1X 105Cells/well. Flow analysis was performed 2d after the supernatant infected cells. The effect of time point for harvesting viral supernatant and cell density of the second plating on adenovirus proliferation was determined.
4) Effect of different doses of drugs on adenovirus proliferation Using 48-well plates
When 48 wells were used for screening, the number of cells plated per well was half that of 24-well plates. Cell supernatants at 3d, 4d and 5d after virus infection were selected to re-infect HEK-293 cells for 2d flow analysis. The time point for collecting viral supernatants was determined when screening was performed using 48-well plates.
Advantageous effects
The method provided by the invention enables simultaneous screening of hundreds of new synthesized compounds, and can screen thousands of compounds in a small molecule library in a short time.
Meanwhile, the method provided by the invention can realize simultaneous screening of 200 compounds each time, and can be completed within 3h during flow cytometry analysis, and the whole experimental process needs 8 d. The TCID50 method, the viral plaque reduction assay, and the IC50 (semi-inhibitory concentration) method can only detect a few compounds at a time of 4-5 days, and if a plurality of compounds are to be detected, a large amount of manpower and material resources are required. The high-throughput detection can be realized by using a fluorescence quantitative method for detection, but primers are required to be designed and probes are required to be synthesized, at most 96 samples can be made in each batch, and 200 samples can be made at least 2 times. Takes more than 3 hours and is expensive.
Drawings
FIG. 1: MTT assay results on HEK-293 cells at different doses of Brincidovir and Cidofovir.
A: MTT assay results for HEK-293 cells with Brincidovir at 5-2000 nM;
b: MTT test results of 10-200 μ M Cidofovir on HEK-293 cells.
FIG. 2: effect of drug addition of Brincidovir 1000nM at different times before and after addition of the virus on adenovirus proliferation.
A: effect of dosing Brincidovir 1000nM at different time points on adenovirus proliferation in HEK-293 cells at 4d after virus addition;
b: at 4d after adding the virus, the result of the proliferation inhibition of the adenovirus in HEK-293 cells by adding Brincidovir 1000nM at different time points;
c: proliferation of virus in HEK-293 cells in culture supernatant supplemented with the drug Brincidovir 1000nM, 4d at different dosing time points;
d: proliferation inhibition of virus in HEK-293 cells by adding the drug Brincidovir 1000nM, 4d culture supernatant at different dosing time points.
FIG. 3: results of various doses of drug inhibiting proliferation of adenovirus.
A: effect of different doses of Brincidovir on adenovirus propagation in HEK-293 cells (4 d).
B: effect of adenovirus propagation in HEK-293 cells in the virus supernatant with different doses of Brincidovir added (2 d).
C: the adenovirus in the virus supernatant was inhibited from proliferating in HEK-293 cells by adding different doses of Brincidovir (2 d).
D: effect of different doses of Cidofovir on adenovirus propagation in HEK-293 cells (4 d).
E: effect of adenovirus propagation in HEK-293 cells in the viral supernatant with the addition of different doses of Cidofovir (2 d).
F: as a result of the inhibition of adenovirus proliferation in HEK-293 cells in the virus supernatant with the addition of various doses of Cidofovir (2 d).
FIG. 4: the effect of different time points for virus supernatant collection and cell density of the second HEK-293 cell plating on the adefovir proliferation with different doses of drug.
A: after adding different doses of Brincidovir and virus infected cells, 100 mu L of culture medium supernatant of the 2 nd day is taken and is paved with 5 x 10 cells 1d ahead of time4Cells HEK-293 cells, proliferation of adenovirus in supernatant in HEK-293 cells.
B: after adding different doses of Brincidovir and virus infected cells, 100 mu L of culture medium supernatant of the 2 nd day is taken and is paved with 1 x 10 cells before infection for 1d5Cells HEK-293 cells, proliferation of adenovirus in supernatant in HEK-293 cells.
C: after adding different doses of Brincidovir and virus infected cells, 100 mu L of culture medium supernatant of the 4d is taken and is paved with 5 x 10 cells 1d ahead of time4Cells HEK-293 cells, proliferation of adenovirus in supernatant in HEK-293 cells.
D: after adding different doses of Brincidovir and virus infected cells, 100 mu L of culture medium supernatant of the 4d is taken and is paved with 1 x 10 cells before infection for 1d5Cells HEK-293 cells, proliferation of adenovirus in supernatant in HEK-293 cells.
E: after adding Cidofovir with different doses and virus infected cells, 100 mu L of culture medium supernatant of 2d is taken and is paved with 5 multiplied by 10 after 1d of infection4Cells HEK-293 cells, proliferation of adenovirus in supernatant in HEK-293 cells.
F: after adding Cidofovir with different doses and virus infected cells, 100 mu L of culture medium supernatant of 2d is taken and is paved with 1 x 10 cells 1d in advance5Cells HEK-293 cells, proliferation of adenovirus in supernatant in HEK-293 cells.
G: after adding Cidofovir with different doses and virus infected cells, 100 mu L of culture medium supernatant of the 4d is taken and is paved with 5 multiplied by 10 after 1d of infection4Cells HEK-293 cells, proliferation of adenovirus in supernatant in HEK-293 cells.
H: after adding Cidofovir with different doses and virus infected cells, 100 mu L of culture medium supernatant of the 4d is taken and is paved with 1 x 10 cells 1d in advance5Cells HEK-293 cells, proliferation of adenovirus in supernatant in HEK-293 cells.
FIG. 5: screening the drug by using a 48-well plate, collecting 50 muL of culture medium supernatant at 3d, 4d and 5d after adding the drug and the virus, and infecting HEK-293 cells (5 multiplied by 10) again4) The proliferation status of (2).
A: adding different doses of Brincidovir, taking the culture medium supernatant 3d after virus infection to infect HEK-293 cells again for 2d, and obtaining the proliferation condition of adenovirus in the supernatant.
B: adding different doses of Brincidovir, taking the culture medium supernatant 4d after virus infection to infect HEK-293 cells again for 2d, and obtaining the proliferation condition of adenovirus in the supernatant.
C: adding different doses of Brincidovir, taking the culture medium supernatant at 5d after virus infection to infect HEK-293 cells for 2d again, and multiplying the adenovirus in the supernatant.
D: adding Cidofovir with different doses, taking the culture medium supernatant after the virus infection for 3d to infect HEK-293 cells for 2d again, and multiplying the adenovirus in the supernatant.
E: adding Cidofovir with different doses, taking the culture medium supernatant 4 days after virus infection to infect HEK-293 cells again for 2d, and obtaining the proliferation condition of adenovirus in the supernatant.
F: adding Cidofovir with different doses, taking the culture medium supernatant after the virus infection for 5 days to infect HEK-293 cells for 2 days again, and proliferating the adenovirus in the supernatant.
FIG. 6: fluorescence patterns of 4d and virus in culture supernatant re-infected HEK-293 cells after addition of different doses of drugs and viruses when drug screening was performed with 48-well plates.
A, a fluorescence map of 4d after adding different doses of drugs and viruses;
b fluorescent map of the HEK-293 cells reinfected with the virus in the culture supernatant (50 μ L) of 4 d.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention. The invention will now be further described with reference to examples and the accompanying drawings.
The following examples are carried out in a conventional manner unless otherwise specified.
Example 1 establishment of a high throughput screening method for determining inhibition of human adenovirus proliferation by drugs
1. Preparation of GFP-integrated human adenovirus
Plasmid pAd-YFP (SEQ ID NO.1) was constructed using pAdTrack-CMV (https:// www.addgene.org/16405 /) and pAdEasy-1 (https:// www.addgene.org/16400 /) plasmids, and plasmid pAd-YFP was transfected into 293FT cells to obtain GFP-integrated human adenovirus, in the specific manner referred to https:// media. addge.org/data/49/60/162 ad396-af64-11e0-90fe-003048dd6500. pdf. Briefly, the method is:
1) the plasmid pAdTrack-CMV was linearized with PmeI restriction endonuclease.
2) The linearized pAdTrack-CMV and the adenovirus backbone vector pAdEasy-1 plasmid were co-electroporated into E.coli BJ5183 cells. And (3) carrying out monoclonal screening by using kanamycin, carrying out enzyme digestion identification on the obtained plasmid, transforming the correctly identified plasmid into DH10B competence, picking out a monoclonal, and extracting to obtain the plasmid pAd-YFP.
3) The plasmid pAd-YFP is linearized by using a restriction enzyme PacI, and transfected into 293FT cells by lipofectamine, and the obtained virus is repeatedly infected into the cells to obtain adenovirus with higher concentration for subsequent experimental study.
4) After infection of the cells, the cells were shown to be green under a fluorescent microscope, and these were determined to be recombinant adenoviruses.
2. Adenovirus titer detection
1) Digesting HEK-293 cells in logarithmic growth phase by trypsin, counting the cells, inoculating the cells into a 96-well plate, and enabling the cell density to be 1 multiplied by 104Cells/well. Culture with DMEM containing 10% FBS.
2) After 24 h, after 10-fold serial dilution of the provirus in DMEM medium containing 2% FBS was added to each well, 100 μ L of the provirus was added to each well, and 6 replicates per concentration were added. Proviral dilution gradient of 10-1，10-2，10-3，10-4，10-5，10-6，10-7，10-8，10-9，10-10. 2d after addition of the virus, the number of wells containing green fluorescent cells was recorded under a fluorescent microscope and the TCID50 of the virus was calculated. The statistical results are shown in Table 1, and the calculated virus TCID50 is 10 7.3 TCID50/mL。
TABLE 1 statistics of results of adenovirus titer detection
Figure 893215DEST_PATH_IMAGE002
3. Toxicity testing of the antiviral drugs Brincidovir (B) and Cidofovir (C) against HEK-293
1) Digesting HEK-293 cells in logarithmic growth phase by trypsin, counting the cells, inoculating the cells into a 96-well plate, and enabling the cell density to be 1 multiplied by 104Cells/well. Culture with DMEM containing 10% FBS.
2) After 24 h, the drug concentrations of Brincidovir were 10 nM, 50 nM, 100nM, 250 nM, 500 nM, 750 nM, 1000nM, 1500 nM, 2000nM, respectively. The drug concentrations added with Cidofovir are respectively 10 muM, 25 muM, 50 muM, 75 muM, 100 muM, 150 muM and 200 muM. 3 replicate wells per concentration, 3 zero-adjusted wells containing medium alone and no cells were set simultaneously, and 3 control wells containing the same number of cells as the test group, the same medium but no drug.
3) And adding 10 muL of MTT solution into each well at 48 h according to the MTT kit specification, incubating for 4 h at 37 ℃, adding 100 muL of Formazan dissolving solution, incubating for 4 h, measuring absorbance at 570 nm, and counting whether the added medicine has toxicity to HEK-293 cells. ,
4) the results of the MTT assay are shown in FIG. 1, from which Brincidovir was found to be non-toxic to HEK-293 cells at concentrations ranging from 10 nM to 2000 nM. Cidofovir is not toxic to HEK-293 cells in the range of 10-200. mu.M.
4. Establishment of high-throughput screening method for inhibiting proliferation of adenovirus by drug
1) Determination of different dosing time points.
1d, trypsinizing HEK-293 cells in logarithmic growth phase, counting the cells and plating them in 24-well plates at a cell density of 5X 104Cells/well. Culture with DMEM containing 10% FBS.
At 2d, the antiviral drug Brincidovir was added at 1000nM at different time points. The adding time points are 8 h, 6 h, 4 h, 2h and 0 h before adding the virus and 2h, 4 h and 6 h after adding the virus. The virus addition was MOI = 0.1. A virus-free and drug-free group (NC) and a virus-free and drug-free group (PC) were set, and the test groups were as follows: the virus, Brincidovir 1000nM, and the drug combination at different time points.
At 6 d, 4d after virus addition, supernatant was aspirated 100 μ L per well into 24-well plates (5X 10) in which HEK-293 cells had been plated4Cells/well, day 5d plating). The first plated cells were subjected to flow analysis, and the results are shown in FIG. 2A, and the inhibition rate is shown in FIG. 2B.
The cells after the addition of the virus supernatant were cultured for 48 hours and then subjected to flow assay. The results are shown in FIG. 2C, and the inhibition results are shown in FIG. 2D.
The test results show that: the influence of the medicine addition at different time points on adenovirus infected cells and virus proliferation is small, however, after the medicine addition, the virus content in the supernatant of the culture medium is obviously changed, and when HEK-293 cells are infected again, the virus content is obviously reduced.
2) Preliminary determination of the concentration of the positive control drug addition.
1d, trypsinizing HEK-293 cells in logarithmic growth phase, counting the cells and plating them in 24-well plates at a cell density of 5X 104Cells/well. Culture with DMEM containing 10% FBS.
And 2d, setting a virus-free drug-free group (NC) and a virus-free drug-free group (PC), wherein the test group comprises: adding virus and adding drug groups with different concentrations; the drug concentration of brincidiofovir was set at 5000 nM; 1000 nM; 500 nM; 100 nM; the drug concentration of Cidofovir is set to 200 mu M; 100 mu M; 50 mu M; 25 μ M. Drugs and viruses were added as per experimental design, with the MOI =0.1 for adenovirus.
At 6 d, 4d after virus addition, supernatant was aspirated 100 μ L per well into 24-well plates (5X 10) in which HEK-293 cells had been plated4Cells/well, day 5d plating). The first plated cells were subjected to flow analysis and the results are shown in FIGS. 3A, 3D.
The cells after the addition of the virus supernatant were cultured for 48 hours and then subjected to flow assay. The results are shown in FIGS. 3B, 3E; the inhibition results are shown in FIGS. 3C and 3F.
The test results show that: the drug with different doses is added, and after the virus infects HEK-293 cells for the first time, the inhibitory action of the drug with different doses on the virus cannot be distinguished obviously by flow cytometry. After the drugs and the viruses are added for 4d, the HEK-293 cells are infected by the culture medium supernatant again, so that whether the antiviral drugs with different doses have the inhibiting effect on the adenoviruses can be obviously distinguished. In addition, the test clearly determines that Brincidovir is used as a positive control drug at a dose of 500 nM or less and Cidofovir is used as a positive control drug at a dose of 50 μ M or less.
3) Time points for virus supernatant collection and determination of cell density of 24-well plate plating after drug and virus addition.
1d, trypsinizing HEK-293 cells in logarithmic growth phase, counting the cells and plating them in 24-well plates at a cell density of 5X 104Cells/well. Culture with DMEM containing 10% FBS.
And 2d, setting a virus-free drug-free group (NC) and a virus-free drug-free group (PC), wherein the test group comprises: adding virus and adding drug groups with different concentrations; the drug concentration of brincidiofovir was set at 500 nM; 100 nM; 50 nM; 10 nM, the drug concentration of Cidofovir is set to 100 μ M; 50 mu M; 25 mu M; 10 mu M. Drugs and viruses were added according to the experimental design, 2h after drug addition, and the MOI of adenovirus = 0.1.
And 4d, namely 2d after adding the virus, sucking 100 mu L of supernatant per well to a 24-well plate paved with HEK-293 cells 1 day earlier. The cell density during plating was 5X 104Cell/well and 1X 105Cells/well.
And 6 d, namely 4d after adding the virus, sucking 100 mu L of supernatant per well to a 24-well plate paved with HEK-293 cells 1 day earlier. The cell density during plating was 5X 104Cell/well and 1X 105Cells/well.
The cells added with the virus supernatant were cultured for 48 hours and then subjected to flow assay. The results are shown in FIG. 4.
The test results show that: adding adenovirus, collecting virus supernatant at 4d, and plating to obtain cell density of 1 × 105The cells/pores can better display the inhibitory effect of drugs with different concentrations on adenovirus.
4) High throughput screening of drugs to inhibit adenovirus proliferation was performed using 48-well plates.
On the 1 st day, HEK-293 cells in logarithmic growth phase were trypsinized, counted and plated in 48-well plates at a cell density of 2.5X 104Cells/well. Culture with DMEM containing 10% FBS.
And 2d, setting a virus-free drug-free group (NC) and a virus-free drug-free group (PC), wherein the test group comprises: adding virus and adding drug groups with different concentrations; the drug concentration of brincidiofovir was set at 500 nM; 250 nM; 100 nM; 50 nM; 25 nM; 10 nM; 5 nM, the drug concentration of Cidofovir is set to 200 μ M; 100 mu M; 50 mu M; 25 mu M; 10 mu M. Drugs and viruses were added according to the experimental design, 2h after drug addition, and the MOI of adenovirus = 0.1.
And 5d, namely 3d after adding the virus, sucking 100 mu L of supernatant per well to a 48-well plate paved with HEK-293 cells 1 day earlier. The cell density during plating was 5X 104Cells/well.
And 6 d, namely 4d after adding the virus, sucking 100 mu L of supernatant per well to a 48-well plate paved with HEK-293 cells 1 day earlier. The cell density during plating was 5X 104Cells/well.
And 7 d, namely 5d after adding the virus, sucking 100 mu L of supernatant per well to a 48-well plate paved with HEK-293 cells 1 day earlier. The cell density during plating was 5X 104Cells/well.
The cells added with the virus supernatant were cultured for 48 hours and then subjected to flow assay. The results are shown in FIG. 5, and the fluorescence of the infection of HEK-293 cells with adenovirus is shown in FIG. 6.
The test results show that: after the adenovirus is added, the virus supernatants are taken at the 4 th and 5 th days for the second virus amplification, so that the inhibition effect of the medicaments with different concentrations on the adenovirus can be well displayed. And selecting and adopting the virus supernatant of the 4d according to a principle of short time consumption.
5) The high throughput screening flow cytometry method for inhibiting adenovirus proliferation by using the medicine comprises the following specific steps:
24-pore plate screening method
No. 1d, 24-well plate, 5X 10 HEK-293 cells per well4(i.e. the1mL of the medium containing 5X 104 cells/mL, pipette 1mL into 24-well plates).
And 2d, adding the medicine 24 hours after the plate is paved. The test should have (1) a blank control group (no drug, no virus group), (2) a positive virus control group (only virus, no drug group), (3) a positive drug group (virus, positive drug, Cidofovir (50 μ M) and Brincidovir (250 nM)), (4) a test group (virus, drug to be screened).
2h after dosing, virus was added, 2 μ L adenovirus per well (MOI = 0.1). The blank control group was virus-free.
5d, after virus addition, 3d, cell plating: 24 well plate, 1X 10 per well5 HEK-293 cells (i.e., 1X 10 in 1mL of medium)5 cells/mL, pipette 1mL into 24-well plates). Consistent with the number of wells in the first plating.
And 6 d, taking 100 mu L of virus supernatant of each hole of the first time of plate paving, and putting the virus supernatant into the cells of the second time of plate paving in the same order as the first time.
And 8d, namely adding virus and then 2d, discarding the supernatant of the 2 nd cell plate, adding 200 muL of culture medium for neutralization, repeatedly blowing and beating, performing flow analysis in a suction flow type pipe, and recording the detection result of each sample.
And (3) comparing the flow result of the drug to be tested with a positive drug group (Cidofovir Cidofovir (50 mu M) and Brincidovir (250 nM)), and analyzing whether the drug to be tested has the effect of inhibiting the adenovirus.
② 48 pore plate screening method
No. 1d, 48-well plate, HEK-293 cells 2.5X 10 cells per well4(i.e., 1mL of the medium containing 5X 104 cells/mL, aspirate 500 μ L into 48-well plates).
And 2d, adding the medicine 24 hours after the plate is paved. The test should have (1) a blank control group (no drug, no virus group), (2) a positive virus control group (only virus, no drug group), (3) a positive drug group (virus, positive drug, Cidofovir (50 μ M) and Brincidovir (250 nM)), (4) a test group (virus, drug to be screened).
2h after dosing, virus was added, 2 μ L adenovirus per well (MOI = 0.1). The blank control group was virus-free.
5d, after virus addition, 3d, cell plating: 48-hole plate with 5X 10 holes4 HEK-293 cells (i.e., 1X 10 in 1mL of medium)5 cells/mL, aspirate 500 μ L into 48-well plates). Consistent with the number of wells in the first plating.
And 6 d, taking 50 mu L of the virus supernatant of each hole of the first time of plate paving, and putting the virus supernatant into the cells of the second time of plate paving in the same order as the first time.
And 8d, namely adding virus and then 2d, discarding the supernatant of the 2 nd cell plate, adding 200 muL of culture medium for neutralization, repeatedly blowing and beating, performing flow analysis in a suction flow type pipe, and recording the detection result of each sample.
And (3) comparing the flow result of the drug to be tested with a positive drug group (Cidofovir Cidofovir (50 mu M) and Brincidovir (250 nM)), and analyzing whether the drug to be tested has the effect of inhibiting the adenovirus.
Using this method, we found the results of the 39 drugs in the natural compound library in Table 2.
Table 2: flow-through results of detection of 39 drugs in natural compound library
Sample name GFP % Sample name GFP % Sample name GFP %
NC
1 0.0 6 27.9 23 17.6
NC 2 0.0 7 57.2 24 50.4
NC 3 0.0 8 44.6 25 61.1
PC 1 50.3 9 50.3 26 39.0
PC 2 56.2 10 17.6 27 30.6
PC 3 65.2 11 12.4 28 38.5
Brincidofovir (250 nM) 3.5 12 62.7 29 15.7
Brincidofovir (250 nM) 4.3 13 36.2 30 40.4
Brincidofovir (250 nM) 3.3 14 35.7 31 62.9
Cidofovir (50 µM) 1.0 15 48.5 32 54.2
Cidofovir (50 µM) 1.1 16 33.2 33 60.4
Cidofovir (50 µM) 1.3 17 41.8 34 59.3
1 45.1 18 36.8 35 2.1
2 30.9 19 39.1 36 40.7
3 19.3 20 1.2 37 42.6
4 7.0 21 13.9 38 4.9
5 20.2 22 14.2 39 1.9
GFP%: the percentage of adenovirus infected HEK-293 cells is indicated.
From the above results, compared with the adenovirus inhibiting effect of the anti-adenovirus positive drugs Cidofovir (50 μ M) and brincididovir (250 nM), the GFP expression levels of the drugs 20, 35, 38 and 39 are lower than or close to the positive control drug, and the proliferation of adenovirus may be inhibited, and subsequently, the screened drug needs to be rescreened and the toxicity of the drug on cell proliferation is observed, and if the drug has obvious toxicity to inhibit cell proliferation, whether the detection can be continued after the drug concentration is reduced can be considered. The method can be used for preliminarily screening the medicaments with obvious inhibiting effect on the proliferation of the adenovirus in high flux.
The applicant states that the present invention is illustrated by the above examples to show the high throughput screening method and application of the drug for inhibiting human adenovirus proliferation of the present invention, but the present invention is not limited to the above examples, i.e., it does not mean that the present invention is implemented only by relying on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Sequence listing
<110> Shanghai animal doctor institute of Chinese academy of agricultural sciences (Shanghai center of Chinese centers of animal health and epidemiology)
High flux screening method for medicine for inhibiting human adenovirus proliferation and its application
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〈170〉PatentInversion3.3
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 37387
<212> pAd-YFP
<213> Artificial Sequence (Artificial Sequence)
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Claims (10)

1. A high-throughput screening method for a medicament for inhibiting the proliferation of human adenoviruses is characterized by comprising the following steps: 1) preparation of GFP-integrated human adenovirus; 2) detection of human adenovirus titer using TCID50Detecting by the method; 3) MTT assay of HEK-293 cells with positive drugs (Brincidovir, Cidofovir) using MTT kit, according to its instructions; 4) high-throughput screening of drugs for inhibiting human adenovirus proliferation.
2. The method of claim 1, wherein: the preparation method of the human adenovirus integrated with GFP comprises the steps of constructing a plasmid pAd-YFP by using pAdTrack-CMV and pAdEasy-1 plasmids, and transfecting the plasmid pAd-YFP to 293FT cells to obtain the human adenovirus integrated with GFP.
3. The method of claim 1, wherein the screening method of step 4) comprises a 24-well plate screening method or a 48-well plate screening method.
4. The method of claim 3, wherein the 24-well plate screening method is specifically:
no. 1d, 24-well plate, 5X 10 HEK-293 cells per well4(i.e., 1mL of the medium containing 5X 104 cells/mL, pipette 1mL into 24-well plates);
2d, adding the medicine 24 hours after the plate is paved; the test should have (1) blank control group (no medicine, no virus group), (2) positive virus control group (only virus, no medicine group), (3) positive medicine group (virus, positive medicine, Cidofovir (50 μ M) and Brincidovir (250 nM)), (4) test group (virus, medicine to be screened);
2h after dosing, adding the virus, and adding 2 muL of human adenovirus (MOI = 0.1) into each well; the blank control group was virus-free;
5d, after virus addition, 3d, cell plating: 24 well plate, 1X 10 per well5 HEK-293 cells (i.e., 1X 10 in 1mL of medium)5 cells/mL, pipette 1mL into 24-well plates); the number of the cells is consistent with that of the cells paved for the first time;
taking 100 mu L of virus supernatant per hole of the first-time plate paving to the cells of the second-time plate paving, wherein the sequence is consistent with that of the first-time plate paving;
8d, namely 2d after adding the virus, discarding the supernatant of the 2 nd paved cell plate, digesting the cell plate by 100 muL of pancreatin, adding 200 muL of culture medium for neutralization, repeatedly blowing and beating, sucking the cell plate into a flow tube for flow analysis, and recording the detection result of each sample;
comparing the flow result of the drug to be tested with a positive drug group (Cidofovir Cidofovir (50 mu M) and Brincidovir (250 nM)), and analyzing whether the drug to be tested has the effect of inhibiting human adenovirus.
5. The method of claim 3, wherein the 48-well plate screening method is specifically:
no. 1d, 48-well plate, HEK-293 cells 2.5X 10 cells per well4(i.e., 1mL of the medium containing 5X 104 Sucking 500 mu L of cells/mL into a 48-hole plate);
2d, adding the medicine 24 hours after the plate is paved; the test should have (1) blank control group (no medicine, no virus group), (2) positive virus control group (only virus, no medicine group), (3) positive medicine group (virus, positive medicine, Cidofovir (50 μ M) and Brincidovir (250 nM)), (4) test group (virus, medicine to be screened);
adding viruses 2h after dosing, and adding 2 muL of adenovirus per well (MOI = 0.1); the blank control group was virus-free;
5d, after virus addition, 3d, cell plating: 48-hole plate with 5X 10 holes4 HEK-293 cells (i.e., 1X 10 in 1mL of medium)5 Sucking 500 mu L of cells/mL into a 48-hole plate); the number of the cells is consistent with that of the cells paved for the first time;
taking 50 mu L of virus supernatant per hole of the first-time plate paving to the cells of the second-time plate paving, wherein the sequence is consistent with that of the first-time plate paving;
8d, namely 2d after adding the virus, discarding the supernatant of the 2 nd paved cell plate, digesting the cell plate by 100 muL of pancreatin, adding 200 muL of culture medium for neutralization, repeatedly blowing and beating, sucking the cell plate into a flow tube for flow analysis, and recording the detection result of each sample;
comparing the flow result of the drug to be tested with a positive drug group (Cidofovir Cidofovir (50 mu M) and Brincidovir (250 nM)), and analyzing whether the drug to be tested has the effect of inhibiting human adenovirus.
6. High throughput screening method according to claim 4, wherein the number of HEK-293 cells of the first 24-well plate plating is 5 x 104Second, 24-well plates, the number of cells plated was 1X 105。
7. The high-throughput screening method for inhibiting proliferation of human adenovirus according to claim 5, wherein the number of HEK-293 cells plated in 48-well plates for the first time is 2.5X 104Second, 48-well plates, the number of cells plated was 5X 104。
8. The high-throughput screening method according to claim 6, wherein the amount of culture medium supernatant is taken as 100 μ L in 24-well plate plating screening.
9. The high-throughput screening method according to claim 7, wherein the amount of culture medium supernatant taken in the 48-well plate plating screening is 50 μ L.
10. High throughput screening method according to claim 8 or 9, wherein the dose of human adenovirus per well is MOI = 0.1; the positive control drug Brincidovir was used at a concentration of 250 nM and Cidofovir at a concentration of 50 μ M.
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