CN112592953B - High-throughput screening method of human adenovirus proliferation inhibiting drug and application thereof - Google Patents

Abstract

The invention discloses a high-throughput screening method of a drug for inhibiting human adenovirus proliferation and application thereof. The human adenovirus labeled 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 anti-human adenovirus drug activity was achieved by detecting changes in adenovirus content in culture medium supernatants of HEK-293 cells after addition of anti-adenovirus drug and human adenovirus using flow cytometry. Specifically, a proper amount of virus supernatant was added again to a certain amount of HEK-293 cells, and the ratio of adenovirus-infected cells (cells with green fluorescence) was measured by flow cytometry to evaluate whether the added drug had a significant inhibitory effect on adenovirus proliferation. The method can be used for rapidly, simply, conveniently, cheaply and effectively screening whether candidate drug molecules have the effect of inhibiting human adenovirus in vitro. The patent accelerates the research and development of novel anti-human adenovirus medicaments.

Description

High-throughput screening method of human adenovirus proliferation inhibiting drug and application thereof

Technical Field

The invention relates to a high-throughput screening method for inhibiting human adenovirus proliferation by using a drug and application thereof, which can screen whether the drug has the effect of inhibiting human adenovirus proliferation in vitro rapidly, simply, conveniently and effectively with high throughput and is beneficial to research and development of novel anti-adenovirus drugs.

Background

Human adenoviruses are linear, double-stranded, icosahedral DNA viruses, and so far 70 human adenoviruses have been identified, and are classified into 7 (A-G) according to their genetic relatives. Different adenovirus species have specific organ preference, and C, E, B viruses infect respiratory tract; some B-type diseasesToxic infection of the urethra; the target organ of infection of a and F is the gastrointestinal tract; class D primarily infects the eye, which may be associated with different adenovirus receptors. Class C ( serology 1, 2, 5) mainly causes minor infections in young children; class B ( serology 3, 7, 14, 21) and class E mainly cause adult infections. Most adenovirus infections may be subclinical, but infections may exhibit 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 the specific treatment of adenovirus infections. Although Cidofovir (C) and brincdofovir are used for adenovirus therapy. Cidofovir has severe renal toxicity. Brincidofovir (B) is a phospholipid conjugate of cidofovir and is mainly used for treating diseases caused by adenovirus infection clinically. Brincidofovir has small toxic and side effects, which are superior to cidofovir, but intestinal toxicity may occur in some patients. Therefore, the development of new, highly effective, low toxic side effects anti-adenovirus drugs remains a clinically significant need for the current treatment of adenovirus diseases. Currently, assays for determining the inhibition of adenovirus by antiviral drugs mainly include immunofluorescence quantification for determining the viral content, immunofluorescence, viral TCID ₅₀ Titer, viral plaque reduction assay, IC ₅₀ (semi-inhibitory concentration). When the method is used for high-throughput screening of anti-adenovirus drugs, the time consumption is long, the workload is large, and the cost is high. How to screen candidate compounds with anti-adenovirus effect in vitro, cheaply, efficiently, rapidly and with high throughput is the key for the development of anti-adenovirus drugs. And a rapid, simple, effective and high-flux screening platform is established to screen whether the medicine has the effect of inhibiting adenovirus proliferation, thereby being beneficial to screening new anti-adenovirus medicines and providing an effective screening platform for developing new anti-adenovirus medicines. The invention uses anti-human adenovirus drugs Cidofovir and brinzidofovir 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 drugs to adenovirus, so as to solve the problems of large workload, long time consumption and high cost when the existing conventional method is used for detecting the inhibition of drugs to adenovirus, facilitate the screening of new anti-adenovirus drugs, and provide an effective screening platform for the research and development of new anti-adenovirus drugs.

Specifically, the invention constructs plasmid pAd-YFP by using pAdTrack-CMV and pAdEasy-1 plasmid, and transfects the plasmid pAd-YFP into 293FT cells to obtain the GFP-integrated human adenovirus, wherein the specific method refers to https:// media. Briefly, the method steps are: plasmid pAdTrack-CMV was linearized using a PmeI restriction endonuclease. The linearized pAdTrack-CMV and adenovirus backbone vector pAdEasy-1 plasmid were co-electrotransferred into E.coli BJ5183 cells. The plasmid pAd-YFP is obtained by carrying out monoclonal screening by kanamycin, carrying out enzyme digestion identification on the obtained plasmid, transforming the plasmid with correct identification into DH10B competence, picking up monoclonal and extracting. The plasmid pAd-YFP was linearized with the restriction enzyme PacI and transfected into 293FT cells with lipofectamine, and the obtained virus infected cells to obtain higher concentrations of adenovirus for subsequent experimental study. After adenovirus infection, the infected cells were green under fluorescent microscopy, which shows that the adenovirus obtained 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 adenovirus proliferation was to re-add the virus supernatant of 4 th d post-infection virus to the laid HEK-293 cells, perform flow cytometry to detect the proportion of green fluorescent cells at 2 nd d post-virus addition, and set up a non-infected non-dosing group (NC), an infected and non-dosing group (PC), and an infected and dosing group (different doses).

Furthermore, the invention provides a high-throughput screening method for simply and conveniently measuring the inhibition of adenovirus proliferation by an anti-adenovirus drug, which mainly comprises the detection of adenovirus titer, and the establishment of the high-throughput screening method for inhibiting adenovirus proliferation by a positive drug (Brincidofovir, cidofovir) on MTT test of HEK-293 cells. The establishment of a high-throughput screening method for inhibiting adenovirus proliferation by using the medicine mainly researches the influence of different adding time points, different dosages, different sampling points and cell densities of the medicine on adenovirus proliferation.

The method comprises the following steps:

1. preparation of GFP-integrated adenoviruses.

2. Adenovirus titer was detected using TCID50 method.

3. MTT assay of HEK-293 cells with positive drug (Brincidofovir, cidofovir) was performed using 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 the drug on adenovirus proliferation

The method mainly designs 8 h,6 h, 4h, 2h, 0 h before virus addition, 2h, 4h, 6 h after virus addition and the drug Brincidofovir 1000nM are added, the number of green cells in cells is measured after 4d of virus infection, and the number of green cells in cells is measured after HEK-293 cells are infected again by culture medium supernatant of 4 d.

2) Effect of different doses of drug on adenovirus proliferation

The drug concentration of Brincidofovir was set at 5000 nM;1000 nM;500 nM;100nM; the drug concentration of Cidofovir is set to 200 μm; 100. a [ mu ] M; 50. a [ mu ] M; 25. and [ mu ] M. After 4d of drug and virus addition, and after 4d of virus supernatant re-infection of HEK-293 cells, the ratio of green cells in the cells was analyzed in a flow format, respectively. And (5) primarily selecting the concentration of the positive drug for inhibiting adenovirus proliferation.

3) Determination of time point at which viral supernatant was collected and cell density of the second plating.

Taking culture medium supernatants of 2d and 4d after adding medicine and virus with 24-well plate, adding into HEK-293 cells laid 1d in advance, and cell densities of 5×10 respectively ⁴ Cell/well and 1×10 ⁵ Cells/wells. Flow assays were performed 2d after infection of the cells with the supernatant. The effect of time point of collection of viral supernatant and cell density of the second plating on adenovirus proliferation was determined.

4) Effect of different doses of drug on adenovirus proliferation with 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 of 3d,4d,5d after virus infection were selected for reinfection with HEK-293 cells 2d for flow analysis. The time point at which the viral supernatant was collected when screening was performed with 48 well plates was determined.

Advantageous effects

The method provided by the invention enables simultaneous screening of hundreds of new compounds synthesized, and thousands of compounds in a small molecule library can be screened in a short time.

Meanwhile, the method provided by the invention can realize simultaneous screening of 200 compounds each time, can be completed within 3 hours during flow cytometry analysis, and needs 8d in the whole experimental process. The TCID50 method, virus plaque reduction assay, IC50 (semi inhibitory concentration) method can only detect a few compounds at a time of 4-5d, and if multiple compounds are to be detected, a lot of manpower and resources are required. The fluorescent quantitative method is used for detection, so that high-throughput detection can be realized, but primers and synthetic probes are required to be designed, at most 96 samples can be prepared in each batch, and 200 samples can be prepared at least 2 times. Takes more than 3 hours and is expensive.

Drawings

Fig. 1: MTT assay results of HEK-293 cells with different doses of Brincidofovir and Cidofovir.

A: MTT assay results of 5-2000nM Brincidofovir on HEK-293 cells;

b: MTT assay results of 10-200. Mu.M Cidofovir on HEK-293 cells.

Fig. 2: effects of the addition of the drug Brincidofovir 1000nM on adenovirus proliferation at different times before and after the addition of the virus.

A: effect of Brincidofovir 1000nM on adenovirus proliferation in HEK-293 cells at 4d after virus addition at different time points;

b: proliferation inhibition results of adenovirus in HEK-293 cells by adding 1000nM Brincidofovir at different time points after virus addition at 4 d;

c: the addition of the drug brinzdofovir 1000nm at various dosing time points, proliferation of virus in HEK-293 cells in 4d medium supernatant;

d: proliferation inhibition results of virus in HEK-293 cells in 4d medium supernatant with the drug Brindofovir 1000nM added at different dosing time points.

Fig. 3: results of inhibition of adenovirus proliferation by different doses of drug.

A: effect of different doses of Brincidofovir on adenovirus proliferation in HEK-293 cells (4 d).

B: the effect of adenovirus proliferation (2 d) in HEK-293 cells in the viral supernatant was added at different doses of Brincidofovir.

C: results of proliferation inhibition of adenovirus in the viral supernatant in HEK-293 cells (2 d) by addition of different doses of Brincidofovir.

D: effect of different doses of Cidofovir on adenovirus proliferation in HEK-293 cells (4 d).

E: effect of adenovirus proliferation in HEK-293 cells (2 d) in viral supernatant by addition of different doses of Cidofovir.

F: results of proliferation inhibition of adenovirus in viral supernatant in HEK-293 cells (2 d) by addition of different doses of Cidofovir.

Fig. 4: the effect of different doses of drug on adenovirus proliferation was affected by the time point at which the virus supernatant was collected and the cell density of the second HEK-293 cell plating.

A: after different doses of Brincidofovir and virus are added to infect cells, 100 mu L of culture medium supernatant of the 2d is taken to spread 5 multiplied by 10 in 1d in advance ⁴ Cell HEK-293 cell, proliferation of adenovirus in supernatant in HEK-293 cell.

B: after different doses of Brincidofovir and virus are added to infect cells, 100 mu L of culture medium supernatant of the 2d is taken to spread 1X 10 in advance of infection for 1d ⁵ Cell HEK-293 cell, proliferation of adenovirus in supernatant in HEK-293 cell.

C: after different doses of Brincidofovir and virus are added to infect cells, 100 mu m of culture medium supernatant of 4d is takenL infection was spread 5X 10 in advance 1d ⁴ Cell HEK-293 cell, proliferation of adenovirus in supernatant in HEK-293 cell.

D: after different doses of Brincidofovir and virus are added to infect cells, 100 MuL of culture medium supernatant of 4d is taken to spread 1X 10 in advance of infection for 1d ⁵ Cell HEK-293 cell, proliferation of adenovirus in supernatant in HEK-293 cell.

E: after adding different doses of Cidofovir and virus to infect cells, the 2d culture medium supernatant is spread with 5×10 at 1d in advance of infection by 100 μl ⁴ Cell HEK-293 cell, proliferation of adenovirus in supernatant in HEK-293 cell.

F: after adding different doses of Cidofovir and virus to infect cells, the 2d culture supernatant is spread with 1×10 in 1d in advance of infection by 100 μl ⁵ Cell HEK-293 cell, proliferation of adenovirus in supernatant in HEK-293 cell.

G: after adding different doses of Cidofovir and virus to infect cells, the culture supernatant of 4d is spread with 5×10 in 1d in advance of infection by 100 μl ⁴ Cell HEK-293 cell, proliferation of adenovirus in supernatant in HEK-293 cell.

H: after adding different doses of Cidofovir and virus to infect cells, the culture supernatant of 4d is spread with 1×10 in 1d in advance of infection by 100 μl ⁵ Cell HEK-293 cell, proliferation of adenovirus in supernatant in HEK-293 cell.

Fig. 5: drug screening was performed using a 48-well plate, and 50 μl of 3d,4d,5d culture medium supernatant after drug and virus addition was collected, and HEK-293 cells were re-infected (5×10 ⁴ ) Is a proliferation condition of (a).

A: different doses of Brincidofovir were added and the culture supernatant 3d after viral infection was taken to reinfect HEK-293 cells for 2d, and the proliferation of adenovirus in the supernatant.

B: different doses of Brincidofovir were added and the culture supernatant 4d after viral infection was taken to re-infect HEK-293 cells for 2d, proliferation of adenovirus in the supernatant.

C: different doses of Brincidofovir were added and the culture supernatant 5d after viral infection was taken to re-infect HEK-293 cells for 2d, proliferation of adenovirus in the supernatant.

D: different doses of Cidofovir were added and culture supernatant 3d after viral infection was used to reinfect HEK-293 cells for 2d and the supernatant was subjected to adenovirus proliferation.

E: different doses of Cidofovir were added and culture supernatant 4d after viral infection was used to reinfect HEK-293 cells for 2d and the supernatant was subjected to adenovirus proliferation.

F: different doses of Cidofovir were added and culture supernatant 5d after viral infection was used to reinfect HEK-293 cells for 2d and the supernatant was subjected to adenovirus proliferation.

Fig. 6: fluorescence images of HEK-293 cells re-infected with the virus 4d and medium supernatant after addition of different doses of drug and virus when drug screening was performed with 48 well plates.

A fluorescence image of 4d after adding different doses of the drug and virus;

fluorescence image of virus re-infected HEK-293 cells in culture medium supernatant (50. Mu.L) from day 4.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof. The invention will now be further described with reference to examples and figures.

The methods used in the examples below are conventional, unless otherwise specified.

Example 1 establishment of a high throughput screening method for assaying drugs to inhibit human adenovirus proliferation

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 /), and plasmid pAd-YFP was transfected into 293FT cells to obtain GFP-integrated human adenovirus, for specific methods reference https:// media. Addgene. Org/data/49/60/162ad396-af64-11e0-90fe-003048dd6500.Pdf. Briefly, the method is:

1) Plasmid pAdTrack-CMV was linearized using a PmeI restriction endonuclease.

2) The linearized pAdTrack-CMV and adenovirus backbone vector pAdEasy-1 plasmid were co-electrotransferred into E.coli BJ5183 cells. The plasmid pAd-YFP is obtained by carrying out monoclonal screening by kanamycin, carrying out enzyme digestion identification on the obtained plasmid, transforming the plasmid with correct identification into DH10B competence, picking up monoclonal and extracting.

3) The plasmid pAd-YFP was linearized with the restriction enzyme PacI and transfected into 293FT cells with lipofectamine, and the obtained virus was repeatedly infected into cells to obtain higher concentrations of adenovirus for subsequent experimental study.

4) After infection of the cells, the cells were visualized as green under a fluorescence microscope, and these defined adenoviruses were recombinant adenoviruses.

2. Adenovirus titer detection

1) HEK-293 cells in logarithmic growth phase were digested with trypsin, counted and inoculated into 96-well plates at a cell density of 1X 10 ⁴ Cells/wells. The cells were cultured with DMEM containing 10% FBS.

2) After 24h, 100 μl of each well was added after 10-fold serial dilution of provirus with DMEM medium containing 2% fbs, and 6 replicates of each concentration were performed. Provirus 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 virus addition, the number of wells containing green fluorescent cells was recorded under a fluorescent microscope and TCID50 of the virus was calculated. The statistical results are shown in Table 1, and the TCID50 of the calculated virus is 10 ^7.3 TCID50/mL。

TABLE 1 statistics of results of adenovirus titre detection

3. Toxicity test of antiviral drug Brincidofovir (B) and Cidofovir (C) on HEK-293

1) By usingTrypsin digested HEK-293 cells in logarithmic growth phase, counted and inoculated in 96-well plate with cell density of 1×10 ⁴ Cells/wells. The cells were cultured with DMEM containing 10% FBS.

2) After 24h, the drug concentrations added to Brincidofovir were 10 nM, 50nM,100 nM,250 nM, 500 nM,750 nM,1000 nM,1500 nM,2000 nM, respectively. The drug concentration of adding Cidofovir is 10 mu M,25 mu M, 50 mu M,75 mu M,100 mu M,150 mu M and 200 mu M respectively. 3 duplicate wells per concentration, 3 zeroing wells with no cells added to the medium, and 3 wells with the same number of cells as the test group, and 3 control groups with the same medium but no drug group were simultaneously set.

3) At 48 and h, 10 mu L of MTT solution is added to each hole according to the MTT kit instruction, after incubation is carried out at 37 ℃ for 4h, 100 mu L of Formazan solution is added, after incubation is carried out for 4h, absorbance is measured at 570 nm, and whether the added medicine has toxicity to HEK-293 cells is counted. ,

4) As shown in FIG. 1, the results of the MTT assay show that Brincidofovir has no toxicity to HEK-293 cells in the concentration range of 10 nM-2000 nM. Cidofovir is not toxic to HEK-293 cells in the range of 10 μm-200 μm.

4. Establishment of high throughput screening method for inhibiting adenovirus proliferation by drug

1) Determination of different dosing time points.

1 st d, HEK-293 cells in logarithmic growth phase were digested with trypsin, counted and seeded in 24-well plates at a cell density of 5X 10 ⁴ Cells/wells. The cells were cultured with DMEM containing 10% FBS.

2d, the antiviral drug Brincidofovir 1000nM was added at various time points. The addition time points are 8 h,6 h, 4h, 2h, 0 h before virus addition and 2h, 4h and 6 h after virus addition. The amount of virus added was moi=0.1. Setting a virus-free drug-free group (NC), a virus-free drug-free group (PC), and a test group: the virus was added and Brincidofovir 1000nM was added at different time points.

6d, 4d after virus addition, 100. Mu.L of supernatant was aspirated from each well into 24 well plates (5X 10) ⁴ Cells/well, plated on day 5 d). Flow assays were performed on the first plated cells, the results are shown in FIG. 2A and the inhibition is shown in FIG. 2B.

Cells after addition of viral supernatant were cultured 48 and h for flow detection. The results are shown in FIG. 2C and the inhibition results are shown in FIG. 2D.

The test results show that: the effect of dosing at different time points on adenovirus-infected cells and virus proliferation is small, however, after dosing, the virus content in the culture medium supernatant is obviously changed, and when HEK-293 cells are infected again, the virus content is obviously reduced.

2) Preliminary determination of additive concentration of positive control drug.

1 st d, HEK-293 cells in logarithmic growth phase were digested with trypsin, counted and seeded in 24-well plates at a cell density of 5X 10 ⁴ Cells/wells. The cells were cultured with DMEM containing 10% FBS.

2d, set no virus addition no drug group (NC), no virus addition no drug group (PC), test group: adding viruses and medicine groups with different concentrations; the drug concentration of Brincidofovir was set at 5000 nM;1000 nM;500 nM;100nM; the drug concentration of Cidofovir is set to 200 μm; 100. a [ mu ] M; 50. a [ mu ] M; 25. and [ mu ] M. Drugs and viruses were added according to the experimental design, with moi=0.1 for adenovirus addition.

6d, 4d after virus addition, 100. Mu.L of supernatant was aspirated from each well into 24 well plates (5X 10) ⁴ Cells/well, plated on day 5 d). The cells from the first plating were subjected to flow analysis and the results are shown in FIGS. 3A and 3D.

Cells after addition of viral supernatant were cultured 48 and h for flow detection. The results are shown in FIGS. 3B and 3E; the inhibition results are shown in FIGS. 3C and 3F.

The test results show that: after HEK-293 cells are infected for the first time by adding different doses of medicines, the inhibition effect of the medicines with different doses on the viruses cannot be distinguished obviously by flow cytometry. After the drug and the virus are added for 4 days, the culture medium supernatant is infected with HEK-293 cells again, so that whether the antiviral drug has an inhibitory effect on adenovirus at different doses can be obviously distinguished. In addition, the experiment clearly determines that the dosage of Brincdofovir serving as a positive control medicament is less than or equal to 500 nM and the dosage of Cidofovir serving as the positive control medicament is less than or equal to 50 mu M.

3) After drug and virus addition, time points of virus supernatant and determination of cell density of 24 well plate plating were collected.

1 st d, HEK-293 cells in logarithmic growth phase were digested with trypsin, counted and seeded in 24-well plates at a cell density of 5X 10 ⁴ Cells/wells. The cells were cultured with DMEM containing 10% FBS.

2d, set no virus addition no drug group (NC), no virus addition no drug group (PC), test group: adding viruses and medicine groups with different concentrations; the drug concentration of Brincidofovir was set at 500 nM;100nM;50 nM;10 nM, drug concentration of Cidofovir set to 100. Mu.M; 50. a [ mu ] M; 25. a [ mu ] M; 10. and [ mu ] M. Drugs and viruses were added according to the experimental design, 2h after drug addition, and adenovirus addition moi=0.1.

4d, 2 nd d after virus addition, each well aspirates 100 μl of supernatant into 24 well plates with HEK-293 cells spread 1 day in advance. Cell density at plating was 5X 10 ⁴ Cell/well and 1×10 ⁵ Two cells/well.

6d, 4d after virus addition, each well aspirates 100 μl of supernatant into 24 well plates with HEK-293 cells spread 1 day in advance. Cell density at plating was 5X 10 ⁴ Cell/well and 1×10 ⁵ Two cells/well.

Flow assays were performed after cell culture 48 h after addition of viral supernatant. The results are shown in FIG. 4.

The test results show that: the 4 th d th virus supernatant after adenovirus addition and the second plating cell density were 1X 10 ⁵ The cells/pores can better show the inhibition effect of different concentrations of drugs on adenovirus.

4) High throughput screening of drugs against adenovirus proliferation was performed using 48-well plates.

1. 1d, HEK-293 cells in logarithmic growth phase were digested with trypsin, counted and seeded in 48-well plates at a cell density of 2.5X10 ⁴ Cells/wells. The cells were cultured with DMEM containing 10% FBS.

2d, set no virus addition no drug group (NC), no virus addition no drug group (PC), test group: adding viruses and medicine groups with different concentrations; the drug concentration of Brincidofovir was set at 500 nM;250 nM;100nM;50 nM;25 nM;10 nM;5 nM, drug concentration of Cidofovir set to 200. Mu.M; 100. a [ mu ] M; 50. a [ mu ] M; 25. a [ mu ] M; 10. and [ mu ] M. Drug and virus were added according to the experimental design, drug 2h was added followed by virus, and adenovirus was added with moi=0.1.

5 th d, 3 rd d th after virus addition, each well aspirates 100 μl of supernatant into a 48-well plate with HEK-293 cells spread 1 day in advance. Cell density at plating was 5X 10 ⁴ Cells/wells.

6d, 4d after virus addition, each well aspirates 100 μl of supernatant into a 48-well plate with HEK-293 cells spread 1 day in advance. Cell density at plating was 5X 10 ⁴ Cells/wells.

7 d,5 th d after virus addition, each well aspirates 100 μl of supernatant into a 48-well plate with HEK-293 cells spread 1 day in advance. Cell density at plating was 5X 10 ⁴ Cells/wells.

Flow assays were performed after cell culture 48 h after addition of viral supernatant. The results are shown in FIG. 5, and the fluorescence of HEK-293 cells infected with adenovirus is shown in FIG. 6.

The test results show that: after adenovirus is added, the 4 th d th and the 5 th d viral supernatants are taken for carrying out secondary virus amplification, so that the inhibition effect of the medicaments with different concentrations on the adenovirus can be better displayed. The viral supernatant from 4 th d was selected according to the principle of short time consumption.

5) The specific steps of the high throughput screening flow cytometry method for inhibiting adenovirus proliferation by the drug are as follows:

(1) 24-well plate screening method

1d,24 th well plate, 5X 10 HEK-293 cells were plated per well ⁴ (i.e., 5X 10 in 1mL of medium) ⁴ cells/mL, 1mL into 24 well plate).

2d, 24h after plating, and dosing. The test should be (1) blank (no drug, no virus), (2) positive virus control (virus only, no drug), (3) positive drug (virus plus positive drug, cidofovir (50 μm) and brincdofovir (250 nM)), (4) test (virus plus drug to be screened).

2. 2h post-dosing, viral addition, 2 μl adenovirus per well (moi=0.1). The blank group was not added with virus.

5d, after virus addition, 3d d, spread cells: 24-well plate, 1X 10 per well ⁵ HEK-293 cells of (i.e.1X 10 in 1mL of medium) ⁵ cells/mL, 1mL into 24 well plate). Consistent with the number of cells wells plated for the first time.

And 6, d, taking 100 mu L of virus supernatant of each hole of the first time of plating, and adding the virus supernatant into cells of the second time of plating, wherein the sequence is consistent with that of the first time.

8d, namely 2 nd d after virus addition, performing neutralization by adding 200 mu L of culture medium to 2 nd-time laid cell plates, discarding supernatant and performing pancreatin digestion by 100 mu L of culture medium, sucking the culture medium into a flow tube for flow analysis after repeated blowing, and recording the detection result of each sample.

Comparing the flow result of the drug to be tested with a positive drug group (Cidofovir (50 mu M) and brinzidofovir (250 nM)) to analyze whether the drug to be tested has the effect of inhibiting adenovirus.

(2) 48-well plate screening method

1d,48 well plates, 2.5X10 HEK-293 cells were plated per well ⁴ (i.e., 5X 10 in 1mL of medium) ⁴ cells/mL, aspirate 500 μl into a 48 well plate).

2d, 24h after plating, and dosing. The test should be (1) blank (no drug, no virus), (2) positive virus control (virus only, no drug), (3) positive drug (virus plus positive drug, cidofovir (50 μm) and brincdofovir (250 nM)), (4) test (virus plus drug to be screened).

2. 2h post-dosing, viral addition, 2 μl adenovirus per well (moi=0.1). The blank group was not added with virus.

5d, after virus addition, 3d d, spread cells: 48-well plate, 5X 1 per well0 ⁴ HEK-293 cells of (i.e.1X 10 in 1mL of medium) ⁵ cells/mL, aspirate 500 μl into a 48 well plate). Consistent with the number of cells wells plated for the first time.

And 6, d, taking 50 mu L of virus supernatant of each hole of the first time of plating, and adding the virus supernatant into cells of the second time of plating, wherein the sequence is consistent with that of the first time.

8d (2 d after virus addition), performing flow analysis on the cell plate paved for the 2 nd time, discarding the supernatant, performing pancreatin digestion on 100 mu L, adding 200 mu L of culture medium for neutralization, repeatedly blowing, and then sucking 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 (50 mu M) and brinzidofovir (250 nM)) to analyze whether the drug to be tested has the effect of inhibiting adenovirus.

Using this method, we have shown the results of the detection of 39 drugs in the natural compound library in Table 2.

Table 2: flow results for detection of 39 drugs in the 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%: represents the percentage of adenovirus infected HEK-293 cells.

From the above results, compared with the effect of anti-adenovirus positive drugs Cidofovir (50 mu M) and Brincidofovir (250 nM) on inhibiting adenovirus, the GFP expression levels of the 20, 35, 38 and 39 drugs are lower than or close to those of positive control drugs, so that proliferation of adenovirus can be inhibited, and the screened drugs need to be re-screened and toxicity of the drugs on cell proliferation observed later, if the drugs have obvious toxicity on inhibiting cell proliferation, whether detection can be continued after the drug concentration is reduced can be considered. The method can be used for preliminarily screening out the drugs with obvious inhibition effect on adenovirus proliferation with high flux.

The applicant states that the present invention is described by way of the above examples as a high throughput screening method and application of the present invention for drugs that inhibit human adenovirus proliferation, but the present invention is not limited to the above examples, i.e. it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Sequence listing

<110> Shanghai veterinary institute of agricultural sciences of China (center for sea division of China center for animal health and epidemiology)

High-throughput screening method of human adenovirus proliferation inhibiting medicine and its application

〈130〉

〈160〉1

〈170〉PatentInversion3.3

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 37387

<212> pAd-YFP

<213> Artificial sequence (Artificial Sequence)

tcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagcctaacttcgatcattggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgccctataccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgctaacggattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcgcgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcagccatgagattatcaaaaaggatcttcacctagatccttttcacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattttgttaaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtccattcgccattcaggatcgaattaattcttaattaacatcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttgtagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactgaataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgcggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcgttccgggtcaaagttggcgttttattattatagtcagtcgagtctagcaagctagcttgatcgcgttaagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatcagttatctagatccggtggatctgagtccggacttgtacagctcgtccatgccgagagtgatcccggcggcggtcacgaactccagcaggaccatgtgatcgcgcttctcgttggggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggcagcagcacggggccgtcgccgatgggggtgttctgctggtagtggtcggcgagctgcacgctgccgtcctcgatgttgtggcggatcttgaagttcaccttgatgccgttcttctgcttgtcggccatgatatagacgttgtggctgttgtagttgtactccagcttgtgccccaggatgttgccgtcctccttgaagtcgatgcccttcagctcgatgcggttcaccagggtgtcgccctcgaacttcacctcggcgcgggtcttgtagttgccgtcgtccttgaagaagatggtgcgctcctggacgtagccttcgggcatggcggacttgaagaagtcgtgctgcttcatgtggtcggggtagcggctgaagcactgcacgccgtaggtcagggtggtcacgagggtgggccagggcacgggcagcttgccggtggtgcagatgaacttcagggtcagcttgccgtaggtggcatcgccctcgccctcgccggacacgctgaacttgtggccgtttacgtcgccgtccagctcgaccaggatgggcaccaccccggtgaacagctcctcgcccttgctcaccatggtggcgaccggtagcgctagcggatctgacggttcactaaaccagctctgcttatatagacctcccaccgtacacgcctaccgcccatttgcgtcaatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaacaaactcccattgacgtcaatggggtggagacttggaaatccccgtgagtcaaaccgctatccacgcccattgatgtactgccaaaaccgcatcaccatggtaatagcgatgactaatacgtagatgtactgccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggccatttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatgggaacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagttatgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattgattactattaatcactagcagatcgtcgataaacctccgcgttaagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatcagttatctagatccggtggatcggatatcttatctagaagcttaggctcgagcggccgcgtcgacggtaccagatctctagcggatctgacggttcactaaaccagctctgcttatatagacctcccaccgtacacgcctaccgcccatttgcgtcaatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaacaaactcccattgacgtcaatggggtggagacttggaaatccccgtgagtcaaaccgctatccacgcccattgatgtactgccaaaaccgcatcaccatggtaatagcgatgactaatacgtagatgtactgccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggccatttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactgccaagtgggcagtttaccgtaaatactccacccattgacgtcaatggaaagtccctattggcgttactatgggaacatacgtcattattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagttatgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattgatsasyscnnchhttactattagacgtttatcactgctagaggatctgggcgtggttaagggtgggaaagaatatataaggtgggggtcttatgtagttttgtatctgttttgcagcagccgccgccgccatgagcaccaactcgtttgatggaagcattgtgagctcatatttgacaacgcgcatgcccccatgggccggggtgcgtcagaatgtgatgggctccagcattgatggtcgccccgtcctgcccgcaaactctactaccttgacctacgagaccgtgtctggaacgccgttggagactgcagcctccgccgccgcttcagccgctgcagccaccgcccgcgggattgtgactgactttgctttcctgagcccgcttgcaagcagtgcagcttcccgttcatccgcccgcgatgacaagttgacggctcttttggcacaattggattctttgacccgggaacttaatgtcgtttctcagcagctgttggatctgcgccagcaggtttctgccctgaaggcttcctcccctcccaatgcggtttaaaacataaataaaaaaccagactctgtttggatttggatcaagcaagtgtcttgctgtctttatttaggggttttgcgcgcgcggtaggcccgggaccagcggtctcggtcgttgagggtcctgtgtattttttccaggacgtggtaaaggtgactctggatgttcagatacatgggcataagcccgtctctggggtggaggtagcaccactgcagagcttcatgctgcggggtggtgttgtagatgatccagtcgtagcaggagcgctgggcgtggtgcctaaaaatgtctttcagtagcaagctgattgccaggggcaggcccttggtgtaagtgtttacaaagcggttaagctgggatgggtgcatacgtggggatatgagatgcatcttggactgtatttttaggttggctatgttcccagccatatccctccggggattcatgttgtgcagaaccaccagcacagtgtatccggtgcacttgggaaatttgtcatgtagcttagaaggaaatgcgtggaagaacttggagacgcccttgtgacctccaagattttccatgcattcgtccataatgatggcaatgggcccacgggcggcggcctgggcgaagatatttctgggatcactaacgtcatagttgtgttccaggatgagatcgtcataggccatttttacaaagcgcgggcggagggtgccagactgcggtataatggttccatccggcccaggggcgtagttaccctcacagatttgcatttcccacgctttgagttcagatggggggatcatgtctacctgcggggcgatgaagaaaacggtttccggggtaggggagatcagctgggaagaaagcaggttcctgagcagctgcgacttaccgcagccggtgggcccgtaaatcacacctattaccggctgcaactggtagttaagagagctgcagctgccgtcatccctgagcaggggggccacttcgttaagcatgtccctgactcgcatgttttccctgaccaaatccgccagaaggcgctcgccgcccagcgatagcagttcttgcaaggaagcaaagtttttcaacggtttgagaccgtccgccgtaggcatgcttttgagcgtttgaccaagcagttccaggcggtcccacagctcggtcacctgctctacggcatctcgatccagcatatctcctcgtttcgcgggttggggcggctttcgctgtacggcagtagtcggtgctcgtccagacgggccagggtcatgtctttccacgggcgcagggtcctcgtcagcgtagtctgggtcacggtgaaggggtgcgctccgggctgcgcgctggccagggtgcgcttgaggctggtcctgctggtgctgaagcgctgccggtcttcgccctgcgcgtcggccaggtagcatttgaccatggtgtcatagtccagcccctccgcggcgtggcccttggcgcgcagcttgcccttggaggaggcgccgcacgaggggcagtgcagacttttgagggcgtagagcttgggcgcgagaaataccgattccggggagtaggcatccgcgccgcaggccccgcagacggtctcgcattccacgagccaggtgagctctggccgttcggggtcaaaaaccaggtttcccccatgctttttgatgcgtttcttacctctggtttccatgagccggtgtccacgctcggtgacgaaaaggctgtccgtgtccccgtatacagacttgagaggcctgtcctcgagcggtgttccgcggtcctcctcgtatagaaactcggaccactctgagacaaaggctcgcgtccaggccagcacgaaggaggctaagtgggaggggtagcggtcgttgtccactagggggtccactcgctccagggtgtgaagacacatgtcgccctcttcggcatcaaggaaggtgattggtttgtaggtgtaggccacgtgaccgggtgttcctgaaggggggctataaaagggggtgggggcgcgttcgtcctcactctcttccgcatcgctgtctgcgagggccagctgttggggtgagtactccctctgaaaagcgggcatgacttctgcgctaagattgtcagtttccaaaaacgaggaggatttgatattcacctggcccgcggtgatgcctttgagggtggccgcatccatctggtcagaaaagacaatctttttgttgtcaagcttggtggcaaacgacccgtagagggcgttggacagcaacttggcgatggagcgcagggtttggtttttgtcgcgatcggcgcgctccttggccgcgatgtttagctgcacgtattcgcgcgcaacgcaccgccattcgggaaagacggtggtgcgctcgtcgggcaccaggtgcacgcgccaaccgcggttgtgcagggtgacaaggtcaacgctggtggctacctctccgcgtaggcgctcgttggtccagcagaggcggccgcccttgcgcgagcagaatggcggtagggggtctagctgcgtctcgtccggggggtctgcgtccacggtaaagaccccgggcagcaggcgcgcgtcgaagtagtctatcttgcatccttgcaagtctagcgcctgctgccatgcgcgggcggcaagcgcgcgctcgtatgggttgagtgggggaccccatggcatggggtgggtgagcgcggaggcgtacatgccgcaaatgtcgtaaacgtagaggggctctctgagtattccaagatatgtagggtagcatcttccaccgcggatgctggcgcgcacgtaatcgtatagttcgtgcgagggagcgaggaggtcgggaccgaggttgctacgggcgggctgctctgctcggaagactatctgcctgaagatggcatgtgagttggatgatatggttggacgctggaagacgttgaagctggcgtctgtgagacctaccgcgtcacgcacgaaggaggcgtaggagtcgcgcagcttgttgaccagctcggcggtgacctgcacgtctagggcgcagtagtccagggtttccttgatgatgtcatacttatcctgtcccttttttttccacagctcgcggttgaggacaaactcttcgcggtctttccagtactcttggatcggaaacccgtcggcctccgaacggtaagagcctagcatgtagaactggttgacggcctggtaggcgcagcatcccttttctacgggtagcgcgtatgcctgcgcggccttccggagcgaggtgtgggtgagcgcaaaggtgtccctgaccatgactttgaggtactggtatttgaagtcagtgtcgtcgcatccgccctgctcccagagcaaaaagtccgtgcgctttttggaacgcggatttggcagggcgaaggtgacatcgttgaagagtatctttcccgcgcgaggcataaagttgcgtgtgatgcggaagggtcccggcacctcggaacggttgttaattacctgggcggcgagcacgatctcgtcaaagccgttgatgttgtggcccacaatgtaaagttccaagaagcgcgggatgcccttgatggaaggcaattttttaagttcctcgtaggtgagctcttcaggggagctgagcccgtgctctgaaagggcccagtctgcaagatgagggttggaagcgacgaatgagctccacaggtcacgggccattagcatttgcaggtggtcgcgaaaggtcctaaactggcgacctatggccattttttctggggtgatgcagtagaaggtaagcgggtcttgttcccagcggtcccatccaaggttcgcggctaggtctcgcgcggcagtcactagaggctcatctccgccgaacttcatgaccagcatgaagggcacgagctgcttcccaaaggcccccatccaagtataggtctctacatcgtaggtgacaaagagacgctcggtgcgaggatgcgagccgatcgggaagaactggatctcccgccaccaattggaggagtggctattgatgtggtgaaagtagaagtccctgcgacgggccgaacactcgtgctggcttttgtaaaaacgtgcgcagtactggcagcggtgcacgggctgtacatcctgcacgaggttgacctgacgaccgcgcacaaggaagcagagtgggaatttgagcccctcgcctggcgggtttggctggtggtcttctacttcggctgcttgtccttgaccgtctggctgctcgaggggagttacggtggatcggaccaccacgccgcgcgagcccaaagtccagatgtccgcgcgcggcggtcggagcttgatgacaacatcgcgcagatgggagctgtccatggtctggagctcccgcggcgtcaggtcaggcgggagctcctgcaggtttacctcgcatagacgggtcagggcgcgggctagatccaggtgatacctaatttccaggggctggttggtggcggcgtcgatggcttgcaagaggccgcatccccgcggcgcgactacggtaccgcgcggcgggcggtgggccgcgggggtgtccttggatgatgcatctaaaagcggtgacgcgggcgagcccccggaggtagggggggctccggacccgccgggagagggggcaggggcacgtcggcgccgcgcgcgggcaggagctggtgctgcgcgcgtaggttgctggcgaacgcgacgacgcggcggttgatctcctgaatctggcgcctctgcgtgaagacgacgggcccggtgagcttgaacctgaaagagagttcgacagaatcaatttcggtgtcgttgacggcggcctggcgcaaaatctcctgcacgtctcctgagttgtcttgataggcgatctcggccatgaactgctcgatctcttcctcctggagatctccgcgtccggctcgctccacggtggcggcgaggtcgttggaaatgcgggccatgagctgcgagaaggcgttgaggcctccctcgttccagacgcggctgtagaccacgcccccttcggcatcgcgggcgcgcatgaccacctgcgcgagattgagctccacgtgccgggcgaagacggcgtagtttcgcaggcgctgaaagaggtagttgagggtggtggcggtgtgttctgccacgaagaagtacataacccagcgtcgcaacgtggattcgttgatatcccccaaggcctcaaggcgctccatggcctcgtagaagtccacggcgaagttgaaaaactgggagttgcgcgccgacacggttaactcctcctccagaagacggatgagctcggcgacagtgtcgcgcacctcgcgctcaaaggctacaggggcctcttcttcttcttcaatctcctcttccataagggcctccccttcttcttcttctggcggcggtgggggaggggggacacggcggcgacgacggcgcaccgggaggcggtcgacaaagcgctcgatcatctccccgcggcgacggcgcatggtctcggtgacggcgcggccgttctcgcgggggcgcagttggaagacgccgcccgtcatgtcccggttatgggttggcggggggctgccatgcggcagggatacggcgctaacgatgcatctcaacaattgttgtgtaggtactccgccgccgagggacctgagcgagtccgcatcgaccggatcggaaaacctctcgagaaaggcgtctaaccagtcacagtcgcaaggtaggctgagcaccgtggcgggcggcagcgggcggcggtcggggttgtttctggcggaggtgctgctgatgatgtaattaaagtaggcggtcttgagacggcggatggtcgacagaagcaccatgtccttgggtccggcctgctgaatgcgcaggcggtcggccatgccccaggcttcgttttgacatcggcgcaggtctttgtagtagtcttgcatgagcctttctaccggcacttcttcttctccttcctcttgtcctgcatctcttgcatctatcgctgcggcggcggcggagtttggccgtaggtggcgccctcttcctcccatgcgtgtgaccccgaagcccctcatcggctgaagcagggctaggtcggcgacaacgcgctcggctaatatggcctgctgcacctgcgtgagggtagactggaagtcatccatgtccacaaagcggtggtatgcgcccgtgttgatggtgtaagtgcagttggccataacggaccagttaacggtctggtgacccggctgcgagagctcggtgtacctgagacgcgagtaagccctcgagtcaaatacgtagtcgttgcaagtccgcaccaggtactggtatcccaccaaaaagtgcggcggcggctggcggtagaggggccagcgtagggtggccggggctccgggggcgagatcttccaacataaggcgatgatatccgtagatgtacctggacatccaggtgatgccggcggcggtggtggaggcgcgcggaaagtcgcggacgcggttccagatgttgcgcagcggcaaaaagtgctccatggtcgggacgctctggccggtcaggcgcgcgcaatcgttgacgctctagcgtgcaaaaggagagcctgtaagcgggcactcttccgtggtctggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgtatccggccgtccgccgtgatccatgcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctccttttggcttccttccaggcgcggcggctgctgcgctagcttttttggccactggccgcgcgcagcgtaagcggttaggctggaaagcgaaagcattaagtggctcgctccctgtagccggagggttattttccaagggttgagtcgcgggacccccggttcgagtctcggaccggccggactgcggcgaacgggggtttgcctccccgtcatgcaagaccccgcttgcaaattcctccggaaacagggacgagccccttttttgcttttcccagatgcatccggtgctgcggcagatgcgcccccctcctcagcagcggcaagagcaagagcagcggcagacatgcagggcaccctcccctcctcctaccgcgtcaggaggggcgacatccgcggttgacgcggcagcagatggtgattacgaacccccgcggcgccgggcccggcactacctggacttggaggagggcgagggcctggcgcggctaggagcgccctctcctgagcggcacccaagggtgcagctgaagcgtgatacgcgtgaggcgtacgtgccgcggcagaacctgtttcgcgaccgcgagggagaggagcccgaggagatgcgggatcgaaagttccacgcagggcgcgagctgcggcatggcctgaatcgcgagcggttgctgcgcgaggaggactttgagcccgacgcgcgaaccgggattagtcccgcgcgcgcacacgtggcggccgccgacctggtaaccgcatacgagcagacggtgaaccaggagattaactttcaaaaaagctttaacaaccacgtgcgtacgcttgtggcgcgcgaggaggtggctataggactgatgcatctgtgggactttgtaagcgcgctggagcaaaacccaaatagcaagccgctcatggcgcagctgttccttatagtgcagcacagcagggacaacgaggcattcagggatgcgctgctaaacatagtagagcccgagggccgctggctgctcgatttgataaacatcctgcagagcatagtggtgcaggagcgcagcttgagcctggctgacaaggtggccgccatcaactattccatgcttagcctgggcaagttttacgcccgcaagatataccataccccttacgttcccatagacaaggaggtaaagatcgaggggttctacatgcgcatggcgctgaaggtgcttaccttgagcgacgacctgggcgtttatcgcaacgagcgcatccacaaggccgtgagcgtgagccggcggcgcgagctcagcgaccgcgagctgatgcacagcctgcaaagggccctggctggcacgggcagcggcgatagagaggccgagtcctactttgacgcgggcgctgacctgcgctgggccccaagccgacgcgccctggaggcagctggggccggacctgggctggcggtggcacccgcgcgcgctggcaacgtcggcggcgtggaggaatatgacgaggacgatgagtacgagccagaggacggcgagtactaagcggtgatgtttctgatcagatgatgcaagacgcaacggacccggcggtgcgggcggcgctgcagagccagccgtccggccttaactccacggacgactggcgccaggtcatggaccgcatcatgtcgctgactgcgcgcaatcctgacgcgttccggcagcagccgcaggccaaccggctctccgcaattctggaagcggtggtcccggcgcgcgcaaaccccacgcacgagaaggtgctggcgatcgtaaacgcgctggccgaaaacagggccatccggcccgacgaggccggcctggtctacgacgcgctgcttcagcgcgtggctcgttacaacagcggcaacgtgcagaccaacctggaccggctggtgggggatgtgcgcgaggccgtggcgcagcgtgagcgcgcgcagcagcagggcaacctgggctccatggttgcactaaacgccttcctgagtacacagcccgccaacgtgccgcggggacaggaggactacaccaactttgtgagcgcactgcggctaatggtgactgagacaccgcaaagtgaggtgtaccagtctgggccagactattttttccagaccagtagacaaggcctgcagaccgtaaacctgagccaggctttcaaaaacttgcaggggctgtggggggtgcgggctcccacaggcgaccgcgcgaccgtgtctagcttgctgacgcccaactcgcgcctgttgctgctgctaatagcgcccttcacggacagtggcagcgtgtcccgggacacatacctaggtcacttgctgacactgtaccgcgaggccataggtcaggcgcatgtggacgagcatactttccaggagattacaagtgtcagccgcgcgctggggcaggaggacacgggcagcctggaggcaaccctaaactacctgctgaccaaccggcggcagaagatcccctcgttgcacagtttaaacagcgaggaggagcgcattttgcgctacgtgcagcagagcgtgagccttaacctgatgcgcgacggggtaacgcccagcgtggcgctggacatgaccgcgcgcaacatggaaccgggcatgtatgcctcaaaccggccgtttatcaaccgcctaatggactacttgcatcgcgcggccgccgtgaaccccgagtatttcaccaatgccatcttgaacccgcactggctaccgccccctggtttctacaccgggggattcgaggtgcccgagggtaacgatggattcctctgggacgacatagacgacagcgtgttttccccgcaaccgcagaccctgctagagttgcaacagcgcgagcaggcagaggcggcgctgcgaaaggaaagcttccgcaggccaagcagcttgtccgatctaggcgctgcggccccgcggtcagatgctagtagcccatttccaagcttgatagggtctcttaccagcactcgcaccacccgcccgcgcctgctgggcgaggaggagtacctaaacaactcgctgctgcagccgcagcgcgaaaaaaacctgcctccggcatttcccaacaacgggatagagagcctagtggacaagatgagtagatggaagacgtacgcgcaggagcacagggacgtgccaggcccgcgcccgcccacccgtcgtcaaaggcacgaccgtcagcggggtctggtgtgggaggacgatgactcggcagacgacagcagcgtcctggatttgggagggagtggcaacccgtttgcgcaccttcgccccaggctggggagaatgttttaaaaaaaaaaaagcatgatgcaaaataaaaaactcaccaaggccatggcaccgagcgttggttttcttgtattccccttagtatgcggcgcgcggcgatgtatgaggaaggtcctcctccctcctacgagagtgtggtgagcgcggcgccagtggcggcggcgctgggttctcccttcgatgctcccctggacccgccgtttgtgcctccgcggtacctgcggcctaccggggggagaaacagcatccgttactctgagttggcacccctattcgacaccacccgtgtgtacctggtggacaacaagtcaacggatgtggcatccctgaactaccagaacgaccacagcaactttctgaccacggtcattcaaaacaatgactacagcccgggggaggcaagcacacagaccatcaatcttgacgaccggtcgcactggggcggcgacctgaaaaccatcctgcataccaacatgccaaatgtgaacgagttcatgtttaccaataagtttaaggcgcgggtgatggtgtcgcgcttgcctactaaggacaatcaggtggagctgaaatacgagtgggtggagttcacgctgcccgagggcaactactccgagaccatgaccatagaccttatgaacaacgcgatcgtggagcactacttgaaagtgggcagacagaacggggttctggaaagcgacatcggggtaaagtttgacacccgcaacttcagactggggtttgaccccgtcactggtcttgtcatgcctggggtatatacaaacgaagccttccatccagacatcattttgctgccaggatgcggggtggacttcacccacagccgcctgagcaacttgttgggcatccgcaagcggcaacccttccaggagggctttaggatcacctacgatgatctggagggtggtaacattcccgcactgttggatgtggacgcctaccaggcgagcttgaaagatgacaccgaacagggcgggggtggcgcaggcggcagcaacagcagtggcagcggcgcggaagagaactccaacgcggcagccgcggcaatgcagccggtggaggacatgaacgatcatgccattcgcggcgacacctttgccacacgggctgaggagaagcgcgctgaggccgaagcagcggccgaagctgccgcccccgctgcgcaacccgaggtcgagaagcctcagaagaaaccggtgatcaaacccctgacagaggacagcaagaaacgcagttacaacctaataagcaatgacagcaccttcacccagtaccgcagctggtaccttgcatacaactacggcgaccctcagaccggaatccgctcatggaccctgctttgcactcctgacgtaacctgcggctcggagcaggtctactggtcgttgccagacatgatgcaagaccccgtgaccttccgctccacgcgccagatcagcaactttccggtggtgggcgccgagctgttgcccgtgcactccaagagcttctacaacgaccaggccgtctactcccaactcatccgccagtttacctctctgacccacgtgttcaatcgctttcccgagaaccagattttggcgcgcccgccagcccccaccatcaccaccgtcagtgaaaacgttcctgctctcacagatcacgggacgctaccgctgcgcaacagcatcggaggagtccagcgagtgaccattactgacgccagacgccgcacctgcccctacgtttacaaggccctgggcatagtctcgccgcgcgtcctatcgagccgcactttttgagcaagcatgtccatccttatatcgcccagcaataacacaggctggggcctgcgcttcccaagcaagatgtttggcggggccaagaagcgctccgaccaacacccagtgcgcgtgcgcgggcactaccgcgcgccctggggcgcgcacaaacgcggccgcactgggcgcaccaccgtcgatgacgccatcgacgcggtggtggaggaggcgcgcaactacacgcccacgccgccaccagtgtccacagtggacgcggccattcagaccgtggtgcgcggagcccggcgctatgctaaaatgaagagacggcggaggcgcgtagcacgtcgccaccgccgccgacccggcactgccgcccaacgcgcggcggcggccctgcttaaccgcgcacgtcgcaccggccgacgggcggccatgcgggccgctcgaaggctggccgcgggtattgtcactgtgccccccaggtccaggcgacgagcggccgccgcagcagccgcggccattagtgctatgactcagggtcgcaggggcaacgtgtattgggtgcgcgactcggttagcggcctgcgcgtgcccgtgcgcacccgccccccgcgcaactagattgcaagaaaaaactacttagactcgtactgttgtatgtatccagcggcggcggcgcgcaacgaagctatgtccaagcgcaaaatcaaagaagagatgctccaggtcatcgcgccggagatctatggccccccgaagaaggaagagcaggattacaagccccgaaagctaaagcgggtcaaaaagaaaaagaaagatgatgatgatgaacttgacgacgaggtggaactgctgcacgctaccgcgcccaggcgacgggtacagtggaaaggtcgacgcgtaaaacgtgttttgcgacccggcaccaccgtagtctttacgcccggtgagcgctccacccgcacctacaagcgcgtgtatgatgaggtgtacggcgacgaggacctgcttgagcaggccaacgagcgcctcggggagtttgcctacggaaagcggcataaggacatgctggcgttgccgctggacgagggcaacccaacacctagcctaaagcccgtaacactgcagcaggtgctgcccgcgcttgcaccgtccgaagaaaagcgcggcctaaagcgcgagtctggtgacttggcacccaccgtgcagctgatggtacccaagcgccagcgactggaagatgtcttggaaaaaatgaccgtggaacctgggctggagcccgaggtccgcgtgcggccaatcaagcaggtggcgccgggactgggcgtgcagaccgtggacgttcagatacccactaccagtagcaccagtattgccaccgccacagagggcatggagacacaaacgtccccggttgcctcagcggtggcggatgccgcggtgcaggcggtcgctgcggccgcgtccaagacctctacggaggtgcaaacggacccgtggatgtttcgcgtttcagccccccggcgcccgcgccgttcgaggaagtacggcgccgccagcgcgctactgcccgaatatgccctacatccttccattgcgcctacccccggctatcgtggctacacctaccgccccagaagacgagcaactacccgacgccgaaccaccactggaacccgccgccgccgtcgccgtcgccagcccgtgctggccccgatttccgtgcgcagggtggctcgcgaaggaggcaggaccctggtgctgccaacagcgcgctaccaccccagcatcgtttaaaagccggtctttgtggttcttgcagatatggccctcacctgccgcctccgtttcccggtgccgggattccgaggaagaatgcaccgtaggaggggcatggccggccacggcctgacgggcggcatgcgtcgtgcgcaccaccggcggcggcgcgcgtcgcaccgtcgcatgcgcggcggtatcctgcccctccttattccactgatcgccgcggcgattggcgccgtgcccggaattgcatccgtggccttgcaggcgcagagacactgattaaaaacaagttgcatgtggaaaaatcaaaataaaaagtctggactctcacgctcgcttggtcctgtaactattttgtagaatggaagacatcaactttgcgtctctggccccgcgacacggctcgcgcccgttcatgggaaactggcaagatatcggcaccagcaatatgagcggtggcgccttcagctggggctcgctgtggagcggcattaaaaatttcggttccaccgttaagaactatggcagcaaggcctggaacagcagcacaggccagatgctgagggataagttgaaagagcaaaatttccaacaaaaggtggtagatggcctggcctctggcattagcggggtggtggacctggccaaccaggcagtgcaaaataagattaacagtaagcttgatccccgccctcccgtagaggagcctccaccggccgtggagacagtgtctccagaggggcgtggcgaaaagcgtccgcgccccgacagggaagaaactctggtgacgcaaatagacgagcctccctcgtacgaggaggcactaaagcaaggcctgcccaccacccgtcccatcgcgcccatggctaccggagtgctgggccagcacacacccgtaacgctggacctgcctccccccgccgacacccagcagaaacctgtgctgccaggcccgaccgccgttgttgtaacccgtcctagccgcgcgtccctgcgccgcgccgccagcggtccgcgatcgttgcggcccgtagccagtggcaactggcaaagcacactgaacagcatcgtgggtctgggggtgcaatccctgaagcgccgacgatgcttctgatagctaacgtgtcgtatgtgtgtcatgtatgcgtccatgtcgccgccagaggagctgctgagccgccgcgcgcccgctttccaagatggctaccccttcgatgatgccgcagtggtcttacatgcacatctcgggccaggacgcctcggagtacctgagccccgggctggtgcagtttgcccgcgccaccgagacgtacttcagcctgaataacaagtttagaaaccccacggtggcgcctacgcacgacgtgaccacagaccggtcccagcgtttgacgctgcggttcatccctgtggaccgtgaggatactgcgtactcgtacaaggcgcggttcaccctagctgtgggtgataaccgtgtgctggacatggcttccacgtactttgacatccgcggcgtgctggacaggggccctacttttaagccctactctggcactgcctacaacgccctggctcccaagggtgccccaaatccttgcgaatgggatgaagctgctactgctcttgaaataaacctagaagaagaggacgatgacaacgaagacgaagtagacgagcaagctgagcagcaaaaaactcacgtatttgggcaggcgccttattctggtataaatattacaaaggagggtattcaaataggtgtcgaaggtcaaacacctaaatatgccgataaaacatttcaacctgaacctcaaataggagaatctcagtggtacgaaacagaaattaatcatgcagctgggagagtcctaaaaaagactaccccaatgaaaccatgttacggttcatatgcaaaacccacaaatgaaaatggagggcaaggcattcttgtaaagcaacaaaatggaaagctagaaagtcaagtggaaatgcaatttttctcaactactgaggcagccgcaggcaatggtgataacttgactcctaaagtggtattgtacagtgaagatgtagatatagaaaccccagacactcatatttcttacatgcccactattaaggaaggtaactcacgagaactaatgggccaacaatctatgcccaacaggcctaattacattgcttttagggacaattttattggtctaatgtattacaacagcacgggtaatatgggtgttctggcgggccaagcatcgcagttgaatgctgttgtagatttgcaagacagaaacacagagctttcataccagcttttgcttgattccattggtgatagaaccaggtacttttctatgtggaatcaggctgttgacagctatgatccagatgttagaattattgaaaatcatggaactgaagatgaacttccaaattactgctttccactgggaggtgtgattaatacagagactcttaccaaggtaaaacctaaaacaggtcaggaaaatggatgggaaaaagatgctacagaattttcagataaaaatgaaataagagttggaaataattttgccatggaaatcaatctaaatgccaacctgtggagaaatttcctgtactccaacatagcgctgtatttgcccgacaagctaaagtacagtccttccaacgtaaaaatttctgataacccaaacacctacgactacatgaacaagcgagtggtggctcccgggctagtggactgctacattaaccttggagcacgctggtcccttgactatatggacaacgtcaacccatttaaccaccaccgcaatgctggcctgcgctaccgctcaatgttgctgggcaatggtcgctatgtgcccttccacatccaggtgcctcagaagttctttgccattaaaaacctccttctcctgccgggctcatacacctacgagtggaacttcaggaaggatgttaacatggttctgcagagctccctaggaaatgacctaagggttgacggagccagcattaagtttgatagcatttgcctttacgccaccttcttccccatggcccacaacaccgcctccacgcttgaggccatgcttagaaacgacaccaacgaccagtcctttaacgactatctctccgccgccaacatgctctaccctatacccgccaacgctaccaacgtgcccatatccatcccctcccgcaactgggcggctttccgcggctgggccttcacgcgccttaagactaaggaaaccccatcactgggctcgggctacgacccttattacacctactctggctctataccctacctagatggaaccttttacctcaaccacacctttaagaaggtggccattacctttgactcttctgtcagctggcctggcaatgaccgcctgcttacccccaacgagtttgaaattaagcgctcagttgacggggagggttacaacgttgcccagtgtaacatgaccaaagactggttcctggtacaaatgctagctaactataacattggctaccagggcttctatatcccagagagctacaaggaccgcatgtactccttctttagaaacttccagcccatgagccgtcaggtggtggatgatactaaatacaaggactaccaacaggtgggcatcctacaccaacacaacaactctggatttgttggctaccttgcccccaccatgcgcgaaggacaggcctaccctgctaacttcccctatccgcttataggcaagaccgcagttgacagcattacccagaaaaagtttctttgcgatcgcaccctttggcgcatcccattctccagtaactttatgtccatgggcgcactcacagacctgggccaaaaccttctctacgccaactccgcccacgcgctagacatgacttttgaggtggatcccatggacgagcccacccttctttatgttttgtttgaagtctttgacgtggtccgtgtgcaccagccgcaccgcggcgtcatcgaaaccgtgtacctgcgcacgcccttctcggccggcaacgccacaacataaagaagcaagcaacatcaacaacagctgccgccatgggctccagtgagcaggaactgaaagccattgtcaaagatcttggttgtgggccatattttttgggcacctatgacaagcgctttccaggctttgtttctccacacaagctcgcctgcgccatagtcaatacggccggtcgcgagactgggggcgtacactggatggcctttgcctggaacccgcactcaaaaacatgctacctctttgagccctttggcttttctgaccagcgactcaagcaggtttaccagtttgagtacgagtcactcctgcgccgtagcgccattgcttcttcccccgaccgctgtataacgctggaaaagtccacccaaagcgtacaggggcccaactcggccgcctgtggactattctgctgcatgtttctccacgcctttgccaactggccccaaactcccatggatcacaaccccaccatgaaccttattaccggggtacccaactccatgctcaacagtccccaggtacagcccaccctgcgtcgcaaccaggaacagctctacagcttcctggagcgccactcgccctacttccgcagccacagtgcgcagattaggagcgccacttctttttgtcacttgaaaaacatgtaaaaataatgtactagagacactttcaataaaggcaaatgcttttatttgtacactctcgggtgattatttacccccacccttgccgtctgcgccgtttaaaaatcaaaggggttctgccgcgcatcgctatgcgccactggcagggacacgttgcgatactggtgtttagtgctccacttaaactcaggcacaaccatccgcggcagctcggtgaagttttcactccacaggctgcgcaccatcaccaacgcgtttagcaggtcgggcgccgatatcttgaagtcgcagttggggcctccgccctgcgcgcgcgagttgcgatacacagggttgcagcactggaacactatcagcgccgggtggtgcacgctggccagcacgctcttgtcggagatcagatccgcgtccaggtcctccgcgttgctcagggcgaacggagtcaactttggtagctgccttcccaaaaagggcgcgtgcccaggctttgagttgcactcgcaccgtagtggcatcaaaaggtgaccgtgcccggtctgggcgttaggatacagcgcctgcataaaagccttgatctgcttaaaagccacctgagcctttgcgccttcagagaagaacatgccgcaagacttgccggaaaactgattggccggacaggccgcgtcgtgcacgcagcaccttgcgtcggtgttggagatctgcaccacatttcggccccaccggttcttcacgatcttggccttgctagactgctccttcagcgcgcgctgcccgttttcgctcgtcacatccatttcaatcacgtgctccttatttatcataatgcttccgtgtagacacttaagctcgccttcgatctcagcgcagcggtgcagccacaacgcgcagcccgtgggctcgtgatgcttgtaggtcacctctgcaaacgactgcaggtacgcctgcaggaatcgccccatcatcgtcacaaaggtcttgttgctggtgaaggtcagctgcaacccgcggtgctcctcgttcagccaggtcttgcatacggccgccagagcttccacttggtcaggcagtagtttgaagttcgcctttagatcgttatccacgtggtacttgtccatcagcgcgcgcgcagcctccatgcccttctcccacgcagacacgatcggcacactcagcgggttcatcaccgtaatttcactttccgcttcgctgggctcttcctcttcctcttgcgtccgcataccacgcgccactgggtcgtcttcattcagccgccgcactgtgcgcttacctcctttgccatgcttgattagcaccggtgggttgctgaaacccaccatttgtagcgccacatcttctctttcttcctcgctgtccacgattacctctggtgatggcgggcgctcgggcttgggagaagggcgcttctttttcttcttgggcgcaatggccaaatccgccgccgaggtcgatggccgcgggctgggtgtgcgcggcaccagcgcgtcttgtgatgagtcttcctcgtcctcggactcgatacgccgcctcatccgcttttttgggggcgcccggggaggcggcggcgacggggacggggacgacacgtcctccatggttgggggacgtcgcgccgcaccgcgtccgcgctcgggggtggtttcgcgctgctcctcttcccgactggccatttccttctcctataggcagaaaaagatcatggagtcagtcgagaagaaggacagcctaaccgccccctctgagttcgccaccaccgcctccaccgatgccgccaacgcgcctaccaccttccccgtcgaggcacccccgcttgaggaggaggaagtgattatcgagcaggacccaggttttgtaagcgaagacgacgaggaccgctcagtaccaacagaggataaaaagcaagaccaggacaacgcagaggcaaacgaggaacaagtcgggcggggggacgaaaggcatggcgactacctagatgtgggagacgacgtgctgttgaagcatctgcagcgccagtgcgccattatctgcgacgcgttgcaagagcgcagcgatgtgcccctcgccatagcggatgtcagccttgcctacgaacgccacctattctcaccgcgcgtaccccccaaacgccaagaaaacggcacatgcgagcccaacccgcgcctcaacttctaccccgtatttgccgtgccagaggtgcttgccacctatcacatctttttccaaaactgcaagatacccctatcctgccgtgccaaccgcagccgagcggacaagcagctggccttgcggcagggcgctgtcatacctgatatcgcctcgctcaacgaagtgccaaaaatctttgagggtcttggacgcgacgagaagcgcgcggcaaacgctctgcaacaggaaaacagcgaaaatgaaagtcactctggagtgttggtggaactcgagggtgacaacgcgcgcctagccgtactaaaacgcagcatcgaggtcacccactttgcctacccggcacttaacctaccccccaaggtcatgagcacagtcatgagtgagctgatcgtgcgccgtgcgcagcccctggagagggatgcaaatttgcaagaacaaacagaggagggcctacccgcagttggcgacgagcagctagcgcgctggcttcaaacgcgcgagcctgccgacttggaggagcgacgcaaactaatgatggccgcagtgctcgttaccgtggagcttgagtgcatgcagcggttctttgctgacccggagatgcagcgcaagctagaggaaacattgcactacacctttcgacagggctacgtacgccaggcctgcaagatctccaacgtggagctctgcaacctggtctcctaccttggaattttgcacgaaaaccgccttgggcaaaacgtgcttcattccacgctcaagggcgaggcgcgccgcgactacgtccgcgactgcgtttacttatttctatgctacacctggcagacggccatgggcgtttggcagcagtgcttggaggagtgcaacctcaaggagctgcagaaactgctaaagcaaaacttgaaggacctatggacggccttcaacgagcgctccgtggccgcgcacctggcggacatcattttccccgaacgcctgcttaaaaccctgcaacagggtctgccagacttcaccagtcaaagcatgttgcagaactttaggaactttatcctagagcgctcaggaatcttgcccgccacctgctgtgcacttcctagcgactttgtgcccattaagtaccgcgaatgccctccgccgctttggggccactgctaccttctgcagctagccaactaccttgcctaccactctgacataatggaagacgtgagcggtgacggtctactggagtgtcactgtcgctgcaacctatgcaccccgcaccgctccctggtttgcaattcgcagctgcttaacgaaagtcaaattatcggtacctttgagctgcagggtccctcgcctgacgaaaagtccgcggctccggggttgaaactcactccggggctgtggacgtcggcttaccttcgcaaatttgtacctgaggactaccacgcccacgagattaggttctacgaagaccaatcccgcccgcctaatgcggagcttaccgcctgcgtcattacccagggccacattcttggccaattgcaagccatcaacaaagcccgccaagagtttctgctacgaaagggacggggggtttacttggacccccagtccggcgaggagctcaacccaatccccccgccgccgcagccctatcagcagcagccgcgggcccttgcttcccaggatggcacccaaaaagaagctgcagctgccgccgccacccacggacgaggaggaatactgggacagtcaggcagaggaggttttggacgaggaggaggaggacatgatggaagactgggagagcctagacgaggaagcttccgaggtcgaagaggtgtcagacgaaacaccgtcaccctcggtcgcattcccctcgccggcgccccagaaatcggcaaccggttccagcatggctacaacctccgctcctcaggcgccgccggcactgcccgttcgccgacccaaccgtagatgggacaccactggaaccagggccggtaagtccaagcagccgccgccgttagcccaagagcaacaacagcgccaaggctaccgctcatggcgcgggcacaagaacgccatagttgcttgcttgcaagactgtgggggcaacatctccttcgcccgccgctttcttctctaccatcacggcgtggccttcccccgtaacatcctgcattactaccgtcatctctacagcccatactgcaccggcggcagcggcagcaacagcagcggccacacagaagcaaaggcgaccggatagcaagactctgacaaagcccaagaaatccacagcggcggcagcagcaggaggaggagcgctgcgtctggcgcccaacgaacccgtatcgacccgcgagcttagaaacaggatttttcccactctgtatgctatatttcaacagagcaggggccaagaacaagagctgaaaataaaaaacaggtctctgcgatccctcacccgcagctgcctgtatcacaaaagcgaagatcagcttcggcgcacgctggaagacgcggaggctctcttcagtaaatactgcgcgctgactcttaaggactagtttcgcgccctttctcaaatttaagcgcgaaaactacgtcatctccagcggccacacccggcgccagcacctgttgtcagcgccattatgagcaaggaaattcccacgccctacatgtggagttaccagccacaaatgggacttgcggctggagctgcccaagactactcaacccgaataaactacatgagcgcgggaccccacatgatatcccgggtcaacggaatacgcgcccaccgaaaccgaattctcctggaacaggcggctattaccaccacacctcgtaataaccttaatccccgtagttggcccgctgccctggtgtaccaggaaagtcccgctcccaccactgtggtacttcccagagacgcccaggccgaagttcagatgactaactcaggggcgcagcttgcgggcggctttcgtcacagggtgcggtcgcccgggcagggtataactcacctgacaatcagagggcgaggtattcagctcaacgacgagtcggtgagctcctcgcttggtctccgtccggacgggacatttcagatcggcggcgccggccgctcttcattcacgcctcgtcaggcaatcctaactctgcagacctcgtcctctgagccgcgctctggaggcattggaactctgcaatttattgaggagtttgtgccatcggtctactttaaccccttctcgggacctcccggccactatccggatcaatttattcctaactttgacgcggtaaaggactcggcggacggctacgactgaatgttaagtggagaggcagagcaactgcgcctgaaacacctggtccactgtcgccgccacaagtgctttgcccgcgactccggtgagttttgctactttgaattgcccgaggatcatatcgagggcccggcgcacggcgtccggcttaccgcccagggagagcttgcccgtagcctgattcgggagtttacccagcgccccctgctagttgagcgggacaggggaccctgtgttctcactgtgatttgcaactgtcctaaccctggattacatcaagatcctctagttaatgtcaggtcgcctaagtcgattaactagagtacccggggatcttattccctttaactaataaaaaaaaataataaagcatcacttacttaaaatcagttagcaaatttctgtccagtttattcagcagcacctccttgccctcctcccagctctggtattgcagcttcctcctggctgcaaactttctccacaatctaaatggaatgtcagtttcctcctgttcctgtccatccgcacccactatcttcatgttgttgcagatgaagcgcgcaagaccgtctgaagataccttcaaccccgtgtatccatatgacacggaaaccggtcctccaactgtgccttttcttactcctccctttgtatcccccaatgggtttcaagagagtccccctggggtactctctttgcgcctatccgaacctctagttacctccaatggcatgcttgcgctcaaaatgggcaacggcctctctctggacgaggccggcaaccttacctcccaaaatgtaaccactgtgagcccacctctcaaaaaaaccaagtcaaacataaacctggaaatatctgcacccctcacagttacctcagaagccctaactgtggctgccgccgcacctctaatggtcgcgggcaacacactcaccatgcaatcacaggccccgctaaccgtgcacgactccaaacttagcattgccacccaaggacccctcacagtgtcagaaggaaagctagccctgcaaacatcaggccccctcaccaccaccgatagcagtacccttactatcactgcctcaccccctctaactactgccactggtagcttgggcattgacttgaaagagcccatttatacacaaaatggaaaactaggactaaagtacggggctcctttgcatgtaacagacgacctaaacactttgaccgtagcaactggtccaggtgtgactattaataatacttccttgcaaactaaagttactggagccttgggttttgattcacaaggcaatatgcaacttaatgtagcaggaggactaaggattgattctcaaaacagacgccttatacttgatgttagttatccgtttgatgctcaaaaccaactaaatctaagactaggacagggccctctttttataaactcagcccacaacttggatattaactacaacaaaggcctttacttgtttacagcttcaaacaattccaaaaagcttgaggttaacctaagcactgccaaggggttgatgtttgacgctacagccatagccattaatgcaggagatgggcttgaatttggttcacctaatgcaccaaacacaaatcccctcaaaacaaaaattggccatggcctagaatttgattcaaacaaggctatggttcctaaactaggaactggccttagttttgacagcacaggtgccattacagtaggaaacaaaaataatgataagctaactttgtggaccacaccagctccatctcctaactgtagactaaatgcagagaaagatgctaaactcactttggtcttaacaaaatgtggcagtcaaatacttgctacagtttcagttttggctgttaaaggcagtttggctccaatatctggaacagttcaaagtgctcatcttattataagatttgacgaaaatggagtgctactaaacaattccttcctggacccagaatattggaactttagaaatggagatcttactgaaggcacagcctatacaaacgctgttggatttatgcctaacctatcagcttatccaaaatctcacggtaaaactgccaaaagtaacattgtcagtcaagtttacttaaacggagacaaaactaaacctgtaacactaaccattacactaaacggtacacaggaaacaggagacacaactccaagtgcatactctatgtcattttcatgggactggtctggccacaactacattaatgaaatatttgccacatcctcttacactttttcatacattgcccaagaataaagaatcgtttgtgttatgtttcaacgtgtttatttttcaattgcagaaaatttcaagtcatttttcattcagtagtatagccccaccaccacatagcttatacagatcaccgtaccttaatcaaactcacagaaccctagtattcaacctgccacctccctcccaacacacagagtacacagtcctttctccccggctggccttaaaaagcatcatatcatgggtaacagacatattcttaggtgttatattccacacggtttcctgtcgagccaaacgctcatcagtgatattaataaactccccgggcagctcacttaagttcatgtcgctgtccagctgctgagccacaggctgctgtccaacttgcggttgcttaacgggcggcgaaggagaagtccacgcctacatgggggtagagtcataatcgtgcatcaggatagggcggtggtgctgcagcagcgcgcgaataaactgctgccgccgccgctccgtcctgcaggaatacaacatggcagtggtctcctcagcgatgattcgcaccgcccgcagcataaggcgccttgtcctccgggcacagcagcgcaccctgatctcacttaaatcagcacagtaactgcagcacagcaccacaatattgttcaaaatcccacagtgcaaggcgctgtatccaaagctcatggcggggaccacagaacccacgtggccatcataccacaagcgcaggtagattaagtggcgacccctcataaacacgctggacataaacattacctcttttggcatgttgtaattcaccacctcccggtaccatataaacctctgattaaacatggcgccatccaccaccatcctaaaccagctggccaaaacctgcccgccggctatacactgcagggaaccgggactggaacaatgacagtggagagcccaggactcgtaaccatggatcatcatgctcgtcatgatatcaatgttggcacaacacaggcacacgtgcatacacttcctcaggattacaagctcctcccgcgttagaaccatatcccagggaacaacccattcctgaatcagcgtaaatcccacactgcagggaagacctcgcacgtaactcacgttgtgcattgtcaaagtgttacattcgggcagcagcggatgatcctccagtatggtagcgcgggtttctgtctcaaaaggaggtagacgatccctactgtacggagtgcgccgagacaaccgagatcgtgttggtcgtagtgtcatgccaaatggaacgccggacgtagtcatatttcctgaagcaaaaccaggtgcgggcgtgacaaacagatctgcgtctccggtctcgccgcttagatcgctctgtgtagtagttgtagtatatccactctctcaaagcatccaggcgccccctggcttcgggttctatgtaaactccttcatgcgccgctgccctgataacatccaccaccgcagaataagccacacccagccaacctacacattcgttctgcgagtcacacacgggaggagcgggaagagctggaagaaccatgtttttttttttattccaaaagattatccaaaacctcaaaatgaagatctattaagtgaacgcgctcccctccggtggcgtggtcaaactctacagccaaagaacagataatggcatttgtaagatgttgcacaatggcttccaaaaggcaaacggccctcacgtccaagtggacgtaaaggctaaacccttcagggtgaatctcctctataaacattccagcaccttcaaccatgcccaaataattctcatctcgccaccttctcaatatatctctaagcaaatcccgaatattaagtccggccattgtaaaaatctgctccagagcgccctccaccttcagcctcaagcagcgaatcatgattgcaaaaattcaggttcctcacagacctgtataagattcaaaagcggaacattaacaaaaataccgcgatcccgtaggtcccttcgcagggccagctgaacataatcgtgcaggtctgcacggaccagcgcggccacttccccgccaggaaccatgacaaaagaacccacactgattatgacacgcatactcggagctatgctaaccagcgtagccccgatgtaagcttgttgcatgggcggcgatataaaatgcaaggtgctgctcaaaaaatcaggcaaagcctcgcgcaaaaaagaaagcacatcgtagtcatgctcatgcagataaaggcaggtaagctccggaaccaccacagaaaaagacaccatttttctctcaaacatgtctgcgggtttctgcataaacacaaaataaaataacaaaaaaacatttaaacattagaagcctgtcttacaacaggaaaaacaacccttataagcataagacggactacggccatgccggcgtgaccgtaaaaaaactggtcaccgtgattaaaaagcaccaccgacagctcctcggtcatgtccggagtcataatgtaagactcggtaaacacatcaggttgattcacatcggtcagtgctaaaaagcgaccgaaatagcccgggggaatacatacccgcaggcgtagagacaacattacagcccccataggaggtataacaaaattaataggagagaaaaacacataaacacctgaaaaaccctcctgcctaggcaaaatagcaccctcccgctccagaacaacatacagcgcttccacagcggcagccataacagtcagccttaccagtaaaaaagaaaacctattaaaaaaacaccactcgacacggcaccagctcaatcagtcacagtgtaaaaaagggccaagtgcagagcgagtatatataggactaaaaaatgacgtaacggttaaagtccacaaaaaacacccagaaaaccgcacgcgaacctacgcccagaaacgaaagccaaaaaacccacaacttcctcaaatcgtcacttccgttttcccacgttacgtcacttcccattttaagaaaactacaattcccaacacatacaagttactccgccctaaaacctacgtcacccgccccgttcccacgccccgcgccacgtcacaaactccaccccctcattatcatattggcttcaatccaaaataaggtatattattgatgatgttaattaacatgcatggatcctacgtctcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagcct

Claims (1)

1. A high throughput screening method for a drug that inhibits human adenovirus proliferation, said method comprising the steps of: 1) Preparation of GFP-integrated human adenovirus; 2) Detection of human adenovirus titres using TCID ₅₀ Detecting by a method; 3) Positive drug, brincidofosir, MTT assay of HEK-293 cells by cidofosir, was tested using MTT kit according to the instructions; 4) High throughput screening of drugs to inhibit human adenovirus proliferation; the preparation method of the GFP-integrated human adenovirus comprises the steps of constructing plasmid pAd-YFP by using pAdTrack-CMV and pAdEasy-1 plasmid, and transfecting the plasmid pAd-YFP into 293FT cells to obtain the GFP-integrated human adenovirus; wherein the screening method of step 4) comprises a 24-well plate screenThe selection method or the 48-orifice plate screening method comprises the following steps:

1d,24 th well plate, 5X 10 HEK-293 cells plated per well ⁴ In other words, 1mL of the culture medium contains 5×10 ⁴ cells/mL, 1mL into 24 well plate;

2d, adding medicines 24h after plating; the test should (1) a blank, namely: no drug and no virus, (2) positive virus control, i.e.: a virus-only, no-drug group, (3) a positive drug group, i.e., a virus-plus positive drug, cidofovir, 50 μm and brincdofovir, 250nM, (4) a test group, i.e.: adding viruses and medicines to be screened;

2h after dosing, 2 μl of human adenovirus per well was added, moi=0.1; the blank control group was not added with virus;

5d, 3d after virus addition, spread cells: 24-well plate, 1X 10 per well ⁵ HEK-293 cells of (A) by 1X 10 in 1mL of medium ⁵ cells/mL, 1mL into 24 well plate; the number of the cell holes paved for the first time is consistent with that of the cell holes paved for the first time;

6d, taking 100 mu L of virus supernatant of each hole plated for the first time, and adding the virus supernatant into cells plated for the second time, wherein the sequence is consistent with that of the first time;

8d, namely 2d after virus addition, removing supernatant, digesting 100 mu L of pancreatin from the cell plate paved for the 2 nd time, adding 200 mu L of culture medium for neutralization, repeatedly blowing, sucking into a flow tube for flow analysis, and recording the detection result of each sample;

comparing the flow result of the medicine to be tested with the positive medicine group, and analyzing whether the medicine to be tested has the effect of inhibiting human adenovirus;

the 48-orifice plate screening method specifically comprises the following steps:

1d,48 well plates, 2.5X10 HEK-293 cells were plated per well ⁴ In other words, 1mL of the culture medium contains 5×10 ⁴ cells/mL, aspirate 500. Mu.L into a 48 well plate;

2d, adding medicines 24h after plating; the test should (1) a blank, namely: no drug and no virus, (2) positive virus control, i.e.: only virus, no drug, and (3) positive drug, namely: add virus, add positive drug, cidofovir 50 μm and brinzidofovir 250nM, (4) test group, i.e.: adding viruses and medicines to be screened;

2h after dosing, 2 μl adenovirus per well with moi=0.1; the blank control group was not added with virus;

5d, 3d after virus addition, spread cells: 48-well plate, 5X 10 per well ⁴ HEK-293 cells of (A) by 1X 10 in 1mL of medium ⁵ cells/mL, aspirate 500. Mu.L into a 48 well plate; the number of the cell holes paved for the first time is consistent with that of the cell holes paved for the first time;

6d, taking 50 mu L of virus supernatant of each hole plated for the first time, and adding the virus supernatant into cells plated for the second time, wherein the sequence is consistent with that of the first time;

8d, namely 2d after virus addition, the cell plate paved for the 2 nd time is discarded, 100 mu L of pancreatin is digested, 200 mu L of culture medium is added for neutralization,

after repeated blowing, sucking the sample into a flow pipe for flow analysis, and recording the detection result of each sample;

comparing the flow result of the medicine to be tested with the positive medicine group, and analyzing whether the medicine to be tested has the effect of inhibiting human adenovirus.

Images