Nrf2-ARE fusion promoter plasmid and its application in screening of anti-aquatic virus drugs

Abstract

The present invention discloses an Nrf2-ARE fusion promoter plasmid and its application in anti-aquatic virus drug screening, wherein the Nrf2-ARE fusion promoter plasmid comprises a promoter fragment of an Nrf2 gene derived from epithelial cells of muscle tissue of blackhead minnow and a partial promoter fragment of an HO-1 gene, wherein the sequence of the partial promoter fragment of the HO-1 gene is shown in SEQ ID NO. 1. The fusion promoter plasmid can widely respond to a variety of Chinese herbal active ingredients such as silybin, curcumin and andrographolide, is superior to a simple promoter in drug screening effect, has stronger tolerance and a wider range, is more conducive to rapid, efficient and economical initial screening of anti-aquatic virus drugs, and provides a target for the development of green and efficient antiviral drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of drug screening, and specifically relates to an Nrf2-ARE fusion promoter plasmid and application thereof in screening of anti-aquatic virus drugs. Background Art

[0002] There are many types of viral diseases in aquatic animals, which are widespread, spread quickly, and have a high mortality rate. The pathogens are tiny and replicate in host cells after infecting animals. Therefore, if you want to use drugs to kill viruses without harming host cells, the drugs must have extremely high selectivity, and drug development is difficult. At the same time, viral diseases are fast-transmitting and have a latent period. Once symptoms appear, they have spread before they can be treated, and the timing of medication is difficult to grasp. Moreover, as a biological macromolecule virus with a simple structure, it is also easy to mutate, and it is difficult to find broad-spectrum drugs to treat it. Therefore, there is currently no specific medicine for viral diseases. The abuse of other drugs will not only reduce the spawning quality of aquatic animal parents such as fish and shrimp, but also reduce the disease resistance of farmed animals themselves, resulting in the accumulation of toxic and harmful substances, and polluting water bodies and the environment. In summary, the lack of antiviral drugs and the limited types of vaccines are still difficulties in the prevention and control of viral diseases in aquatic animals.

[0003] Traditional drug development has problems such as high cost, long cycle, low safety, and unknown mechanism. The use of natural or synthetic compounds to screen antiviral active ingredients can guide new antiviral targets, clarify antiviral mechanisms, and then develop new antiviral drugs. Studies in recent years have shown that some Chinese herbal medicines and their active ingredients have shown certain effects on the prevention and treatment of aquatic viral diseases. Astragalus polysaccharide can inhibit the viral replication of SVCV; Isatis root and berberine have anti-grass carp reovirus effects; Robinia seeds can induce the production of host interferon and thus provide antiviral immune protection for the body. Therefore, green drugs with the same origin as medicine and food have gradually become a hot spot for antiviral drug screening.

[0004] From a technical perspective, there are currently traditional, high-throughput, high-content and computer virtual screening technology methods for drug screening. From a targeting perspective, there are two drug screening methods: virus-targeted and host-targeted. Among them, traditional drug screening methods based on in vivo experiments and the degree of cytopathic effects have a long cycle, low efficiency and subjective influences; screening methods such as high-content and computer virtual screening are limited by professional instruments, software and technology, and are costly, making it difficult for basic laboratories to meet the needs of this drug screening method; drug screening methods targeting viral genes are limited by the type of virus, are not broad-spectrum, and are prone to drug resistance. Therefore, how to efficiently and quickly screen green, broad-spectrum antiviral drugs will become an important issue that needs to be solved in the screening of anti-aquatic virus drugs. Summary of the invention

[0005] In view of the problems in the prior art, the purpose of the present invention is to provide a new screening method for anti-aquatic virus drugs, which is specifically based on a fusion promoter plasmid targeting the antioxidant pathway and has the advantages of broad spectrum, simple operation, and short experimental cycle.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention first provides an Nrf2-ARE fusion promoter plasmid, which comprises a promoter fragment of an Nrf2 gene derived from epithelial cells of muscle tissue of blackhead minnow and a partial promoter fragment of an HO-1 gene, wherein the partial promoter fragment of the HO-1 gene is marked as ARE and its sequence is shown in SEQ ID NO.1, and the promoter fragment of the Nrf2 gene is shown in SEQ ID NO.2.

[0008] Nuclear transcription factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) are related to the antioxidant pathway of fathead minnow muscle cells (FHM). Based on bioinformatics analysis of the promoter region of the Nrf2 gene and the promoter region of the HO-1 gene, a fusion promoter plasmid with promoter activity was designed and constructed.

[0009] In the above technical solution, ARE may appear repeatedly. For example, in a specific embodiment, the plasmid contains 5 tandem AREs and the fragments are connected by CCC. In this case, the sequence of the fusion promoter fragment is shown in SEQ ID NO.4.

[0010] In the above technical solution, the plasmid also contains a reporter gene for detecting the activity of the fusion promoter, such as a luciferase reporter gene.

[0011] The present invention further verifies through in vitro experiments that the Nrf2-ARE fusion promoter plasmid with the above structure is responsive to drugs, and thus can be used for screening of anti-aquatic animal virus drugs.

[0012] When the activity of the Nrf2-ARE fusion promoter is activated by drugs, the activation of the fusion promoter activity can further activate the body's endogenous antioxidant and anti-inflammatory defense pathways, thereby resisting the damage caused by viral infection. Therefore, the Nrf2-ARE fusion promoter can be used to screen antiviral drugs based on the above principle.

[0013] In the above technical solution, the aquatic animal virus should be an aquatic animal virus that can cause oxidative stress in the host, such as spring viremia of carp virus (SVCV), rana grylioiridovirus (RGV), etc.

[0014] Furthermore, the method for screening anti-aquatic animal virus drugs based on the Nrf2-ARE fusion promoter plasmid is: first transfect the Nrf2-ARE fusion promoter plasmid into cells, then incubate the drug with the cells, detect the promoter activity, and screen the drug according to the strength of the promoter activity. When the reporter gene in the Nrf2-ARE fusion promoter plasmid is a luciferase reporter gene, the strength of the promoter activity can be quantified according to the intensity of the fluorescence signal, which is more intuitive and clear.

[0015] Experimental results have shown that the Nrf2-ARE fusion promoter can be activated by active ingredients of various Chinese herbal medicines, such as andrographolide, silybin, curcumin, etc., and the activity of the fusion promoter can still be significantly activated when the drug concentration is as low as 6 μg / mL.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The anti-aquatic virus drug screening method based on the activity of the Nrf2-ARE fusion promoter provided by the present invention changes the phenomenon that a single promoter has a blind spot in drug screening, is more intensive and convenient, has a short experimental cycle, is simple to operate and easy to repeat, saves the cost of batch screening, and reduces the subjectivity of drug effect judgment;

[0018] (2) The drug screening method provided by the present invention targets the host's antioxidant signaling pathway and is applicable to all aquatic animal viruses that can cause oxidative stress in the host. It has a broad spectrum and is not prone to drug resistance. It provides a new solution for drug resistance caused by drug abuse in aquaculture and large-scale screening of green drugs;

[0019] (3) The fusion promoter provided by the present invention can respond to a variety of active ingredients of Chinese herbal medicines. The initial screening results of the drugs showed that 10 μg / mL of andrographolide / silybin / curcumin can significantly activate the activity of the fusion promoter. Further optimal concentration screening showed that drug concentrations of 6 to 50 μg / mL can almost all significantly activate the activity of the fusion promoter. Compared with the screening of a single promoter, not only is the screening range wider, but the response effect of each concentration of the drug is also more obvious.

[0020] (4) The fusion promoter plasmid provided by the present invention can be amplified in large quantities in E. coli and is easy to obtain; and when the reporter gene used to detect the promoter activity is a luciferase reporter system, it not only has high sensitivity and a wide detection linear range, but also can quantitatively indicate the strength of the promoter activity by the intensity of the fluorescent signal, and the result is more intuitive and clear;

[0021] (5) Through experimental detection, the positive drugs screened by the screening method provided by the present invention can inhibit the replication of SVCV and RGV, which verifies the effectiveness of the drug screening method and is beneficial to the prevention and control of related aquatic viral diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The sequence of the promoter region of the Nrf2 gene and the transcription factor binding site map in Example 1 of the present invention;

[0023] Figure 2 The sequence of the promoter region of the HO-1 gene and the transcription factor binding site map in Example 1 of the present invention, wherein the sequence marked with a box line is the selected ARE sequence;

[0024] Figure 3 This is a structural diagram of the pGL3-Nrf2-ARE fusion promoter plasmid in Example 1 of the present invention;

[0025] Figure 4 The cytotoxicity test results of andrographolide, silybin and curcumin in Example 2 of the present invention are shown;

[0026] Figure 5 The response of the pGL3-Nrf2-ARE plasmid in Example 2 of the present invention to 10 μg / mL of andrographolide, silybin and curcumin;

[0027] Figure 6 The responses of pGL3-Nrf2 and pGL3-HO-1 plasmids in Example 2 of the present invention to 10 μg / mL of andrographolide, silybin and curcumin;

[0028] Figure 7 The response of the pGL3-Nrf2-ARE plasmid in Example 2 of the present invention to different concentrations of the initially screened positive drugs;

[0029] Figure 8 This is a graph showing the antiviral activity test results of the positive drugs screened in Example 3 of the present invention against SVCV and RGV. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0031] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0032] Example 1

[0033] In this example, a plasmid containing a pGL3-Nrf2-ARE fusion promoter was constructed, and the construction process is as follows:

[0034] (1) Acquisition of Nrf2 and HO-1 promoter sequences

[0035] The genomic DNA sequences and mRNA sequences of Nrf2 (Gene ID: 120459756, NCBI Reference Sequence: XM_039647189.1) and HO-1 (Gene ID: 120467971, NCBI Reference Sequence: XM_039656609.1) of FHM were obtained using the NCBI database (National Center for Biotechnology Information (nih.gov)). The transcription factor binding sites were predicted using AnimalTFDB3.0 (AnimalTFDB3 (hust.edu.cn)) and JASPAR (JASPAR-A database of transcription factor binding profiles (genereg.net)) online software. The prediction results are shown in the figure. Figure 1 and Figure 2 As shown, Figure 1 To predict the specific sequence and transcription factor binding sites of Nrf2 promoter region, Figure 2 The specific sequence of the HO-1 promoter region and the prediction of the transcription factor binding site. The promoter region sequence of the Nrf2 gene is shown in SEQ ID NO.2, and the promoter region sequence of the HO-1 gene is shown in SEQ ID NO.3.

[0036] (2) Selection and synthesis of ARE and construction of fusion plasmid

[0037] According to the prediction results of the above transcription factor binding sites, the specific sequence of the antioxidant element (ARE) enriched with transcription factors such as JUN / FOS / Nrf2 / Maf was determined: 5'-TGGTTAGCGCGTTCATGCTGACTCACGGTTTCAACATCGG-3' (SEQ ID NO.1). The five ARE element sequences were connected in series, and the two were connected by CCC. They were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the total length of the target sequence fragment was 212bp. The synthesized tandem ARE fragment was inserted into the pGL3-Nrf2 promoter plasmid that had been constructed and verified to be active to obtain the pGL3-Nrf2-ARE fusion promoter plasmid. The structural schematic diagram of the pGL3-Nrf2-ARE fusion promoter plasmid is shown in the figure. Figure 3As shown, wherein the sequence of the fusion promoter fragment is shown as SEQ ID NO.4.

[0038] Example 2

[0039] This example verifies the response of the pGL3-Nrf2-ARE fusion promoter to the screened drugs, wherein the drugs selected in this example are andrographolide, silybin and curcumin. The specific operation of the verification process is as follows:

[0040] (1) To confirm the concentration of the drug, CCK-8 was used to detect the cytotoxicity of the drug. The CCK-8 detection reagent is based on WST-8, which is a compound similar to MTT. In the presence of the electron coupling reagent 1-Methoxy PMS, it can be reduced to generate orange-yellow water-soluble formazan; the more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color is linearly related to the number of cells. The detailed steps are as follows:

[0041] ① Transfer cells to a 96-well plate, add 100 μL of culture medium to each well, the cell density should be between 1 000 and 5 000 cells / well, and fill the edge wells with sterile PBS;

[0042] ②5% CO2, 28°C incubated until the cells adhered to the wall (about 6-8h), then treated the cells with drugs in culture medium containing different concentrations of drugs (0, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg / mL), with 6 replicate wells for each gradient, continued incubation for 24h, and observed the cell status under an inverted microscope;

[0043] ③ Add 10 μL CCK-8 solution to each well and continue culturing for 2.5 h;

[0044] ④ Use a microplate reader to measure the absorbance of each well at OD 450nm and calculate the cell viability.

[0045] The cytotoxicity results of the three drugs are as follows Figure 4 As shown in the figure, except for curcumin, the cell survival rates of the other two drugs at a concentration of 50 μg / mL were all greater than 70%. Therefore, in subsequent experiments, a concentration of 10 μg / mL was selected for preliminary drug screening.

[0046] (2) 24 hours after transfection of the fusion promoter plasmid and the pRL-TK plasmid, andrographolide, silybin and curcumin were incubated with the cells at a concentration of 10 μg / mL at 28°C for 24 hours, and then luciferase activity was detected according to the operating instructions of the dual-luciferase reporter gene assay kit.

[0047] Activity test results such as Figure 5As shown in Figure 2, the promoter activity of pGL3-Nrf2-ARE was significantly upregulated after drug treatment, and it responded to all three drugs. Figure 6 -a) and pGL3-HO-1( Figure 6 -b, i.e., containing only the promoter sequence of the HO-1 gene) can respond to two of the three drugs in the screening, which results in a blind spot in the screening and reduces the efficiency of drug screening. It can be seen that the fusion promoter provided by the present invention improves the disadvantages of a single promoter in drug screening.

[0048] (3) The fusion promoter plasmid and pRL-TK plasmid were transfected again. After 24 h, the effective drugs screened above were gradiently diluted, and 6 drug concentrations (0, 3.125, 6.25, 12.5, 25, and 50 μg / mL) determined based on the drug cytotoxicity experiment were selected to treat the cells. After 24 h at 28°C, the effect of the drug concentration gradient on the promoter activity was detected.

[0049] Test results such as Figure 7 It was shown that the three drugs at 6.25-50 μg / mL could significantly activate the promoter activity of pGL3-Nrf2-ARE.

[0050] Example 3

[0051] Oxidative stress is one of the main pathological mechanisms of various viral infections, such as spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). As an endogenous defense mechanism, the antioxidant pathway not only maintains the body's oxidation-reduction balance homeostasis, but also antagonizes various viruses such as hepatitis C virus (HCV) and SVCV.

[0052] In order to detect the antiviral ability of the drug obtained by screening based on the activity of the Nrf2-ARE fusion promoter, this example selects RNA virus SVCV and DNA virus RGV to verify the antiviral effect of andrographolide, and the specific operation is as follows:

[0053] Cells were pretreated with andrographolide for 6 hours, and then infected with SVCV and RGV at 0.1 MOI, respectively. After incubation at 28 ° C for 1 hour, the culture medium containing the corresponding drug concentration was replaced and cultured. Cell supernatant and cell samples were collected, and the virus titer and virus replication level were determined to verify the drug screening strategy based on promoter activity. The collected cell culture supernatant was diluted 10 times in a row with 6 replicates per gradient, added to the cell culture plate, 100 μL per well, and the diluent was used as a negative control. After 7 days of culture, the cell lesions of all wells were observed and recorded, and the virus titer was calculated according to the Reed-Muench method.

[0054] The results are as follows Figure 8 As shown, drug treatment can reduce the replication of SVCV and RGV and the number of viral particles in the supernatant, which indicates that the method of screening anti-aquatic virus drugs based on promoter activity is feasible.

[0055] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Nrf2-ARE fusion promoter plasmid, characterized in that: The plasmid comprises a fusion promoter fragment whose sequence is shown as SEQ ID NO.

4.

2. The Nrf2-ARE fusion promoter plasmid according to claim 1, characterized in that The plasmid contains a luciferase reporter gene fragment.

3. Use of the Nrf2-ARE fusion promoter plasmid as described in claim 1 or 2 in screening of anti-aquatic animal virus drugs.

4. The use according to claim 3, characterized in that: First, the Nrf2-ARE fusion promoter plasmid was transfected into the cells, and then the drugs were incubated with the cells to detect the activity of the Nrf2-ARE fusion promoter.

5. The use according to claim 3, characterized in that: The medicine is a Chinese herbal medicine.

6. The use according to claim 5, characterized in that: The drug is andrographolide, silybin or curcumin.

7. The use according to claim 4, characterized in that: The drug concentration in the incubation system is ≥6 μg / mL.

8. The use according to claim 3, characterized in that: The aquatic animal virus is specifically an aquatic animal virus that can cause oxidative stress in the host.

9. The use according to claim 8, characterized in that: The aquatic animal viruses include SVCV and RGV.

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