Application of ABHD6-targeted inhibitor in preparation of products for resisting porcine reproductive and respiratory syndrome virus and anti-virus product of ABHD6-targeted inhibitor

Abstract

The invention discloses application of a targeted ABHD6 inhibitor in preparation of a product for resisting porcine reproductive and respiratory syndrome virus and an antiviral product thereof. The inhibitor for targeting ABHD6 is a compound WWL70 for inhibiting the activity of host ABHD6, and / or a small interfering RNA for silencing or down-regulating the expression level of a host ABHD6 gene, the small interfering RNA is selected from SI-ABHD6-2 and / or SI-ABHD6-3, and the antisense strand of SI-ABHD6-2 is as shown in SEQ ID NO: 8; and the antisense strand of the SI-ABHD6-3 is as shown in SEQ ID NO: 10. Experiments prove that the compound WWL70 and the small interfering RNA (SI-ABHD6-2 or SI-ABHD6-3) are respectively used for treating cells and then infecting the cells with the porcine reproductive and respiratory syndrome virus, so that the replication of the porcine reproductive and respiratory syndrome virus can be obviously inhibited, and the effect of resisting the infection of the porcine reproductive and respiratory syndrome virus is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal virus biological prevention and treatment, and more particularly relates to application of an inhibitor targeting ABHD6 in preparation of a product against porcine reproductive and respiratory syndrome virus and an antiviral product thereof. BACKGROUND

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a porcine infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which leads to problems such as reproductive disorders in sows, respiratory diseases in piglets and high mortality, and causes significant economic losses to the pig industry. Vaccines or drugs targeting the virus itself are prone to cause virus recombination and variation, increasing the prevention and control pressure. Therefore, it is necessary to develop new virus prevention and control strategies. Studies have found that a large number of host factors are involved in the PRRSV infection process, and these host factors are highly conserved. Anti-viral drugs or feed developed based on these host factors have broad-spectrum and good application prospects. For example, it is found in patent CN118516408A that overexpression of PEBP4 protein promotes PRRSV replication, which can be used as a host target point related to PRRSV replication. However, it is not completely clear which host factors can be used as an independent target point for anti-PRRSV infection, because there are many host factors involved in PRRSV infection.

[0003] Alpha / beta hydrolase domain 6 (ABHD6) is a multifunctional lipid hydrolase that participates in metabolic-immune cross-regulation by regulating lipid raft assembly and degrading anti-inflammatory lipids (such as PGD2-G).

[0004] Studies have shown that ABHD6 and its substrates are involved in the regulation of various (patho) physiological processes, including neurotransmission, insulin secretion, fat browning, and neurological and metabolic diseases, making the enzyme a promising therapeutic target for treating a variety of diseases. However, there is no related research indicating that the host factor ABHD6 is related to porcine reproductive and respiratory syndrome. Therefore, it is necessary to study whether regulating the expression level of the host ABHD6 gene or the activity of the ABHD6 protein has an impact on the infection of porcine reproductive and respiratory syndrome virus, so as to provide a new approach and method for the prevention or treatment of porcine reproductive and respiratory syndrome. SUMMARY

[0005] In view of the above defects or improvement needs of the prior art, the application provides an application of an ABHD6-targeting inhibitor in the preparation of an anti-porcine reproductive and respiratory syndrome virus product and an anti-virus product thereof, which aims to first find that a host factor ABHD6 is an important host target point related to the replication of a porcine reproductive and respiratory syndrome virus (PRRSV), and that the inhibition of the activity or silencing of ABHD6 in a host cell and the down-regulation of the expression level of an ABHD6 gene by using an ABHD6-targeting inhibitor can inhibit the replication of the porcine reproductive and respiratory syndrome virus in the host cell, and can play an anti-porcine reproductive and respiratory syndrome virus effect, thereby solving the technical problems that the existing prevention or treatment means for the porcine reproductive and respiratory syndrome are limited, and the vaccine prevention or the drug targeting the virus itself is easy to cause the recombination and variation of the virus.

[0006] To achieve the above-mentioned purpose, according to one aspect of the present application, there is provided an application of an ABHD6-targeting inhibitor in the preparation of an anti-porcine reproductive and respiratory syndrome virus product, wherein the ABHD6-targeting inhibitor is a compound for inhibiting the activity of ABHD6 in a host and / or a small interfering RNA for silencing or down-regulating the expression level of an ABHD6 gene in a host. The compound for inhibiting the activity of ABHD6 in a host is WWL70 for selectively inhibiting the activity of ABHD6, and the chemical formula of WWL70 is C 27 H 23 N3O3. The small interfering RNA for silencing or down-regulating the expression level of an ABHD6 gene in a host is selected from SI-ABHD6-2 and / or SI-ABHD6-3, wherein the antisense strand of SI-ABHD6-2 is shown in SEQ ID NO: 8, and the antisense strand of SI-ABHD6-3 is shown in SEQ ID NO: 10.

[0007] Preferably, in the application, the ABHD6-targeting inhibitor comprises SI-ABHD6-3, and the sense strand of SI-ABHD6-3 is shown in SEQ ID NO: 9.

[0008] Preferably, in the application, the ABHD6-targeting inhibitor further comprises SI-ABHD6-2, and the sense strand of SI-ABHD6-2 is shown in SEQ ID NO: 7.

[0009] Preferably, in the application, the ABHD6-targeting inhibitor is used for preparing a drug or feed for resisting the infection of a porcine reproductive and respiratory syndrome virus.

[0010] Preferably, in the application, the ABHD6-targeting inhibitor is WWL70, and the concentration of WWL70 is ≥1 µmol / L or ≥1 µmol / g; preferably, the concentration of WWL70 is 1-60 µmol / L or 1-60 µmol / g.

[0011] According to another aspect of the present application, there is also provided a product against porcine reproductive and respiratory syndrome virus infection, the effective component of which comprises a compound WWL70 and / or small interfering RNA for inhibiting the replication of porcine reproductive and respiratory syndrome virus; The small interfering RNA is selected from SI-ABHD6-2 and / or SI-ABHD6-3, wherein the antisense strand of SI-ABHD6-2 is shown as SEQ ID NO: 8; and the antisense strand of SI-ABHD6-3 is shown as SEQ ID NO: 10.

[0012] Preferably, the product, the small interfering RNA of which is SI-ABHD6-3, the sense strand of which is shown as SEQ ID NO: 9.

[0013] Preferably, the product, the product of which is a medicine or feed against porcine reproductive and respiratory syndrome virus infection, the dose of SI-ABHD6-3 is ≥ 30 pmol.

[0014] Preferably, the product, the product of which is a medicine or feed against porcine reproductive and respiratory syndrome virus infection, the concentration of WWL70 is ≥ 1 µmol / L or ≥ 1 µmol / g.

[0015] Preferably, the product, the concentration of WWL70 is 1-60 µmol / L or 1-60 µmol / g.

[0016] Overall, compared with the prior art, the above technical solutions conceived by the present application can achieve the following beneficial effects: The present application finds that ABHD6 protein is an important host target point related to the replication of porcine reproductive and respiratory syndrome virus (PRRSV), and that by using an inhibitor targeting ABHD6 to inhibit host ABHD6, respectively using compound WWL70 to inhibit the activity of ABHD6 in host cells, and designing small interfering RNA targeting ABHD6 gene to silence and down-regulate the expression level of ABHD6 gene, it is proved that both can inhibit the replication of porcine reproductive and respiratory syndrome virus in host cells, and can have an effect against porcine reproductive and respiratory syndrome virus infection, and the inhibitor targeting ABHD6 can be applied to the preparation of a product against porcine reproductive and respiratory syndrome virus. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the effect of different concentrations of compound WWL70 on cell proliferation detected by CCK-8 in Example 1; Figure 2 is the result of compound WWL70 against porcine reproductive and respiratory syndrome virus in Example 2; Figure 3 is the result of the immunoblotting experiment in Example 2 to verify that the compound WWL70 can inhibit the replication of the porcine reproductive and respiratory syndrome virus; Figure 4 is the result of the interference RNA targeting silencing and down-regulating ABHD6 to resist the porcine reproductive and respiratory syndrome virus in Example 3; Figure 5 is the result of the immunoblotting experiment in Example 3 to verify that the interference RNA targeting silencing and down-regulating ABHD6 can inhibit the replication of the porcine reproductive and respiratory syndrome virus. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be made to the present application in combination with the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0019] The present application finds that the expression of ABHD6 gene in the pig infected with the porcine reproductive and respiratory syndrome virus (PRRSV) is down-regulated compared with the pig not infected with the porcine reproductive and respiratory syndrome virus. In order to further study whether the host factor ABHD6 will affect the host infected with the porcine reproductive and respiratory syndrome virus, we explore the effect of the host cell ABHD6 gene expression level on the infection of the porcine reproductive and respiratory syndrome virus by regulating the host cell ABHD6 gene expression level. The results show that the inhibition of the activity of the host cell ABHD6 by using the compound WWL70 can inhibit the replication of the porcine reproductive and respiratory syndrome virus in the cell, and has significant anti-porcine reproductive and respiratory syndrome virus activity.

[0020] In addition, based on the small interfering RNA technology, three small interfering RNAs are used to silence and down-regulate the expression of ABHD6 gene. The results show that when the small interfering RNA SI-ABHD6-1 fails to silence and down-regulate the expression of ABHD6 gene, the treatment by SI-ABHD6-1 does not have the effect of inhibiting the replication of the porcine reproductive and respiratory syndrome virus in the cell, while after the small interfering RNA SI-ABHD6-2 or SI-ABHD6-3 successfully silences and down-regulates the expression of ABHD6 gene, it can significantly inhibit the replication of the porcine reproductive and respiratory syndrome virus in the cell. It is speculated that the host factor ABHD6 is a host target point related to the replication of the porcine reproductive and respiratory syndrome virus (PRRSV).

[0021] The present application adopts an inhibitor targeting ABHD6 to inhibit the activity of host ABHD6 or silence, down-regulate the expression level of ABHD6 gene, and proves that the inhibitor targeting ABHD6 can significantly inhibit the infection of porcine reproductive and respiratory syndrome virus to host cells, thereby playing a role in preventing or treating porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) infection, and indicating that the host factor ABHD6 is an important host target point related to the replication of porcine reproductive and respiratory syndrome virus (PRRSV). In the present application, the inhibitor targeting ABHD6 can be an existing compound (such as compound WWL70) that inhibits the activity of ABHD6 at the protein level, can be a small interfering RNA that inhibits the expression of ABHD6 at the gene expression level, or can be a combination of the two.

[0022] Based on this finding, the present application provides an application of an inhibitor targeting ABHD6 in the preparation of an anti-porcine reproductive and respiratory syndrome virus product, wherein the inhibitor targeting ABHD6 includes a compound for inhibiting the activity of host ABHD6, and / or a small interfering RNA (siRNA) for silencing or down-regulating the expression level of host ABHD6 gene. The small interfering RNA for silencing or down-regulating the expression level of host ABHD6 gene is selected from SI-ABHD6-2 and / or SI-ABHD6-3, wherein the sense strand of SI-ABHD6-2 is shown as SEQ ID NO: 7, and the antisense strand is shown as SEQ ID NO: 8; the sense strand of SI-ABHD6-3 is shown as SEQ ID NO: 9, and the antisense strand is shown as SEQ ID NO: 10; in some embodiments, the small interfering RNA is preferably SI-ABHD6-3.

[0023] In some embodiments, the inhibitor targeting ABHD6 is compound WWL70, which has a chemical formula of C 27 H 23 N3O3, a molecular weight of 437.49, and a CAS number of 947669-91-2.

[0024] The inhibitor targeting ABHD6 is used for preparing a medicine or feed or feed additive for resisting the infection of porcine reproductive and respiratory syndrome virus.

[0025] For use in preparing a drug or feed for inhibiting porcine reproductive and respiratory syndrome virus replication, the concentration of WWL70 is ≥1µmol / L or ≥1µmol / g, and the total amount of SI-ABHD6-2 and / or SI-ABHD6-3 is ≥30pmol. In some embodiments, for inhibiting the replication of porcine reproductive and respiratory syndrome virus (PRRSV-HB), the concentration of WWL70 is 1-60µmol / L or 1-60µmol / g; the total amount of SI-ABHD6-3 is ≥30pmol.

[0026] In addition, the present invention also provides an anti-porcine reproductive and respiratory syndrome virus product, the active ingredients of which include compound WWL70 and / or small interfering RNA for inhibiting the replication of porcine reproductive and respiratory syndrome virus; the small interfering RNA is used to silence or downregulate the expression level of the ABHD6 gene, and is selected from SI-ABHD6-2 or SI-ABHD6-3, wherein the positive chain of SI-ABHD6-2 is shown as SEQ ID NO:7, and the antisense chain is shown as SEQ ID NO:8; the positive chain of SI-ABHD6-3 is shown as SEQ ID NO:9, and the antisense chain is shown as SEQ ID NO:10; preferably, the small interfering RNA is SI-ABHD6-3.

[0027] In some embodiments, the anti-PRRS virus product is a drug, feed, or feed additive that inhibits the replication of PRRS virus, wherein the active ingredient is WWL70. Within the safe use range, the concentration of WWL70 is ≥1µmol / L or ≥1µmol / g. In some embodiments, the concentration of WWL70 is preferably 1-60µmol / L or 1-60µmol / g, and can be used to prevent or treat PRRS.

[0028] The product against porcine reproductive and respiratory syndrome virus is a medicine, feed or feed additive for inhibiting the replication of porcine reproductive and respiratory syndrome virus, and the effective component is small interfering RNA (SI-ABHD6-2 and / or SI-ABHD6-3) for silencing or down-regulating the ABHD6 gene. In some embodiments for inhibiting the replication of porcine reproductive and respiratory syndrome virus PRRSV-HB, the small interfering RNA is SI-ABHD6-3, the concentration of SI-ABHD6-3 is ≥ 30 pmol, the sense strand of SI-ABHD6-3 is shown as SEQ ID NO: 9, and the antisense strand is shown as SEQ ID NO: 10. The product preferably further comprises an agent (such as a nuclease inhibitor) or component for enhancing the stability of small interfering RNA in vivo. In addition, in the present application, the stability of small interfering RNA in vivo can be enhanced by reducing the degradation rate through chemical modification (such as modification of phosphate groups, modification of bases), targeted delivery (use of carriers such as lipid nanoparticles, polymer carriers such as polyethyleneimine, viral vectors such as adeno-associated virus), and intracellular sustained-release design (optimization of double-stranded structure such as introduction of stem-loop structure, targeted modification such as connection of a targeting ligand to the surface of siRNA).

[0029] In the present application, the dosage form of the medicine for inhibiting the replication of porcine reproductive and respiratory syndrome virus is not limited, and the medicine can be selected from tablets, capsules, granules, dispersions, injections or sprays. The medicine preferably further comprises a component for avoiding the degradation of small interfering RNA in vivo, and can be used for preventing or treating porcine reproductive and respiratory syndrome.

[0030] The following is an example In the following examples, the experimental methods are existing procedures of molecular biology, cell biology or virology, and the experimental methods used are conventional methods unless otherwise specified; the materials, reagents and the like used in the examples can be obtained from commercial channels unless otherwise specified.

[0031] In the following examples, porcine reproductive and respiratory syndrome virus (PRRSV-HB) and African green monkey embryo kidney cells (MARC145) are provided by the Animal Genetic Breeding and Precision Breeding Laboratory of Wuhan Light Industry University or selected from the market; the alpha / beta hydrolase domain 6 (ABHD6) inhibitor WWL70 is purchased from GLP BIO Company, USA, and small interfering RNA (Small interfering RNA; siRNA) is synthesized by Suzhou Jimagene Company.

[0032] Example 1 Toxicity evaluation of WWL70 on cells MARC145 In this example, the alpha / beta hydrolase domain 6 (ABHD6) inhibitor is WWL70, and its chemical formula is C 27 H 23N3O3 was purchased from GLPBIO, USA, and African green monkey embryonic kidney MARC145 cells were used as target cells.

[0033] African green monkey embryonic kidney (MARC145) cells were plated in the inner circle of a 96-well culture plate (200ul PBS was added to the outer wells to prevent the "edge effect" caused by evaporation). 100uL of DMEM culture medium (containing 10% fetal bovine serum) was added to each well as the control group. The experimental group was added with DMEM culture medium (containing 10% fetal bovine serum) containing different concentrations of compound WWL70 (1µmol / L, 10µmol / L, 20µmol / L, 40µmol / L, 60µmol / L), and 3 replicates were set for each concentration. After incubation at 37°C, 5% CO2 for 24 hours, the culture supernatant was discarded and the cells were washed twice with PBS. 100 μL of culture medium containing 10% CCK8 reagent was added to each well as a replacement medium (to avoid bubbles). Three blank wells were set up (for background deduction) and only 100 μL of culture medium containing 10% CCK8 reagent (without MARC145 cells) was added. The cells were incubated at 37°C, 5% CO2 for 1 hour. The test samples were taken and tested on a microplate reader to read the OD450 readings. The data were processed using Graphpad. The experimental results are shown in Figure 2. Figure 1 shown.

[0034] Depend on Figure 1 The results showed that compared with the cell activity of the blank control group without WWL70 treatment (cell activity was 100%), the addition of different concentrations of compound WWL70 (1~60µmol / L) had no significant effect on the activity of African green monkey embryonic kidney cells MARC145, indicating that compound WWL70 has no toxicity to African green monkey embryonic kidney cells MARC145 and can be used in subsequent experiments.

[0035] Example 2 α / β Hydrolase Domain 6 Inhibitors Inhibit Porcine Reproductive and Respiratory Syndrome Virus Replication (1) Effect of WWL70 on viral RNA content in MARC145 cells infected with PRRSV African green monkey embryonic kidney MARC145 cells were plated in 6-well culture plates and randomly divided into the following groups for treatment: Blank control group: African green monkey embryonic kidney cells MARC145 were plated and 2 ml of DMEM culture medium (containing 10% fetal bovine serum) was added to each well. The cells were incubated at 37°C, 5% CO2 incubator for 24 hours, and the culture supernatant was discarded. The cells were washed twice with phosphate buffered saline (PBS).

[0036] WWL70 group: After the African green monkey embryonic kidney cells MARC145 were plated, 2 ml of DMEM culture solution containing 1 μmol / L of compound WWL70 (containing 10% fetal bovine serum) was added per well, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 h. After that, the culture supernatant was discarded, and the cells were washed twice with PBS.

[0037] PRRSV group: After the African green monkey embryonic kidney cells MARC145 were plated, 2 ml of DMEM culture solution (containing 10% fetal bovine serum) was added per well, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 h. After that, the culture supernatant was discarded, and the cells were washed twice with PBS. Then, the cells were infected with PRRSV-HB at a virus infection multiplicity (MOI) of 1 (i.e., the ratio of virus particles to the number of cells was 1), and the cells were incubated at 37°C in a 5% CO2 incubator for 24 h to inoculate porcine reproductive and respiratory syndrome virus.

[0038] WWL70+PRRSV group: After the African green monkey embryonic kidney cells MARC145 were plated, 2 ml of DMEM culture solution containing 1 μmol / L of compound WWL70 (containing 10% fetal bovine serum) was added per well, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 h. After that, the culture supernatant was discarded, and the cells were washed twice with PBS. Then, the cells were infected with PRRSV-HB at a virus infection multiplicity (MOI) of 1, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 h to inoculate porcine reproductive and respiratory syndrome virus.

[0039] After each group was incubated at 37°C in a 5% CO2 incubator for 24 h, the supernatant was discarded, and the cells were washed twice with PBS. Then, lysis solution RZ was added, and total RNA was extracted using a total RNA extraction kit from Tiangen. The copy number of virus after PRRSV infection of MARC145 cells was detected by qRT-PCR, with GAPDH as an internal reference gene. The experimental detection results based on gene expression levels are shown in Figure 2 Figure 2 The left side is the relative mRNA expression level of PRRSV ORF5, and the right side is the relative mRNA expression level of PRRSV ORF7.

[0040] Although porcine reproductive and respiratory syndrome virus (PRRSV) is prone to variation and recombination, ORF5 and ORF7 are relatively conserved regions in the viral genome and are involved in encoding the main structural proteins of the virus. Therefore, by detecting the mRNA content of PRRSV ORF5 (primer 1) and ORF7 (primer 2) in host cells in different treatment groups, the replication of virus (PRRSV) in host cells can be reflected at the gene level.

[0041] From Figure 2 ​The results show that compared with the blank control group, the mRNA content of ORF5 (primer 1) and ORF7 (primer 2) in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV-HB) in the PRRSV group increased significantly, indicating that the porcine reproductive and respiratory syndrome virus successfully infected the host cells. Then, the activity of the α / β hydrolase domain 6 in the host cells was inhibited by using the compound WWL70 first, and it was found that after the cells were infected with the virus, the mRNA expression level of ORF5 and ORF7 in the viral genome was significantly reduced, indicating that the replication of the porcine reproductive and respiratory syndrome virus in the host cells was significantly inhibited. Among them, the concentration of compound WWL70 was 1 µmol / L, which could show a very significant anti-porcine reproductive and respiratory syndrome virus infection effect.

[0042] (2) Verify the antiviral effect of ABHD6 inhibitor WWL70 based on protein level Repeat the experiment to verify it. After the African green monkey embryo kidney cells MARC145 were plated in a 6-well culture plate, they were randomly divided into the following groups (two duplicate wells were set for each group): Blank control group: After the African green monkey embryo kidney cells MARC145 were plated, 2 ml of DMEM culture medium (containing 10% fetal bovine serum) was added per well, and then incubated in a 37°C, 5% CO2 incubator for 24 h. After that, the culture supernatant was discarded and washed with PBS for 2 times.

[0043] WWL70 group: After the African green monkey embryo kidney cells MARC145 were plated, 2 ml of DMEM culture medium containing 1 µmol / L of compound WWL70 (containing 10% fetal bovine serum) was added per well, and then incubated in a 37°C, 5% CO2 incubator for 24 h. After that, the culture supernatant was discarded and washed with PBS for 2 times.

[0044] PRRSV group: After the African green monkey embryo kidney cells MARC145 were plated, 2 ml of DMEM culture medium (containing 10% fetal bovine serum) was added per well, and then incubated in a 37°C, 5% CO2 incubator for 24 h. After that, the culture supernatant was discarded and washed with PBS for 2 times. Then, the MARC145 cells were infected with PRRSV-HB at a multiplicity of infection (MOI) of 1, and then incubated in a 37°C, 5% CO2 incubator for 24 h to inoculate porcine reproductive and respiratory syndrome virus.

[0045] WWL70+PRRSV group: After the African green monkey embryo kidney cells MARC145 were plated, 2 ml of DMEM culture solution containing 1 µmol / L of compound WWL70 was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 h. Then the culture supernatant was discarded, and the cells were washed twice with PBS. Then the cells were infected with PRRSV-HB at a virus infection multiplicity (MOI) of 1, and incubated at 37°C in a 5% CO2 incubator for 24 h. The cells were infected with porcine reproductive and respiratory syndrome virus.

[0046] After treatment, the culture supernatant of each group was discarded, and the cells of different treatment groups were collected and placed on ice. An appropriate amount of SDS-PAGE loading buffer was added, and the cells were fully stirred and lysed. The metal bath was placed for 10 min for denaturation. After centrifugation, the supernatant was subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to a PVDF membrane by wet transfer. After transfer, the PVDF membrane was blocked with 5% skim milk powder, and then incubated with anti-porcine reproductive and respiratory syndrome virus GP5 protein antibody and ABHD6 protein antibody overnight. The membrane was washed with TBST for 3 times, and then incubated with HRP secondary antibody for 1 h. The antigen-antibody complex was detected, and the Western Blot detection results are shown in Figure 3 Figure 3 A is the Western Blot detection of the expression changes of ABHD6 protein and PRRSV GP5 protein in PRRSV infected MARC145 cells in different treatment groups; and B is the gray value ratio analysis result of the target protein and the internal reference protein (ABHD6 / GAPDH, PRRSV / GAPDH) based on the Western Blot results.

[0047] From Figure 3 As shown in the results in A, compared with the blank control group, the expression amount of viral antigen GP5 protein in the host cells was significantly increased after the cells were infected with porcine reproductive and respiratory syndrome virus in the PRRSV group, indicating that the porcine reproductive and respiratory syndrome virus successfully infected the cells. Compared with the PRRSV group, the expression amount of viral antigen GP5 protein in the host cells was reduced (the band corresponding to 40KD became narrower) after the cells were treated with compound WWL70 and then infected with the virus in the WWL70+PRRSV group, indicating that the replication of porcine reproductive and respiratory syndrome virus in the host cells was inhibited after treatment with compound WWL70, i.e., the compound WWL70 has an anti-porcine reproductive and respiratory syndrome virus effect.

[0048] ​By standardizing (dividing the gray value of the target protein by the gray value of the internal reference protein (such as GAPDH or β-actin)), the influence of the difference in loading amount can be eliminated. In the Western Blot (WB) experiment, by analyzing the gray value ratio of the target protein to the internal reference protein, the relative expression amount of the target protein can be evaluated. If the gray value ratio of the target protein is significantly higher than that of the control group, it indicates that the protein is up-regulated under the experimental conditions, and vice versa.

[0049] By Figure 3 As can be seen from the results of the B group, compared with the blank control group, the gray value ratio of the target protein (GP5 protein) in the PRRSV group was significantly higher than that in the control group, indicating that the expression amount of the porcine reproductive and respiratory syndrome virus antigen GP5 protein in the PRRSV group was increased. In the WWL70+PRRSV group, the cells were treated with WWL70 at a concentration of 1 µmol / L, and then infected with porcine reproductive and respiratory syndrome virus. The gray value ratio of the target protein (GP5 protein) in the cells was significantly lower than that in the PRRSV group, indicating that the use of WWL70 can inhibit the expression of porcine reproductive and respiratory syndrome virus antigen GP5 protein in host cells. It can be seen that the inhibitor targeting the α / β hydrolase domain 6 has the effect of inhibiting the replication of porcine reproductive and respiratory syndrome virus, and can be used for the preparation of anti-porcine reproductive and respiratory syndrome virus drugs. However, the mechanism of how the inhibitor targeting the α / β hydrolase domain 6 inhibits the replication of porcine reproductive and respiratory syndrome virus after inhibiting the activity of ABHD6 still needs further study.

[0050] Example 3 Effect of small interfering RNA targeting and inhibiting the expression of ABHD6 gene on porcine reproductive and respiratory syndrome virus In this embodiment, the inhibitor targeting the α / β hydrolase domain 6 (ABHD6) is small interfering RNA, which is synthesized by Suzhou Jimake Gene Co., Ltd. Among them, three small interfering RNAs targeting the ABHD6 gene are synthesized, namely ABHD6-green monkey-775, ABHD6-green monkey-824 and ABHD6-green monkey-1066, which are denoted as SI-ABHD6-1, SI-ABHD6-2 and SI-ABHD6-3, respectively. The sequence of the small interfering RNA used in the experiment is shown in Table 1.

[0051] Table 1 Sequence of small interfering RNA used

[0052] According to the "Standard for the Presentation of Nucleotide and Amino Acid Sequence Listings in Patent Applications" (WIPO ST.25 standard) and WIPO ST.26 standard, "u" in RNA needs to be replaced with "t" in the sequence listing. Therefore, the nucleotide "U" of RNA in Table 1 is replaced with "T" before the sequence listing is made.

[0053] In RNA interference (RNAi), the sense strand and the antisense strand are two strands that constitute small interfering RNA (siRNA). The sense strand usually does not directly participate in the degradation of the target mRNA, but serves as a complementary pair of the antisense strand; the antisense strand is the core executor of RNAi, which guides the RISC complex to recognize and cut the target mRNA by base complementary pairing with the target mRNA, so as to achieve gene silencing or down-regulation of gene expression.

[0054] ABHD6-green monkey-775-ss in Table 1 is the sense strand of SI-ABHD6-1, and ABHD6-green monkey-775-as is the antisense strand of SI-ABHD6-1; ABHD6-green monkey-824-ss is the sense strand of SI-ABHD6-2, and ABHD6-green monkey-824-as is the antisense strand of SI-ABHD6-2; ABHD6-green monkey-1066-ss is the sense strand of SI-ABHD6-3, and ABHD6-green monkey-1066-as is the antisense strand of SI-ABHD6-3.

[0055] (1) Real-time fluorescent quantitative PCR detection based on gene expression level After the African green monkey embryo kidney cells MARC145 were plated in 12-hole culture plates, they were randomly divided into groups and 1 mL of DMEM culture solution (containing 10% fetal bovine serum) was added to each hole, and small interfering RNA (siRNA) in Table 1 was added, two duplicate holes were set for each small interfering RNA, and siRNA / plus complexes were prepared according to the instructions of the transfection reagent (Gimaa gene) and added to the cells (the amount of siRNA was 30 pmol), and then the cells were incubated in a 37°C, 5% CO2 incubator for 24 h. One of the holes was inoculated with porcine reproductive and respiratory syndrome virus (PRRSV-HB) at a virus infection multiplicity (MOI) of 0.1, and then incubated in a 37°C, 5% CO2 incubator for 24 h. After the virus was inoculated, the holes were respectively recorded as NC+PRRSV, SI-ABHD6-1+PRRSV, SI-ABHD6-2+PRRSV, and SI-ABHD6-3+PRRSV. The hole into which negative control small interfering RNA was added was used as a negative control group (NC), and the hole into which negative control FAM small interfering RNA was added was used to verify that the small interfering RNA was successfully transferred into the cells.

[0056] After each group was incubated in a 37°C, 5% CO2 incubator for 24 h, the culture supernatant was discarded, and PBS was used for washing twice, and lysis solution RZ was added. Total RNA was extracted using a total RNA extraction kit from Tiangeng, and the copy number of the virus after PRRSV infection of MARC145 cells was detected by qRT-PCR, with GAPDH as an internal reference gene. The detection results of each group are shown in Figure 4 .

[0057] From Figure 4 It can be seen from the results that, compared with the NC group, the expression level of the virus in the NC+PRRSV group was extremely significantly increased, indicating that the porcine reproductive and respiratory syndrome virus successfully infected the cells. The cells in the experimental groups were treated with SI-ABHD6-1, SI-ABHD6-2, and SI-ABHD6-3, respectively, and then infected with porcine reproductive and respiratory syndrome virus. The mRNA expression levels of the virus in the cells treated with SI-ABHD6-2 and SI-ABHD6-3 were extremely significantly lower than those in the NC+PRRSV group, indicating that the two small interfering RNAs SI-ABHD6-2 and SI-ABHD6-3 had a significant inhibitory effect on the replication of porcine reproductive and respiratory syndrome virus in host cells, and SI-ABHD6-3 had a better inhibitory effect on porcine reproductive and respiratory syndrome virus.

[0058] The cells in the experimental group were treated with three small interfering RNAs (SI-ABHD6-1, SI-ABHD6-2, and SI-ABHD6-3) targeting the ABHD6 gene. The ABHD6 mRNA expression level in the cells treated with SI-ABHD6-1 was not statistically different from that in the NC group, while the ABHD6 mRNA expression level in the cells treated with SI-ABHD6-2 or SI-ABHD6-3 was significantly lower than that in the NC group, indicating that the two small interfering RNAs (SI-ABHD6-2 and SI-ABHD6-3) could successfully silence and down-regulate the expression of the ABHD6 gene and significantly inhibit the expression of the ABHD6 gene, and SI-ABHD6-3 had a better effect on inhibiting the expression of the ABHD6 gene.

[0059] The use of SI-ABHD6-2 and SI-ABHD6-3 in the cells could significantly inhibit the replication of the porcine reproductive and respiratory syndrome virus in the cells, and SI-ABHD6-3 had a better effect on inhibiting the porcine reproductive and respiratory syndrome virus. Therefore, we speculate that the ABHD6 gene regulation may affect the replication process of the porcine reproductive and respiratory syndrome virus, and the mechanism needs further research. We speculate that the inhibitor targeting ABHD6 may interfere with the invasion of the virus by changing the integrity of the host cell membrane lipid raft, or alleviate the immune pathological damage by enhancing the anti-inflammatory lipid signal, thereby playing a role in resisting the infection of the porcine reproductive and respiratory syndrome virus (PRRSV).

[0060] Based on the above experimental results, we found that the use of the inhibitor targeting ABHD6 to inhibit the activity of ABHD6 in the host cells or silence and down-regulate the expression of the ABHD6 gene could inhibit the replication of the porcine reproductive and respiratory syndrome virus in the cells and had an effect on resisting the infection of the porcine reproductive and respiratory syndrome virus, indicating that the inhibitor targeting ABHD6 could be used to prepare a drug, feed or feed additive for resisting the porcine reproductive and respiratory syndrome virus, and applied to prevent or treat the porcine reproductive and respiratory syndrome. The inhibitor targeting ABHD6 is one or a combination of WWL70, SI-ABHD6-2, and SI-ABHD6-3.

[0061] (2) Verification of the effect of small interfering RNA (SI-ABHD6-3) on resisting the porcine reproductive and respiratory syndrome virus based on the protein level Repeat the experiment to verify the African green monkey embryo kidney cells MARC145 flat 6-hole culture plate, each small interfering RNA set two duplicate wells, each well added 2 mL of DMEM culture solution (containing 10% fetal bovine serum), according to Seven High Trans™ siRNA non-liposome transfection reagent instructions, preparation of transfection reagent mixture and siRNA diluent mixture after adding to the cells (siRNA dosage is 30 pmol), placed in 37°C, 5% CO2 incubator for 24 h, one of the holes according to the virus infection multiplicity (MOI) is 1 inoculated with porcine reproductive and respiratory syndrome virus (PRRSV-HB), placed in 37°C, 5% CO2 incubator for 24 h, inoculated with virus respectively recorded as NC+PRRSV, SI-ABHD6+PRRSV. One of the holes is replaced by DMEM culture solution (containing 10% fetal bovine serum) instead of the test solution, and the other hole is replaced by negative control small interfering RNA as negative control group (NC), and the other hole is replaced by negative control FAM small interfering RNA for verifying that the small interfering RNA is successfully transferred into the cells, and the experimental group is added with small interfering RNA SI-ABHD6-3 for verification.

[0062] Each group was placed in a 37°C, 5% CO2 incubator for 24 h, the culture supernatant was discarded, and PBS was used to wash twice. The cells treated differently were collected and placed on ice, and appropriate SDS-PAGE loading buffer was added to fully stir and lyse. The metal bath was placed in a denaturing bath for 10 min, and after centrifugation, the supernatant was subjected to SDS-PAGE. After electrophoresis, wet transfer was used to transfer to PVDF membrane, and after transfer, 5% skim milk powder was used for blocking, and then overnight incubation with anti-swine reproductive and respiratory syndrome virus GP5 protein and ABHD6 protein antibody, TBST washing 3 times, and then incubation with HRP secondary antibody for 1 h, antigen-antibody complex detection, and the detection results of each group are shown in Figure 5 Figure 5 A is the Western Blot detection of PRRSV infected MARC145 cell ABHD6 protein and PRRSV GP5 protein expression changes, and B is the gray analysis of ABHD6 / GAPDH and PRRSV / GAPDH based on the Western Blot detection results.

[0063] From Figure 5 ​It can be known from the results that compared with the NC group, the expression amount of the virus antigen GP5 protein in the host cells infected with the porcine reproductive and respiratory syndrome virus in the NC+PRRSV group is significantly increased, which indicates that the porcine reproductive and respiratory syndrome virus successfully infects the cells. After the cells are treated by the small interfering RNA SI-ABHD6-3 and then infected with the virus, the expression amount of the ABHD6 protein in the host cells is significantly reduced, which indicates that the small interfering RNA SI-ABHD6-3 treatment down-regulates the expression of the ABHD6 gene. The expression amount of the virus antigen GP5 protein in the host cells is also significantly reduced, which indicates that the use of the SI-ABHD6-3 to down-regulate the expression of the ABHD6 gene can inhibit the replication of the porcine reproductive and respiratory syndrome virus in the host cells, and can be used for preparing products against the porcine reproductive and respiratory syndrome virus, such as drugs or feed.

[0064] Those skilled in the art will easily understand that the above description is only the preferred embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of an inhibitor targeting ABHD6 in the preparation of an anti-porcine reproductive and respiratory syndrome virus product, characterized in that: The inhibitor targeting ABHD6 is a compound used to inhibit the activity of host ABHD6, and / or a small interfering RNA used to silence or downregulate the expression level of the host ABHD6 gene; The compound that inhibits the activity of host ABHD6 is WWL70 that selectively targets and inhibits the activity of ABHD6, and its chemical formula is C 27 H 23 N3O3; The small interfering RNA for silencing or downregulating the expression level of the host ABHD6 gene is selected from SI-ABHD6-2 and / or SI-ABHD6-3, wherein the antisense strand of SI-ABHD6-2 is shown as SEQ ID NO: 8; the antisense strand of SI-ABHD6-3 is shown as SEQ ID NO:

10.

2. The use according to claim 1, characterized in that The inhibitor targeting ABHD6 includes SI-ABHD6-3, and the positive chain of the SI-ABHD6-3 is shown in SEQ ID NO:

9.

3. The use according to claim 2, characterized in that The inhibitor targeting ABHD6 also includes SI-ABHD6-2, and the positive chain of the SI-ABHD6-2 is shown in SEQ ID NO:

7.

4. The use according to any one of claims 1 to 3, characterized in that The inhibitor targeting ABHD6 is used to prepare medicine or feed for resisting porcine reproductive and respiratory syndrome virus infection.

5. The use according to claim 4, characterized in that The inhibitor targeting ABHD6 is WWL70, and its concentration is ≥1µmol / L or ≥1µmol / g; preferably, the concentration of WWL70 is 1~60µmol / L or 1~60µmol / g.

6. A product for resisting porcine reproductive and respiratory syndrome virus infection, characterized in that: Its active ingredients include compound WWL70 and / or small interfering RNA for inhibiting the replication of porcine reproductive and respiratory syndrome virus; The small interfering RNA is selected from SI-ABHD6-2 and / or SI-ABHD6-3, wherein the antisense strand of SI-ABHD6-2 is shown as SEQ ID NO: 8; and the antisense strand of SI-ABHD6-3 is shown as SEQ ID NO:

10.

7. The product according to claim 6, characterized in that The small interfering RNA is SI-ABHD6-3, and the positive strand of the SI-ABHD6-3 is shown in SEQ ID NO:

9.

8. The product according to claim 7, characterized in that The product is a medicine or feed for resisting porcine reproductive and respiratory syndrome virus infection, and the dosage of the SI-ABHD6-3 is ≥30 pmol.

9. The product according to claim 6, characterized in that The product is a drug or feed for resisting porcine reproductive and respiratory syndrome virus infection, and the concentration of the WWL70 is ≥1µmol / L or ≥1µmol / g.

10. The product according to claim 9, characterized in that The concentration of the WWL70 is 1-60 µmol / L or 1-60 µmol / g.

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