Application of inhibitors of fatty acid synthase gene FASN in the preparation of drugs for preventing and / or treating HSV-1 virus infection

Abstract

The present invention relates to the use of inhibitors of fatty acid synthase gene FASN in the preparation of drugs for preventing and / or treating HSV-1 virus infection, and belongs to the field of biological gene technology. The inhibitor of fatty acid synthase gene FASN is β-ketoacyl synthase domain inhibitor C75 or small interfering RNA; the small interfering RNA includes the sequence shown below: si-FASN F: GCUCGAUACCUUCCUGAAUTT; si-FASN R: AUUCAGGAAGGUAUCGAGCTT. The present invention determines the relationship between FASN gene and HSV-1 infection, which can be used as a therapeutic target for its infection. The replication and proliferation of HSV-1 virus can be inhibited by knocking out FASN gene, and its inhibitor C75 can be used for the treatment of HSV-1 infection. The present invention provides an effective new approach for the treatment of HSV-1 infection.

Description

Technical Field

[0001] The present invention belongs to the field of biological gene technology, and specifically relates to the use of an inhibitor of the fatty acid synthase gene FASN in the preparation of a drug for preventing and / or treating HSV-1 virus infection. Background Art

[0002] Herpes simplex virus (HSV) can be divided into type 1 and type 2. Humans are the only natural host of HSV. HSV infection is extremely common in the population. HSV infection has become the fourth largest infectious disease in the world and is the most vulnerable virus to humans. Herpes simplex virus type 1 (HSV-1) is a common pathogen in humans. It belongs to the subfamily of the herpesviridae family and is an enveloped double-stranded DNA virus with a genome of approximately 152kb. The incubation period after HSV-1 infection is usually 2-12 days. Infected people may experience systemic symptoms such as fever, headache, muscle aches, and local skin or mucous membrane herpes-like lesions. Herpes can rupture and erode to form ulcers, accompanied by pain. HSV-1 infection can cause a variety of complications, including herpes encephalitis, herpes keratitis, herpes pneumonia, etc., which can be life-threatening in severe cases. HSV-1 is a type of virus that can coexist with the host for a long time. It can interact with host cells to simulate the transcriptional regulation function of host cells, and is of great significance in the fields of virology and molecular biology. The process of HSV-1 virus expressing its own genes after contacting the host has a strict temporal sequence and exhibits the characteristics of cascade regulation. Therefore, the HSV-1 genome is divided into immediate-early genes (IE), early genes (E) and late genes (L), also known as α, β, and γ genes. Most HSV-1 infections occur in childhood, and the body cannot completely eliminate HSV-1 virus infection, often showing asymptomatic lifelong latency. The current analysis of HSV-1 antibody positive conversion rate data shows that with the development of social economy and medical conditions, the infection rate of HSV-1 has a downward trend worldwide, and the comparative analysis of infection rates for people of different age groups also shows that the middle-aged and elderly are the group with the highest HSV-1 infection rate. HSV-1 can cause two different forms of infection: lytic infection (often called proliferative infection) and latent infection. After HSV-1 causes lytic infection in the epithelial cells of peripheral mucosal tissues, it can enter sensory neurons and establish latent infection. Latently infected HSV-1 viruses can also re-enter the replication cycle through reactivation and return to peripheral tissues for recurrent infection. This ability to both evade host immune attacks through latency and spread during activation is a superb survival strategy of HSV-1 and the fundamental reason why no current drug can completely eliminate the virus. At present, there is no specific treatment for HSV-1 infection. Since there is no safe and reliable vaccine for vaccination and prevention, the commonly used therapeutic drugs in clinical practice are antiviral drugs and analgesics. Antiviral drugs can inhibit viral replication, thereby alleviating symptoms and shortening the course of the disease. Commonly used antiviral drugs include acyclovir, valacyclovir, famciclovir, etc.Analgesics can relieve pain symptoms. Commonly used analgesics include ibuprofen, acetaminophen, etc. The effect of drug treatment is general and there are problems such as drug resistance, incomplete removal and recurrence. In summary, diseases caused by HSV-1 have a considerable burden on global healthcare.

[0003] Fatty acid synthase (FASN) is a multifunctional enzyme. In higher animals, FASN is a multifunctional enzyme composed of a polypeptide chain, usually in the form of a dimer. Each subunit contains an ACP domain that can catalyze the synthesis of fatty acids. Studies have found that intracellular metabolic processes may participate in the host's antiviral innate immune response and may affect all stages of the viral replication cycle, including virus-host interaction, fusion of the capsule and cell membrane, viral assembly and budding. Lipids are not only a source of cellular energy and a component of the cell membrane, but also a signaling molecule involved in the regulation of gene expression. However, there is currently no research on the fatty acid synthase gene FASN in anti-HSV-1 virus. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide an inhibitor of the fatty acid synthase gene FASN for use in the preparation of a drug for preventing and / or treating HSV-1 virus infection.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] Use of an inhibitor of the fatty acid synthase gene FASN in the preparation of a drug for preventing and / or treating HSV-1 virus infection.

[0007] Further, preferably, the inhibitor of fatty acid synthase gene FASN is β-ketoacyl synthase domain inhibitor C75 or small interfering RNA;

[0008] The small interfering RNA comprises the sequence shown below:

[0009] si-FASN-F:GCUCGAUACCUUCCUGAAUTT;

[0010] si-FASN-R:AUUCAGGAAGGUAUUCGAGCTT.

[0011] The present invention also provides a drug for preventing and / or treating HSV-1 virus infection, comprising a pharmaceutically acceptable carrier and an inhibitor of the fatty acid synthase gene FASN.

[0012] Further, preferably, the inhibitor of fatty acid synthase gene FASN is β-ketoacyl synthase domain inhibitor C75 or small interfering RNA;

[0013] The small interfering RNA comprises the sequence shown below:

[0014] si-FASN-F:GCUCGAUACCUUCCUGAAUTT;

[0015] si-FASN-R:AUUCAGGAAGGUAUUCGAGCTT.

[0016] The present invention also provides an in vitro non-therapeutic method for inhibiting HSV-1 virus infection of cells. HSV-1 virus infection cells are cultured in the presence of an inhibitor of the fatty acid synthase gene FASN, thereby inhibiting the HSV-1 virus.

[0017] Finally, the present invention provides the application of the fatty acid synthase gene FASN overexpression cell line in the preparation of the HSV-1 virus vaccine production cell line.

[0018] The present invention has found through research that the fatty acid synthase gene FASN is a therapeutic target for HSV-1 virus infection, and its inhibitor is used to prepare a drug that has a preventive or therapeutic effect on HSV-1 virus infection.

[0019] The present invention determines the relationship between FASN and HSV-1 virus infection, provides a target gene for the treatment of HSV-1 virus infection, and then uses FASN for the prevention or treatment of HSV-1 virus infection, and uses its inhibitor to screen or prepare drugs that have a protective or preventive effect on HSV-1 virus infection. The present invention mainly explores: (1) knocking out the FASN gene can inhibit the expression and replication of HSV-1; (2) overexpression of FASN can promote the expression of HSV-1 virus; (3) FASN β-ketoacyl synthase domain inhibitor C75 can inhibit HSV-1 virus infection.

[0020] According to the research of the present invention, knocking out FASN can resist HSV-1 virus infection, which was confirmed in RAW264.7 cells. Inhibiting FASN can resist HSV-1 virus infection, which was confirmed in A549 cells. Overexpression of FASN can promote the expression and replication of herpes simplex virus HSV-1, which was confirmed in A549 cells.

[0021] Knocking out the FASN gene can inhibit the replication of HSV-1 virus: FASN knockout and wild-type RAW264.7 cells were infected with HSV-1, and the replication of HSV-1 virus was inhibited in FASN knockout cells.

[0022] Using siRNA to interfere with FASN gene expression or using the β-ketoacyl synthase domain inhibitor C75 can inhibit the replication of HSV-1 virus: knocking down FASN using siRNA inhibited the replication of HS V-1 in A549 cells; in A549 cells, the replication of HSV-1 virus was inhibited after treatment with C75.

[0023] Overexpression of FASN can promote HSV-1 viral replication: A549 cells transfected with the FASN overexpression plasmid pCMV-FASN-3×FLAG-SV40-Neo and the control plasmid pCMV-3×FLAG-SV40-Ne o were infected with HSV-1. The replication of HSV-1 virus was promoted in cells overexpressing FASN.

[0024] The sequence of the fatty acid synthase gene FASN involved in the present invention has been published in NCBI, and the sequence number is: NM_004104.5.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention determines the relationship between FASN and HSV-1 infection, which can be used as a therapeutic target for its infection. The replication and proliferation of HSV-1 virus can be inhibited by knocking out the FASN gene, and its inhibitor C75 can be used for the treatment of HSV-1 infection. The present invention provides an effective new approach for the treatment of HSV-1 infection;

[0027] (2) Currently, the treatment of herpes simplex virus infection mainly relies on acyclovir (ACV) and penciclovir (PCV) and their respective prodrugs valacyclovir and famciclovir. These therapeutic drugs target the HSV-1 genome in the human body. Virus mutations are prone to drug resistance, and the drug resistance rate is higher in patients with immune deficiency. The anti-HSV-1 therapeutic target FASN determined by the present invention is derived from the host, and the gene sequence is conservative, so it is not easy to develop drug resistance to drugs designed for this target;

[0028] (3) The present invention has determined the feasibility of fatty acid synthase FASN as a target for treating HSV-1 at the cellular level. In addition to the FASN inhibitor C75 mentioned in the present invention, more FASN inhibitory drugs can be developed for use against HSV-1 virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 RAW264.7 (mouse mononuclear macrophage leukemia cells) wild-type and FASN knockout cells were infected with HSV-1 (MOI = 1), and the cell supernatant was collected after 24 hours to determine the virus titer using the CCID50 method; Figure 1 WT-Fasn represents wild-type RAW264.7 cells, and KO-Fasn represents FASN-knocked-out RAW264.7 cells; the results showed that knocking out the FASN gene in RAW264.7 cells could inhibit the expression and replication of herpes simplex virus HSV-1;

[0030] Figure 2 Wild-type RAW264.7 cells and FASN gene knockout RAW264.7 cells were infected with HSV-1 (MOI=1), and the collected proteins were analyzed by western blotting 24 hours later; the expression of HSV-1 late protein ICP5 was detected, and FASN and β-Actin proteins were also detected; Figure 2 WT represents wild-type RAW264.7 cells, KO represents FASN knocked-out RAW264.7 cells, and "1, 2, 3" in the figure represent the first biological replicate, the second biological replicate, and the third biological replicate under different conditions, respectively; the results show that knocking out the FASN gene in RAW264.7 cells can inhibit the expression and replication of herpes simplex virus HSV-1;

[0031] Figure 3 RAW264.7 wild-type and FASN knockout cells were infected with HSV-1 (MOI=1), and the cell slides were analyzed for immunofluorescence results 24 hours later; FASN gene and HSV-1 late protein ICP5 were detected; Figure 3 Wild represents wild-type RAW264.7 cells, Fasn - / - RAW264.7 cells represent FASN knockout. The results show that knockout of the FASN gene in RAW264.7 cells can inhibit the expression and replication of herpes simplex virus HSV-1.

[0032] Figure 4 To overexpress the FASN gene in wild-type A549 cells using plasmid pCMV-FASN-3×FLAG-SV40-Neo, OE-NC cells were used as the experimental control group using empty plasmid pCMV-3×FLAG-SV40-Neo; 48 hours later, HSV-1 was infected (MOI=1), and 24 hours later, the cell supernatant was collected and the virus titer was determined by CCID50 method; Figure 4 OE-NC represents the control group, and OE-Fasn represents the overexpression group; the results show that overexpression of FASN in A549 cells can promote the expression and replication of herpes simplex virus HSV-1;

[0033] Figure 5To overexpress the FASN gene in wild-type A549 cells using plasmid pCMV-FASN-3×FLAG-SV40-Neo, OE-NC was used as an experimental control group using empty plasmid pCMV-3×FLAG-SV40-Neo, and infected with HSV-1 (MOI=1) 48 h later, and the proteins were collected 6 h later for western blotting results; Figure 5 The expression of HSV-1 immediate early protein ICP27 was detected in the samples. In addition, FAS N and β-Actin proteins were also detected. Figure 5 OE-NC represents the control group, OE-Fasn represents the overexpression group, and "1, 2, 3" in the figure represent the first biological replicate, the second biological replicate, and the third biological replicate under different conditions, respectively; the results show that overexpression of FASN in A549 cells can promote the expression and replication of herpes simplex virus HSV-1;

[0034] Figure 6 A549 cells were pretreated with 40 μM FASN inhibitor C75, and the control group was added with DMSO of the same volume as the inhibitor; 12 h later, HSV-1 was infected (MOI=1), and 24 h later, the cell supernatant was collected for CCID50 determination of virus titer results; Figure 6 DMSO represents the solvent control group with DMSO added, and C75 represents the drug group with 40 μM C75 added; the results showed that the expression and replication of HSV-1 were inhibited after A549 cells were treated with C75, an inhibitor of the β-ketoacyl synthase domain of FASN;

[0035] Figure 7 A549 cells were pretreated with 40 μM FASN inhibitor C75, and the control group was added with DMSO of the same volume as the inhibitor; 12 h later, HSV-1 was infected (MOI = 1), and 6 h later, proteins were collected for western blotting results; among them, the expression of HSV-1 immediate early protein ICP27 was detected, and FASN and β-Actin proteins were also detected; Figure 7 DMSO in the figure represents the solvent control group with DMSO added, C75 represents the drug group with 40 μM C75 added, and "1, 2, 3" in the figure represent the first biological repeat, the second biological repeat, and the third biological repeat under different conditions, respectively; the results show that after A549 cells were treated with C75, an inhibitor of the β-ketoacyl synthase domain of FASN, the expression and replication of HSV-1 were inhibited;

[0036] Figure 8To knock down the FASN gene in A549 cells using siRNA, the control group was si-NC without knockdown site; 48h later, HSV-1 was infected (MOI=1), and 24h later, the cell supernatant was collected for CCID50 determination of virus titer results; Figure 8 In the figure, si-NC represents the control group transfected with negative control siRNA, and si-Fasn represents the experimental group transfected with FASN knockdown. The results showed that after knocking down the FASN gene in A549 cells, the expression and replication of HSV-1 were inhibited.

[0037] Fig. 9 To knock down the FASN gene in A549 cells using siRNA, the control group was si-NC without knockdown site; 48h later, the cells were infected with HSV-1 (MOI=1), and 6h later, the proteins were collected for western blotting results; among them, the expression of HSV-1 immediate early protein ICP27 was detected, and FAS N and β-Actin proteins were also detected; Fig. 9 In the figure, si-NC represents the control group transfected with negative control siRNA, si-Fasn represents the experimental group transfected with FASN knockdown, and "1, 2, 3" in the figure represent the first biological repeat, the second biological repeat, and the third biological repeat under different conditions, respectively; the results showed that after knocking down the FASN gene of A549 cells, the expression and replication of HSV-1 were inhibited. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with embodiments.

[0039] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0040] All culture medium formulas used in the present invention are:

[0041] Complete culture medium: Commercial DMEM (Vivacell) was supplemented with 1% of the double antibody and 10% of the fetal bovine serum (v%).

[0042] Maintenance medium: add 1% of the total volume of double antibody and 2% of the total volume of fetal bovine serum (v%) to commercial DMEM (Vivacell);

[0043] Serum-free culture medium: commercial DMEM culture medium (Vivacell).

[0044] The virus involved in the present invention: HSV-1 virus (strain: Strain-17) is provided by the Vaccine Research Laboratory of the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0045] The siRNA sequences involved in the present invention are shown in Table 1.

[0046] Table 1

[0047]

[0048] The antibodies involved in the present invention are shown in Table 2.

[0049] Table 2

[0050]

[0051]

[0052] The reagents involved in the present invention are shown in Table 3.

[0053] Table 3

[0054]

[0055] The preparation method of the corresponding reagents in the present invention is as follows: (1) The components of the transfer buffer are shown in Table 4.

[0056] Table 4

[0057]

[0058] Add 600 mL of ultrapure water to the above ingredients, stir to dissolve and then adjust the volume to 1 L.

[0059] (2) The composition of 10×TBS buffer is shown in Table 5.

[0060] Table 5

[0061]

[0062] Add 400 mL of ultrapure water to the above ingredients, stir to dissolve, adjust the pH value to 7.5, and make up to 500 mL.

[0063] (3) The composition of 1×TBST is shown in Table 6.

[0064] Table 6

[0065]

[0066] Mix well and use immediately.

[0067] (4) The composition of the blocking solution is shown in Table 7.

[0068] Table 7

[0069]

[0070] Mix thoroughly and use immediately.

[0071] (5) 0.5% TritonX-100. The ingredients are shown in Table 8.

[0072] Table 8

[0073]

[0074] Shake well.

[0075] (6) 0.25% pancreatic enzyme composition as shown in Table 9.

[0076] Table 9

[0077]

[0078] After mixing, store in aliquots at -40℃.

[0079] The corresponding Chinese and English names of the English abbreviations involved in the present invention are shown in Table 10.

[0080] Table 10

[0081]

[0082] Example 1: Determination of HSV-1 virus titer in the culture supernatant 24 hours after HSV-1 infection of wild-type RAW264.7 cells and FASN knockout RAW264.7 cells.

[0083] 1. Prepare cells. Take well-growing RAW264.7 cells for subculture and inoculate the cells in a six-well plate. Add 2 ml of complete medium to each well. The starting cell amount is 2.0×10 6 The cells were cultured in a 37°C 5% CO2 incubator for 16 hours.

[0084] 2. Infect with HSV-1 virus. Take out the 6-well plate, remove the complete culture medium, wash the cells twice with 1×PBS, and add 1.0 ml of HSV-1 virus solution diluted with maintenance solution to each well (MOI=1). Incubate in a 37℃ 5% CO2 incubator for 1 hour, remove the virus solution, wash twice with 1×PBS, add 2 ml of maintenance solution to each well, and continue to culture in a 37℃ 5% CO2 incubator for 24 hours.

[0085] 3. Harvest the virus supernatant. Take out 1 ml of the culture supernatant and put it into a 1.5 ml centrifuge tube and store it at -80 degrees Celsius.

[0086] 4. Determine the virus titer using the CCID50 method.

[0087] 1) Prepare Vero cells. Mix the Vero cells with the maintenance solution and plate them on a 96-well plate. The volume of each well is 100 μl, containing approximately 5×10 4 cells.

[0088] 2) Dilute the virus solution to be tested. Take out the HSV-1 virus supernatant harvested in step 3 from the -80°C refrigerator and wait for it to thaw. Dilute the virus stock solution 10 times with the maintenance solution in an EP tube, i.e., take 100 μl of the virus stock solution and mix it with 900 μl of the maintenance solution to make 10 -1 Dilution gradient, take 100μl 10 -1 The virus solution of the dilution gradient was mixed with 900 μl of the maintenance solution to make 10 -2 Dilution gradient. 11 virus gradients are formed in sequence, namely 10 -1 -10 -11 .

[0089] 3) Inoculation. Take a 96-well plate with Vero cells and add the diluted virus solution to the 96-well plate. Add 8 wells for each dilution gradient and add 100 μl to each well. Add high dilution first and then low dilution. Add 100 μl of maintenance solution to the remaining 8 wells as a blank control. Place in a 37°C 5% CO2 incubator and culture for 5-7 days.

[0090] 4) Observation and statistics. During the culture process, observe the cytopathic condition under a microscope every day and record the number of cytopathic wells in each gradient until the number of cytopathic wells no longer increases after continuous observation, and then count the results.

[0091] 5) Calculate the results. Calculate the virus titer using the Karber method. That is, lgCCID50 / ml = the negative logarithm of the highest dilution of complete lesions + the sum of the lesion wells of each dilution of incomplete lesions × 0.125 + 0.5 + 1;

[0092] 5. Experimental results are as follows Figure 1 As shown. RAW264.7 (mouse mononuclear macrophage leukemia cells) wild-type and FASN gene knockout cells were infected with HSV-1 (MOI=1), and the cell supernatant was collected after 24 hours to determine the virus titer by CCID50 method. The results showed that knocking out the FASN gene in RAW264.7 cells could inhibit the expression and replication of herpes simplex virus HSV-1.

[0093] Example 2: HSV-1 infection of wild-type RAW264.7 cells and FASN knockout RAW264.7 cells, and Western blotting to detect the expression of HSV-1 late protein ICP5

[0094] 1. Prepare cells. Take well-growing RAW264.7 cells for subculture and inoculate the cells in a six-well plate. Add 2 ml of complete medium to each well. The starting cell amount is 2.0×10 6 The cells were cultured in a 37°C 5% CO2 incubator for 16 hours.

[0095] 2. Infect with HSV-1 virus. Take out the 6-well plate, remove the complete culture medium, wash the cells twice with 1×PBS, and add 1.0 ml of HSV-1 virus solution diluted with maintenance solution to each well (MOI=1). Incubate in a 37℃ 5% CO2 incubator for 1 hour, remove the virus solution, wash twice with 1×PBS, add 2 ml of maintenance solution to each well, and continue to culture in a 37℃ 5% CO2 incubator for 24 hours.

[0096] 3. Harvest protein samples. Remove the cell culture supernatant, add 4°C pre-cooled 1×PBS to wash twice, discard the 1×PBS and place the 6-well plate on ice. Take 1.5ml RIPA lysis buffer on ice and add 15μl PMSF (100mM), mix well, and obtain a lysis buffer containing PMSF. Add 200μl of lysis buffer containing PMSF to each well and lyse on ice for 30 minutes. To fully lyse the cells, shake the culture plate once at the same interval. After lysis, use a pipette to transfer the cell fragments and lysis buffer to a 1.5ml centrifuge tube. Centrifuge at 12000rpm for 5 minutes at 4°C, transfer the supernatant after centrifugation into 0.5ml centrifuge tubes and store at -40°C.

[0097] 4. Protein immunoblotting was used to detect the expression of FASN, β-Actin protein and HSV-1 late protein ICP5.

[0098] 1) Sample preparation. Take 80 μL of the protein sample collected in step 3 and add 20 μL of 5× Loading buffer to a final concentration of 1×. Mix by pipetting repeatedly, and boil in a 100°C metal bath for 10 minutes to denature the protein. Centrifuge at 10,000 g for 5 minutes, and take the supernatant as a sample and place on ice for later use.

[0099] 2) SDS-polyacrylamide gel electrophoresis. Assemble the electrophoresis tank, add 1×MOPES electrophoresis buffer (GenScript), the inner side covers the sample wells, and the outer side reaches the 2Gels position. Tear off the precast gel (GenScript) strip and carefully pull out the comb of the precast gel. Slowly add the sample, use 1×Loa ding buffer to fill the sample wells without samples to avoid electrophoresis deviation, and finally add the protein marker. After adding the sample, cover the top cover, connect the electrophoresis instrument, turn on the electrophoresis instrument, and run the electrophoresis at 120V for 60 minutes. Stop the electrophoresis when the bromophenol blue indicator migrates to 1 to 2 cm away from the front.

[0100] 3) Transfer. After the electrophoresis is completed, use a thin plate to gently pry open the two glass plates of the gel plate, cut the gel containing the target protein with a control protein marker, and then carefully spread the gel on the filter paper. Cover the cut and marked PVDF membrane on the corresponding gel, and be careful not to have bubbles between the two. Assemble the sandwich splint, with the black (gel) facing the black negative electrode and the transparent (membrane) facing the red positive electrode. Pour the transfer solution (wet transfer solution) to fill the entire transfer tank, add ice cubes and transfer the membrane in an ice bath. Set the transfer current to a constant current of 300mA. The transfer time is determined according to the size of the protein. The transfer time for FASN and ICP5 proteins is 120 minutes, and the transfer time for β-Actin protein is 60 minutes.

[0101] 4) Blocking. After the transfer, remove the membrane, place the protein side upward, rinse with 1×TBST for 5 seconds, and then place in 5 mL of blocking solution (TBST containing 5% skim milk, v%) and block on a horizontal shaker at room temperature for 2 hours.

[0102] 5) Incubate the primary antibody. Dilute the primary antibody (the primary antibodies used include ICP5, FASN, and β-Actin) with blocking solution (TBST containing 5% skim milk, v%) according to the dilution multiple recommended in the antibody manual. After absorbing the blocking solution, rinse with 1×TBST for 5 seconds, immediately add the diluted primary antibody, incubate at room temperature for 1 hour on a horizontal shaker, and then incubate in a 4°C refrigerator for 16 hours. The dilution ratio of each antibody: ICP5 (1:2000); FASN (1:2000); β-Actin (1:25000);

[0103] 6) Wash the membrane. After the incubation is completed, remove the primary antibody and recover it (it can be reused 3-4 times within one month at 4°C). Add 5 mL of 1×TBST to a square fresh-keeping box and place the membrane in it so that the TBST covers the PVDF membrane. Shake it at low speed (70 rpm) on a shaker for 10 minutes and repeat the washing 3 times.

[0104] 7) Incubate with secondary antibody. Dilute the secondary antibody (including goat anti-mouse IgG and goat anti-rabbit IgG, the dilution ratio of the secondary antibody is 1:8000) with 5 mL of blocking solution (TBST containing 5% skim milk, v%), where the secondary antibody for FASN is goat anti-rabbit IgG, and the secondary antibody for ICP5 and β-Actin protein is goat anti-mouse IgG. Immerse the protein-transferred PVDF membrane in a square fresh-keeping box filled with skim milk containing the secondary antibody, and incubate on a horizontal shaker (70 rpm) for 2 h.

[0105] 8) Wash the membrane. After the incubation is completed, discard the secondary antibody, add 1×TBST to a square fresh-keeping box, put the membrane in it, make the TBST cover the PVDF membrane, shake it on a shaker at low speed (70 rpm) for 10 minutes, and repeat the washing 4 times.

[0106] 9) Development. Prepare the developer (mix the developer A and B in the Bio-Yun Tian Extreme Supersensitive ECL Chemiluminescence Kit in a 1:1 ratio before use). When developing, add the developer to the membrane until it is completely covered. After reacting in the dark for 1 minute, place it in the UVP instrument to expose the protein bands and take photos for record.

[0107] 5. Experimental results are as follows Figure 2 As shown. RAW264.7 wild-type and FASN gene knockout cells were infected with HSV-1 (MOI=1), and the collected proteins were subjected to western blotting 24 hours later to detect the expression of HSV-1 late protein ICP5. In addition, FASN and β-Actin proteins were also detected. The results showed that knocking out the FASN gene in RAW264.7 cells could inhibit the expression and replication of herpes simplex virus HSV-1.

[0108] Example 3: After HSV-1 infected FASN knockout and wild-type RAW264.7 cells, the expression of HSV-1 late protein ICP5 was detected by immunofluorescence.

[0109] 1. Cell slides. Place sterile cell slides in 24-well plates. Take well-growing FASN knockout and wild-type RAW264.7 cells for subculture, inoculate cells into 24-well plates, add 0.5 ml of complete culture medium to each well, and the starting cell amount is 5.0×10 5 Each well was shaken evenly and cultured in a 37°C 5% CO2 incubator for 16 hours.

[0110] 2. Infect with HSV-1 virus. Take out the 24-well plate, remove the complete medium and wash the cells twice with 1×PBS, add 0.2ml serum-free medium to each well with diluted HSV-1 virus solution (MOI=1). Incubate in a 37℃ 5% CO2 incubator for 1h, remove the virus solution, wash twice with 1×PBS, add 0.5ml maintenance solution to each well and continue to culture in a 37℃ 5% CO2 incubator for 24 hours.

[0111] 3. Immunofluorescence.

[0112] 1) Take out the cells, discard the original culture medium in the well plate, add 1×PBS to the well plate and wash the cells three times, each time for 3 minutes.

[0113] 2) Fix the slides with 4% tissue cell fixative (Solabol, v%) at room temperature for 15 minutes, and wash the slides with 1×PBS for 3 times, each time for 3 minutes.

[0114] 3) 0.5% TritonX-100 (prepared in 1×PBS, v%) was used for permeabilization at room temperature for 20 minutes.

[0115] 4) Wash the slides with 1×PBS for 3 times, 3 minutes each time, dry the slides with absorbent paper, add 200 μL of 5% BSA (Solabol, v%) on the slides, and block at room temperature for 30 minutes.

[0116] 5) Add FASN and ICP5 primary antibodies to each slide (the dilution ratio of FASN and ICP5 primary antibodies is 1:500), mix the antibodies with 5% BSA (Solabol, v%) in step 4), put them in a wet box, and incubate at 4°C for 16 hours.

[0117] 6) Add fluorescent secondary antibody: wash the slides 3 times with 1×PBST for 3 minutes each time, dry the excess liquid on the slides with absorbent paper, add 200μL 5% BSA (Solabo, v%), then add 1μL CoraLite488-conjugated GoatAnti-Mouse IgG(H+L)(proteintech) and 1μL CoraLite594-conjugated Goat Anti-Rabbit IgG(H+L)(proteintech) fluorescent secondary antibodies to each well and incubate in a wet box at 37℃ for 1h, wash the slides 3 times with 1×PBST for 3 minutes each time (all experimental steps starting from step 6 should be carried out in the dark).

[0118] 7) Use absorbent paper to absorb the liquid on the slide, seal the slide with a sealing solution containing anti-fluorescence quencher (DAPI) (Abcam, ab104139), and then observe and collect images under a fluorescence microscope.

[0119] 4. Experimental results are as follows Figure 3 As shown. RAW264.7 wild-type and FASN gene knockout cells were infected with HSV-1 (MOI=1), and 24 hours later, the cell slides were subjected to immunofluorescence to detect the FASN gene and the late protein ICP5 of HSV-1. The results showed that knocking out the FASN gene in RAW264.7 cells could inhibit the expression and replication of herpes simplex virus HSV-1.

[0120] Example 4: Determination of HSV-1 virus titer in the culture supernatant after HSV-1 infected A549 cells overexpressing the FASN gene.

[0121] 1. Prepare cells. Take well-growing A549 cells for subculture and inoculate the cells in a six-well plate. The volume of complete culture medium is 2 ml and the starting cell amount is 1.5×10 6 The cells were cultured in a 37°C 5% CO2 incubator for 16 hours.

[0122] 2. Transfect cells using liposome method.

[0123] 1) Dilute Lipofectamine 3000. Take 720 μL of Opti-MEM medium and add 30 μL of Lipofectamine 3000.

[0124] 2) Dilute the plasmid. Take 720 μL Opti-MEM medium, add 30 μL P3000, mix well and divide equally into two EP tubes, add 7.5 μg pCMV-FASN-3×FLAG-SV40-Neo to one tube, and add 7.5 μg pCMV-3×FLAG-SV40-Neo (i.e., pCMV vector, an empty plasmid for control) to the other tube.

[0125] 3) Mix and incubate. Divide the diluted Lipofectamine 3000 evenly into two diluted plasmid tubes, mix Lipofectamine 3000 evenly with the first tube (containing 7.5 μg pCMV-FASN-3×FLAG-SV40-Neo) and the second tube (containing 7.5 μg pCMV-3×FLAG-SV40-Neo), and incubate at room temperature for 15 minutes.

[0126] 4) Transfect cells. The supernatant of the two tubes of DNA-lipid complex 6-well plate cells obtained in step 3) was transfected with pCMV-FASN-3×FLAG-SV40-Neo in 3 wells, and the other 3 wells were transfected with pCMV-3×FLAG-SV40-Neo as a control group, 250 μL of complex / well. After mixing with a pipette, the mixture was placed in a 37°C 5% CO2 incubator for 48 hours.

[0127] 3. Virus infection. Take out the 6-well plate, remove the culture supernatant, wash the cells twice with 1×PBS, and add 1.0 ml of HSV-1 virus solution diluted with maintenance solution to each well (MOI=1). Incubate in a 37℃ 5% CO2 incubator for 1 hour, remove the HSV-1 virus solution, wash twice with 1×PBS, add 2 ml of maintenance solution to each well, and continue to culture in a 37℃ 5% CO2 incubator for 24 hours.

[0128] 4. Harvest the virus supernatant using the same method as in Example 1.

[0129] 5. The virus titer was determined by CCID50 method, which was the same as that in Example 1.

[0130] 6. Experimental results are as follows Figure 4As shown. The FASN gene was overexpressed in wild-type A549 cells using plasmid pCMV-FASN-3×FLAG-SV40-Neo, and HSV-1 was infected (MOI=1) 48 hours later. After 24 hours, the cell supernatant was collected and the virus titer was determined by CCID50 method. The results showed that overexpression of FASN in A549 cells can promote the expression and replication of herpes simplex virus HSV-1.

[0131] Example 5: After HSV-1 infected A549 cells overexpressing the FASN gene, protein immunoblotting was used to detect the expression of HSV-1 immediate early protein ICP27.

[0132] 1. Prepare cells. Take well-growing A549 cells for subculture and inoculate the cells in a six-well plate. The volume of complete culture medium is 2 ml, the initial cell amount is 1.5×10^6 cells / well, and culture in a 37℃ 5% CO2 incubator for 16 hours.

[0133] 2. Liposome transfection of cells, the method used is the same as that in Example 4.

[0134] 3. Virus infection. Take out the 6-well plate, remove the culture supernatant, wash the cells twice with 1×PBS, and add 1.0 ml of HSV-1 virus solution diluted with maintenance solution to each well (MOI=1). Incubate in a 37℃ 5% CO2 incubator for 1 hour, remove the HSV-1 virus solution, wash twice with 1×PBS, add 2 ml of maintenance solution to each well, and continue to culture in a 37℃ 5% CO2 incubator for 6 hours.

[0135] 4. Harvest protein samples using the same method as in Example 2.

[0136] 5. The expression of FASN, β-Actin protein and HSV-1 early protein ICP27 was detected by western blotting, and the method used was the same as that in Example 2.

[0137] 6. Experimental results are as follows Figure 5 As shown. The FASN gene was overexpressed in wild-type A549 cells using plasmid pCMV-FASN-3×FLAG-SV40-Neo, and HSV-1 was infected (MOI=1) 48 hours later. After 6 hours, the protein was collected for protein immunoblotting to detect the expression of HSV-1 immediate early protein ICP27. The results showed that overexpression of FASN in A549 cells can promote the expression and replication of herpes simplex virus HSV-1.

[0138] Example 6: A549 cells were treated with the β-ketoacyl synthase domain inhibitor C75 of FASN and then infected with HSV-1. The titer of HSV-1 virus in the supernatant was determined by CCID50 method, and the expression of HSV-1 immediate early protein ICP27 was detected by protein immunoblotting.

[0139] 1. Prepare cells. Take well-growing A549 cells for subculture and inoculate the cells in two six-well plates. The volume of complete culture medium is 2 ml and the starting cell amount is 1.5×10 6 The cells were cultured in a 37°C 5% CO2 incubator for 16 hours.

[0140] 2. Pre-treat cells with C75. Use DMSO as solvent to prepare C75 into a 40mM stock solution. Take out a 6-well plate, add C75 stock solution with a final concentration of 40μM to 3 wells, and add DMSO with the same volume as 40μM C75 to 3 wells as negative control. Mix gently and place in a 37℃ 5% CO2 incubator for 12 hours. Perform the same operation on another 6-well plate.

[0141] 3. Virus infection. Take out the 6-well plate, remove the culture supernatant, wash the cells twice with 1×PBS, and add 1.0 ml of HSV-1 virus solution diluted with maintenance solution to each well (MOI=1). Incubate in a 37°C 5% CO2 incubator for 1 hour, remove the virus solution, wash twice with 1×PBS, add 2 ml of maintenance solution to each well, continue to add DMSO and C75 mother solution with a final concentration of 40 μM to the corresponding wells, mix slightly, continue to culture one 6-well plate in a 37°C 5% CO2 incubator for 6 hours to harvest protein samples, and continue to culture another 6-well plate in a 37°C 5% CO2 incubator for 24 hours to harvest the virus supernatant and detect the virus titer.

[0142] After 4 and 6 hours, the protein samples were harvested using the same method as in Example 2.

[0143] 5. The expression of FASN, β-Actin protein and HSV-1 early protein ICP27 was detected by western blotting, and the method used was the same as that in Example 2.

[0144] 6. After 24 hours, the virus supernatant was harvested using the same method as in Example 1.

[0145] 7. The virus titer was determined by CCID50 method, which was the same as that in Example 1.

[0146] 8. Experimental results are as follows Figure 6-7 shown.

[0147] 1) If Figure 6As shown, A549 cells were pretreated with 40 μM concentration of FASN inhibitor C75, infected with HSV-1 (MOI=1) 12 hours later, and the cell supernatant was collected 24 hours later for CCID50 determination of virus titer. The results showed that after A549 cells were treated with FASN β-ketoacyl synthase domain inhibitor C75, the expression and replication of HSV-1 were inhibited;

[0148] 2) If Figure 7 As shown, A549 cells were pretreated with 40 μM concentration of FASN inhibitor C75, infected with HSV-1 (MOI=1) 12 h later, and the collected proteins were subjected to protein immunoblotting 6 h later to detect the expression of HSV-1 immediate early protein ICP27. The results showed that after A549 cells were treated with C75, an inhibitor of the β-ketoacyl synthase domain of FASN, the expression and replication of HSV-1 were inhibited.

[0149] Example 7: After knocking down the expression of FASN gene in A549 cells by RNAi technology, the cells were infected with HSV-1, the titer of HSV-1 virus in the supernatant was determined by CC ID50 method, and the expression of HSV-1 immediate early protein ICP27 was detected by protein immunoblotting.

[0150] 1. Prepare cells. Take well-growing A549 cells for subculture and inoculate the cells in two 12-well plates. Inoculate 6 wells of cells in each 12-well plate for the experiment. The volume of complete culture medium is 1 ml and the starting cell amount is 5.0×10 5 The cells were cultured in a 37°C 5% CO2 incubator for 16 hours.

[0151] 2. Transfect cells using liposome method.

[0152] 1) Dilute Lipofectamine 3000. Take 150 μL of Opti-MEM medium and add 9 μL of Lipofectamine 3000.

[0153] 2) Dilute siRNA (si-NC is used as a negative control, and si-FASN is used to knock down FASN). Prepare two 1.5 mL Eppendorf tubes, numbered 1 and 2, add 150 μL Opti-MEM serum-free medium to the first tube, and add 4.5 μL NC-siRNA (containing si-NC-F and si-NC-R, with a concentration of 30 pmol, Jima Gene). Add 150 μL Opti-MEM serum-free medium to the second tube, and add 4.5 μL si-FASN (containing si-FASN-F and si-FASN-R, with a concentration of 30 pmol, Jima Gene), and gently pipette to mix.

[0154] 3) Mix and incubate. Add 75 μL of Lipofectamine 3000 diluted in step 1) to the Eppendorf tubes No. 1 and 2 respectively, mix well, and incubate at room temperature for 15 minutes.

[0155] 4) Transfect cells. Add the two tubes of siRNA-lipid complexes obtained in step 3) to the cell culture supernatant of a 12-well plate, transfect 3 wells with si-FASN, and 3 wells as a control group with si-NC, 100 μL of complex / well. Mix gently and culture in a 37°C 5% CO2 incubator for 48 hours.

[0156] 3. Virus infection: the method used is the same as that in Example 5.

[0157] After 4 and 6 hours, the protein samples were harvested using the same method as in Example 2.

[0158] 5. The expression of FASN, β-Actin protein and HSV-1 early protein ICP27 was detected by western blotting, and the method used was the same as that in Example 2.

[0159] 6. After 24 hours, the virus supernatant was harvested using the same method as in Example 1.

[0160] 7. The virus titer was determined by CCID50 method, which was the same as that in Example 1.

[0161] 8. Experimental results are as follows Figure 8-Figure 9 shown.

[0162] 1) If Figure 8 As shown, si-FASN was used to knock down the FASN gene in A549 cells, and the control group was si-NC without knockdown sites. After 48 hours, HSV-1 was infected (MOI = 1), and the cell supernatant was collected 24 hours later for CCID50 determination of viral titer results; the results showed that after knocking down the FASN gene in A549 cells, the expression and replication of HSV-1 were inhibited.

[0163] 2) If Fig. 9 As shown, si-FASN was used to knock down the FASN gene in A549 cells, and the control group was si-NC without knockdown sites. After 48 hours, HSV-1 was infected (MOI = 1), and protein was collected for protein immunoblotting after 6 hours. The results showed that after knocking down the FASN gene in A549 cells, the expression and replication of HSV-1 were inhibited.

[0164] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of an inhibitor of fatty acid synthase gene FASN in the preparation of a drug for preventing and / or treating HSV-1 virus infection, characterized in that: The inhibitor of the fatty acid synthase gene FASN is small interfering RNA; The small interfering RNA has the sequence shown below: si-FASN-F:GCUCGAUACCUUCCUGAAUTT; si-FASN-R:AUUCAGGAAGGUAUUCGAGCTT.

2. A drug for preventing and / or treating HSV-1 virus infection, characterized in that: It comprises a pharmaceutically acceptable carrier and an inhibitor of fatty acid synthase gene FASN, wherein the inhibitor of fatty acid synthase gene FASN is a small interfering RNA; The small interfering RNA has the sequence shown below: si-FASN-F:GCUCGAUACCUUCCUGAAUTT; si-FASN-R:AUUCAGGAAGGUAUUCGAGCTT.

3. Application of fatty acid synthase gene FASN overexpressing cell line in the preparation of HSV-1 virus vaccine producing cell line.

Images