A shRNA that targets and knocks down the HDAC5 gene and its applications
By constructing a recombinant lentiviral expression plasmid that targets and knocks down HDAC5 in Vero cells and screening stable cell lines, the shortcomings in the study of HDAC5 gene regulation of viral replication were addressed, and effective inhibition of PPRV proliferation was achieved, providing a new method for the development of anti-PPRV drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LANZHOU VETERINARY RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES(LANZHOU BRANCH CENTER OF CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENTER)
- Filing Date
- 2026-01-26
- Publication Date
- 2026-05-26
AI Technical Summary
There is limited research on the regulation of viral replication by the HDAC5 gene in Vero cell models, and there is a lack of effective methods to inhibit viral proliferation, especially in the development of drugs targeting PPRV infection, which lacks new ideas.
We designed shRNAs that target and knock down the HDAC5 gene, constructed recombinant lentiviral expression plasmids using lentivirus-mediated RNAi technology, and infected Vero cells. We then screened for stable HDAC5 knockdown cell lines with puromycin to verify their inhibitory effect on PPRV proliferation.
A stable Vero cell line with HDAC5 knockdown was successfully constructed, significantly reducing the expression level of PPRV N protein and providing a new approach for anti-PPRV infection drugs.
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Figure CN122081318A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology and relates to a shRNA that targets and knocks down the HDAC5 gene and its applications. Background Technology
[0002] RNA interference (RNAi) is a highly conserved evolutionary phenomenon involving the efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). RNAi is widespread in the biological world. Due to its high sequence specificity and efficient interference capabilities, it can target and inhibit the expression of specific genes, making it a popular and powerful tool for gene function research. Many methods exist for silencing target gene expression using RNAi technology. Vector-based methods involve cloning an oligonucleotide sequence containing an siRNA sequence, a stem-loop of approximately 9 nucleotides, and the inverse complementary sequence of the siRNA into a plasmid or viral vector. This vector is then transfected into cells, where it is expressed in the cell nucleus to produce endogenous short hairpin RNA (shRNA). Finally, the shRNA exits the nucleus and enters the cytoplasm, where it is cleaved by enzymes such as Dicer to generate siRNA, thus exerting the RNAi effect.
[0003] Histone deacetylase 5 (HDAC5) belongs to the class IIa HDAC family. Its carboxyl terminus contains a deacetylase active domain (DAC) and a nuclear export sequence (NES), while its amino terminus contains a transcription factor MEF2 binding domain and a nuclear localization sequence (NLS). It plays a crucial role in regulating viral replication. It is closely related to various human diseases caused by viral infections, such as human immunodeficiency virus (HIV) infection, hepatitis B virus (HBV) infection, and Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Current research on the function of HDAC5 in responding to viral infections has primarily been conducted in human or mouse cells and mouse animal models.
[0004] The Vero cell line (derived from African green monkey kidney epithelial cells) is a widely used susceptible cell line in virology research due to the lack of the type I interferon-1 (IFN-I) signaling pathway. Various human viruses, animal viruses, and zoonotic viruses can replicate effectively in Vero cells and produce typical cytopathic effects (CPE). However, studies on HDAC5 gene regulation of viral replication in Vero cell models are still rare. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides a shRNA that targets and knocks down the HDAC5 gene. Using lentivirus-mediated RNAi technology, a Vero cell line with stable HDAC5 gene knockdown was obtained, demonstrating that knocking down HDAC5 gene expression can inhibit PPRV proliferation, providing a new approach for the development of drugs against PPRV infection.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] In a first aspect, the present invention provides an shRNA that targets and knocks down the HDAC5 gene, wherein the sense strand of the shRNA is as shown in SEQ ID NO.4 and the antisense strand is as shown in SEQ ID NO.4, and the sense strand and antisense strand are annealed to form a double-stranded DNA with sticky ends.
[0008] Secondly, the present invention provides a recombinant lentiviral expression plasmid that targets and knocks down the HDAC5 gene, wherein the recombinant lentiviral expression plasmid is constructed by inserting the above-mentioned double-stranded DNA downstream of the U6 promoter of a lentiviral vector.
[0009] In one technical solution, the lentivirus vector is pLKO.1.
[0010] Thirdly, the present invention provides a recombinant lentiviral expression vector that targets and knocks down the HDAC5 gene, obtained by co-transfecting packaging cells with the above-mentioned recombinant lentiviral expression plasmid that targets and knocks down the HDAC5 gene and an helper plasmid.
[0011] In one technical solution, the auxiliary plasmids are psPAX2 and pMD2.G.
[0012] Fourthly, the present invention provides a Vero cell line that targets and knocks down the HDAC5 gene. The Vero cell line is obtained by infecting Vero cells with the above-mentioned recombinant lentiviral expression vector that targets and knocks down the HDAC5 gene and then screening them.
[0013] Fifthly, the present invention provides the above-mentioned shRNA targeting and knocking down the HDAC5 gene, the above-mentioned recombinant lentiviral expression plasmid targeting and knocking down the HDAC5 gene, the above-mentioned recombinant lentiviral expression vector targeting and knocking down the HDAC5 gene, and the application of the above-mentioned Vero cell line targeting and knocking down the HDAC5 gene in inhibiting HDAC5 gene expression.
[0014] In a sixth aspect, the present invention provides the application of the above-mentioned shRNA targeting and knocking down the HDAC5 gene, the above-mentioned recombinant lentiviral expression plasmid targeting and knocking down the HDAC5 gene, the above-mentioned recombinant lentiviral expression vector targeting and knocking down the HDAC5 gene, and the above-mentioned Vero cell line targeting and knocking down the HDAC5 gene in screening anti-PPRV drug targets.
[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0016] This invention utilizes lentivirus-mediated RNAi technology to design shRNA targeting and knocking down the HDAC5 gene by selecting a conserved sequence of HDAC5 in Vero cells. A recombinant lentivirus expression plasmid targeting and knocking down the HDAC5 gene is constructed. This recombinant lentivirus expression plasmid is co-transfected with a helper plasmid into packaging cells to obtain a recombinant lentivirus expression vector, which is then used to infect Vero cells. Vero cell lines with stable HDAC5 gene knockdown are obtained through puromycin pressure selection.
[0017] This invention utilizes PPRV infection to stably knock down the HDAC5 gene in Vero cell lines, and finds that the expression level of PPRV N protein is reduced in the HDAC5 knockdown Vero cell lines, demonstrating that knocking down the expression of the HDAC5 gene can inhibit the proliferation of PPRV, providing a new approach for the development of anti-PPRV drugs. Attached Figure Description
[0018] Figure 1 The results are shown in the gel electrophoresis of the enzyme digestion products.
[0019] Figure 2 The interference effect of recombinant expression plasmids obtained with different interference sequences on the target gene HDAC5 is shown.
[0020] Figure 3 Cell morphology and fluorescence expression after co-transfection of HEK-293T cells with packaged lentiviral plasmids.
[0021] Figure 4 The fluorescence expression and HDAC5 protein expression of HEK-293T cells were investigated after infection with concentrated crude lentivirus solution.
[0022] Figure 5 The cytotoxicity of different concentrations of polybrene and puromycin on Vero cells was investigated.
[0023] Figure 6 The results show the screening of positive cells after Vero cells were infected with the recombinant lentiviral expression vector.
[0024] Figure 7 To determine the transcriptional level and protein expression of HDAC5 in stable HDAC5 cell lines.
[0025] Figure 8 To determine the copy number of the PPRV N gene and the expression of the PPRV N protein in HDAC5 stable cell lines. Detailed Implementation
[0026] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following embodiments are conventional methods.
[0027] Example 1: Construction of Recombinant Lentiviral Expression Plasmid
[0028] Based on the predicted sequence of the HDAC5 gene of the African green monkey (Chlorocebus sabaeus) published in the NCBI database (XM_073005246.1), two interfering siRNA sequences were screened out, and an invalid interfering sequence shNC was designed as a control group, as shown in Table 1.
[0029] Table 1. siRNAs for knocking down the HDAC5 gene
[0030]
[0031] The above interfering sequence siRNAs were combined in both forward and reverse directions, with a loop structure added in the middle, so that the oligonucleotides could form hairpin-structured shRNAs. A Taq DNA polymerase promoter and protective bases were added to the 5' end, and six Ts were added to the 3' end as a transcription terminator to form double-stranded oligo DNA of the target sequence, with corresponding AgeI and EcoRI digestion sticky ends at both ends (see Table 2).
[0032] Table 2. shRNA sequences of HDAC5 gene knockdown
[0033]
[0034] Primer dilution: Add 172 μL of sterile water to each of the synthesized sense and antisense strands and mix by shaking.
[0035] Annealing: Add 5 μL of each of the diluted positive and negative strands to a PCR tube, vortex to mix, briefly centrifuge, collect the liquid at the bottom of the tube, place it in the PCR instrument, and execute the procedure shown in Table 3.
[0036] Dilution of annealing product: Take 1 μL of annealing product and add 166 μL of sterile water to dilute it. Mix thoroughly and set aside.
[0037] Table 3 PCR reaction procedure
[0038]
[0039] Using the lentiviral vector pLKO.1 as the starting vector, the enzyme was double-digested with AgeI and EcoRI enzymes (Thermo Fisher Scientific) (the digestion reaction system is shown in Table 4). The digestion system was incubated at 37°C for 1 h. The digested product (7872 bp, see Table 4) was recovered by gel excision using an agarose gel purification and recovery kit (manufacturer: Qingke Biotechnology, catalog number: TSP602-200). Figure 1 ).
[0040] Table 4. AgeI and EcoRI enzyme digestion reaction system
[0041]
[0042] Construction of the ligation system: Take 1 μL of the diluted annealing product and add it to a PCR tube. Add the digested pLKO.1 vector, T4 DNase, sterile water, and 2×T4 Buffer. Shake well and centrifuge briefly. The specific components and corresponding volumes are shown in Table 5.
[0043] Table 5 Connection System
[0044]
[0045] Connection: Place the connection system in a 30℃ constant temperature incubator and connect for 1 h.
[0046] Transformation: Remove Stbl3 competent cells from -80℃ and immediately place them on ice. After 5 minutes, wait for the bacterial block to thaw. Add all the ligation product to the competent cells and gently mix by tapping the bottom of the EP tube (avoid pipetting). Incubate on ice for 25 minutes. Heat shock at 42℃ for 45 seconds, then immediately return to ice and incubate for 2 minutes. Shaking will reduce transformation efficiency. Add 0.9 mL of LB medium to a centrifuge tube and incubate at 30℃ and 225 rpm for 90 minutes. Centrifuge at 5000 rpm for 1 minute to collect the bacteria. Collect approximately 100 μL of supernatant, gently resuspend the bacterial block by pipetting, and spread it onto LB medium containing ampicillin. Incubate the plate upside down at 30℃ overnight. Randomly select 8 single clones from the plate for sequencing. Expand the sequencing results of the correctly sequenced single clones and prepare glycerol culture for preservation.
[0047] Plasmid extraction: The target plasmid was extracted using an endotoxin-free plasmid medium-quantity extraction kit (manufacturer: TIANGEN, catalog number: DP108), resulting in the following recombinant lentiviral expression plasmids: pLKO.1-U6-shRNA-1-CMV-copGFP-PURO (denoted as pLKO.1-H5-1), pLKO.1-U6-shRNA-2-CMV-copGFP-PURO (denoted as pLKO.1-H5-2), and pLKO.1-U6-shNC-CMV-copGFP-PURO control plasmid (denoted as pLKO.1-NC).
[0048] Example 2: Screening for the siRNA sequence with the best interference effect on the HDAC5 gene.
[0049] Vero cells were digested with trypsin to prepare a cell suspension, which was then seeded into 6-well plates for culture, so that the cell density could reach about 60-70% by the time of transfection the next day.
[0050] Before transfection, discard the old culture medium in the well plate and add fresh complete culture medium. Take three sterile 1.5 mL centrifuge tubes, add 125 μL of Opti-MEM® culture medium to each tube, then add 2.5 μg of the three plasmids obtained in Example 1 above. Gently pipette several times, add 4 μL of Lipo8000™ transfection reagent (manufacturer: Beyotime, catalog number: C0533-7.5 ml), gently pipette to mix, and then slowly drop the mixture into the corresponding cell wells. Incubate at 37°C and 5% CO2 for approximately 72 h.
[0051] The transfected Vero cells were collected and subjected to Western blot analysis to determine HDAC5 protein expression. The results are as follows: Figure 2 As shown, compared with pLKO.1-NC, plasmid pLKO.1-H5-1 has the most significant effect on the downregulation of the target gene HDAC5.
[0052] Example 3: Packaging Lentiviral Viruses to Construct Recombinant Lentiviral Expression Vectors
[0053] HEK-293T cells in good condition were inoculated in 10 cm cell culture dishes, and the cell density reached 70-90% before transfection the next day.
[0054] Take a sterile 1.5 mL centrifuge tube and add 750 μL of Opti-MEM® medium (manufacturer: Gibco, catalog number: 31985070). Mix pLKO.1-NC and pLKO.1-H5-1 plasmids with helper plasmids (packaging plasmid psPAX2 and envelope plasmid pMD2.G) at a mass ratio of pLKO.1:psPAX2:pMD2.G = 4:3:1 (total mass of mixed plasmids is 15 μg) in the centrifuge tube, gently pipette to mix, add 24 μL of Lipo8000™ transfection reagent, gently pipette to mix, and slowly drop the mixture into cell culture dishes. Gently shake well and incubate at 37°C, 5% CO2. Replace with fresh complete medium and continue culturing 6–8 h after transfection.
[0055] Observe cell morphology and fluorescence expression 48-72 h after transfection. Figure 3 As shown, the lentiviral particles obtained by packaging the pLKO.1-NC plasmid are denoted as LV-NC, and the lentiviral particles obtained by packaging the pLKO.1-H5-1 plasmid are denoted as LV-H5-1.
[0056] When most cells became rounded and detached, the cell supernatant (containing lentiviral particles) was collected, filtered through a 0.45 μm filter membrane, and cell debris was removed. The crude lentiviral solution was concentrated using a lentiviral concentrator (manufacturer: Bio-Long, catalog number: BF06205), and then aliquoted into 1 mL tubes and stored at -80°C.
[0057] HEK-293T cells in good seeding condition were cultured in 6-well plates at 37°C with 5% CO2 until complete cell adhesion. The optimal cell density before infection was 40-60%. Before infection, the culture medium was replaced with 1 mL of DMEM medium containing 2% serum and no antibiotics. 1 mL of concentrated crude lentivirus solution was added to each well using the half-volume infection method. The proportion of positive cells was observed under a fluorescence microscope 48-72 h after infection. Figure 4 As shown, HEK-293T cells infected with lentiviral particle LV-NC are represented as HEK-NC, and HEK-293T cells infected with lentiviral particle LV-H5-1 are represented as HEK-H5-1. Fluorescence was observed at both 48 h and 72 h after infection. Figure 4 A), Western blot experiments showed that the knockdown of HDAC5 in HEK293T cells was also significant ( Figure 4 B) indicates that the recombinant lentiviral expression vector was successfully constructed.
[0058] Example 4: Construction of a Vero cell line with stable HDAC5 gene knockdown
[0059] (1) Screening Vero cell lines with HDAC5 gene knockdown
[0060] Polybrene is a positively charged small molecule that binds to anions on the cell surface, enhancing the adsorption and infection efficiency of lentiviruses. Polymycin is commonly used for screening stable cell lines; cells successfully expressing the puromycin resistance gene (pac gene) develop puromycin resistance and can survive and proliferate in puromycin-containing media, eventually forming stable cell lines. To screen Vero cell lines with stable HDAC5 gene knockdown, the optimal concentrations of polybrene and puromycin in Vero cells were first determined using a CCK-8 assay. Figure 5 As shown, the optimal concentration of polybrene is 6 μg / mL. Figure 5 A), the optimal concentration of puromycin is 5 μg / mL ( Figure 5 B).
[0061] Healthy Vero cells were seeded into 6-well plates, ensuring a confluence of approximately 30-40% at infection on the second day. The culture medium was replaced with 1 mL of DMEM medium containing 2% serum and free of antibiotics. The concentrated crude lentivirus solution obtained in Example 3 was added at 1 mL / well using the half-volume infection method (three replicates were set up for the control and knockdown groups). Simultaneously, 6 μg / mL of polybrene was added to each well to enhance lentivirus infection efficiency. The proportion of positive cells was observed under a fluorescence microscope 48-72 h after infection. When the positive cell count exceeded 70%, the cells were digested and resuspended, transferred to T25 cell flasks, and selected by adding 5 μg / mL of puromycin. Due to the significant cell death following the addition of puromycin, the medium was changed every 24 h, and cell growth was observed daily.
[0062] When the cell confluence reaches approximately 80%, continue subculturing and expanding the culture, and continue selecting with puromycin until the proportion of positive cells approaches 95%, indicating that complete selection has been essentially achieved (by day 13 of subculturing, the proportion of positive cells is close to 95%), forming a stable cell line. Figure 6 As shown (Ve-NC represents Vero cells infected with LV-NC control group lentivirus, and Ve-H5-1 represents Vero cells infected with LV-H5-1 knockdown group lentivirus), the cells were aliquoted and cryopreserved.
[0063] (2) Identification of stable Vero cell lines
[0064] Take approximately 1×10⁻⁶ Ve-NC and Ve-H5-1 cells respectively 6Total RNA was extracted using the TRIzol method, and the transcription of the HDAC5 gene was detected by qRT-PCR. The concentration of extracted RNA was measured using a NanoDrop 8000 spectrophotometer. 1 μg of total RNA was used for reverse transcription to obtain cDNA according to the instructions of the PrimeScript RT Reverse Transcription Kit (manufacturer: TaKaRa, catalog number: RR047A). The qRT-PCR reaction system was prepared according to the instructions of the 2×RealStar Fast SYBR qPCR Mix Kit (manufacturer: Genstar, catalog number: A301). The qRT-PCR reaction system and amplification program are shown in Tables 6 and 7, respectively.
[0065] Table 6 qRT-PCR reaction system
[0066]
[0067] Table 7 Two-step qRT-PCR amplification program
[0068]
[0069] The qRT-PCR primer sequences are shown in Table 8. The relative expression levels of the HDAC5 gene were determined by 2... –△△Ct calculate.
[0070] Table 8 qRT-PCR primer sequences
[0071]
[0072] qRT-PCR results are as follows Figure 7 As shown in Figure A, the HDAC5 transcription level in the Ve-H5-1 cell line decreased by about half.
[0073] Approximately 2 × 10⁶ Ve-NC and Ve-H5-1 cells were taken respectively. 6 One sample was subjected to a Western blot experiment to determine the protein expression of HDAC5. The results are as follows: Figure 7 As shown in Figure B, protein expression decreased by approximately 40%, indicating the successful construction of a stable Vero cell line with HDAC5 knockdown.
[0074] Example 5: Effect of HDAC5 gene knockdown on PPRV proliferation in stable transgenic cell lines
[0075] Stable cell lines were seeded into 6-well plates, with three replicates for both Ve-NC and Ve-H5-1 cell lines, and approximately 2 × 10⁶ cells per well. 5After the cells had fully adhered to the culture vessel, they were inoculated with 0.1 MOI PPRV (Petty Ruminant Disease Virus, preserved in our laboratory). Samples were collected 24 h after infection for viral RNA level detection. qRT-PCR results showed that knocking down HDAC5 reduced the copy number of the PPRV N gene by approximately 12% (e.g., ...). Figure 8 As shown in Figure A). Western blot analysis of HDAC5 knockdown revealed that PPRV N protein expression decreased after HDAC5 knockdown (as shown in Figure A). Figure 8 As shown in Figure B), knocking down HDAC5 is detrimental to PPRV proliferation.
[0076] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.
Claims
1. An shRNA targeting knockdown of HDAC5 gene, characterized in that, The sense strand of the shRNA is shown as SEQ ID NO. 4, and the antisense strand is shown as SEQ ID NO. 5, and the sense strand and the antisense strand are annealed to form a double-stranded DNA with sticky ends.
2. A recombinant lentiviral expression plasmid targeting knockdown of HDAC5 gene, characterized in that, The recombinant lentiviral expression plasmid is constructed by inserting the double-stranded DNA of claim 1 downstream of the U6 promoter of a lentiviral vector. 3.The recombinant lentiviral expression plasmid targeting and knocking down HDAC5 gene of claim 2, characterized in that, The lentiviral vector is pLKO.
1.
4. A recombinant lentiviral expression vector targeting knockdown of HDAC5 gene, characterized in that, The recombinant lentiviral expression plasmid for targeting and knocking down the HDAC5 gene of claim 2 or 3 is co-transfected with a helper plasmid into a packaging cell to obtain. 5.The recombinant lentiviral expression vector targeting and knocking down HDAC5 gene of claim 4, characterized in that, The helper plasmid is psPAX2 and pMD2.G.
6. A Vero cell line targeting knockdown of HDAC5 gene, characterized in that, The Vero cell line is obtained by infecting Vero cells with the recombinant lentiviral expression vector for targeting and knocking down the HDAC5 gene of claim 4 or 5 and screening.
7. The shRNA for targeting and knocking down the HDAC5 gene of claim 1, the recombinant lentiviral expression plasmid for targeting and knocking down the HDAC5 gene of claim 2 or 3, the recombinant lentiviral expression vector for targeting and knocking down the HDAC5 gene of claim 4 or 5, or the Vero cell line for targeting and knocking down the HDAC5 gene of claim 6 are used for inhibiting the expression of the HDAC5 gene.
8. The shRNA for targeting and knocking down the HDAC5 gene of claim 1, the recombinant lentiviral expression plasmid for targeting and knocking down the HDAC5 gene of claim 2 or 3, the recombinant lentiviral expression vector for targeting and knocking down the HDAC5 gene of claim 4 or 5, or the Vero cell line for targeting and knocking down the HDAC5 gene of claim 6 are used for screening an anti-PPRV drug target.