A lentivirus recombinant vector interfering with PRKAA1 gene, a lentivirus construction method and application thereof

Abstract

This invention discloses a lentiviral recombinant vector for interfering with the PRKAA1 gene, a method for lentiviral construction, and its applications, belonging to the field of biochemistry. This invention uses bioinformatics tools to predict shRNA target gene sequences with high specificity and low off-target effects, obtaining shRNAs with target gene sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4, and constructs a lentiviral recombinant vector. Using this lentiviral recombinant vector, lentiviral packaging efficiency is high, viral titer is stable, and infection effect is good. It can stably knock down PRKAA1 in mammalian cells for a long period, making it suitable for long-term experiments and animal studies.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically a lentiviral recombinant vector that interferes with the PRKAA1 gene, a lentiviral construction method, and its application. Background Technology

[0002] In the fields of molecular biology and disease mechanism research, the PRKAA1 gene (encoding the AMPKα1 subunit) is a core target regulating energy metabolism, cell proliferation, and inflammatory responses, and its functional study has significant scientific and clinical value. Currently, mainstream loss-of-function methods for this gene mainly include transient siRNA transfection, CRISPR-Cas9 gene knockout, and shRNA lentiviral interference. However, these existing technologies have revealed certain limitations in long-term stable knockdown and in-depth biological function studies.

[0003] (1) siRNA transient transfection technology: Although this technology has the advantages of simple operation and high initial interference efficiency, its effect is short-lived, the knockdown effect is easily decayed and unstable. This method is only suitable for short-term in vitro cell experiments and cannot meet the needs of long-term stable transfection studies or in vivo animal experiments; in addition, the delivery efficiency of siRNA in vivo is poor and it is easy to degrade, which limits its in vivo application.

[0004] (2) CRISPR-Cas9 gene knockout technology: Although this technology can achieve efficient gene knockout, the process is irreversible; once knocked out, it cannot be restored. Furthermore, the CRISPR system is prone to off-target effects. More importantly, for key genes maintaining basal cell metabolism, such as PRKAA1, complete knockout may lead to cell death or induce the activation of other intracellular alternative compensatory mechanisms, thereby masking the gene's true phenotype and severely interfering with the accuracy of research results.

[0005] (3) Traditional shRNA lentiviral interference technology: Although lentivirus-mediated shRNA can integrate exogenous genes into the host genome to achieve long-term silencing, it is currently a common method for establishing stable knockdown cell lines. However, existing shRNA systems targeting PRKAA1 often suffer from technical bottlenecks such as unoptimized target sequence design, low viral packaging titers, and poor cell infection efficiency, resulting in low and unstable actual knockdown efficiency and difficulty in obtaining stable cell lines. In addition, existing studies often lack a standardized and complete process from shRNA construction to validation, which greatly limits the promotion and application of this technology in PRKAA1 functional studies.

[0006] In summary, existing technologies still have significant limitations in the stable silencing and functional verification of the PRKAA1 gene. Therefore, there is an urgent need in this field to develop a highly efficient, well-defined, and clearly validated PRKAA1-targeting shRNA lentiviral system to overcome the shortcomings of existing technologies and provide a reliable tool for stable interference with PRKAA1 and in-depth biological functional studies. Summary of the Invention

[0007] To address the issues of low knockdown efficiency and instability in existing shRNA lentiviral systems targeting PRKAA1, a method for constructing and identifying lentiviruses that interfere with the PRKAA1 gene is provided.

[0008] Technical solution:

[0009] (i) A lentiviral recombinant vector that interferes with the PRKAA1 gene, said lentiviral recombinant vector containing shRNA targeting the gene sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0010] (II) A method for constructing a lentiviral recombinant vector that interferes with the PRKAA1 gene, comprising the following steps:

[0011] Based on the target gene sequence, two complementary oligonucleotide single strands were designed and synthesized. The two complementary DNA oligonucleotides were annealed to generate a DNA insert fragment, which was then inserted into a lentiviral transfer vector to obtain a lentiviral recombinant vector containing shRNA, i.e., a lentiviral recombinant vector that interferes with the PRKAA1 gene.

[0012] (III) A method for constructing a lentivirus that interferes with the PRKAA1 gene, comprising the following steps:

[0013] Step 1: shRNA Design and Screening

[0014] Based on the mRNA sequence of the PRKAA1 gene, bioinformatics tools were used to predict shRNA target sequences with high specificity and low off-target effects, resulting in multiple candidate target gene sequences; the candidate target gene sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0015] Step 2: Construction of shuttle plasmids:

[0016] Based on the target gene sequence, two complementary oligonucleotide single strands were designed and synthesized. The two complementary DNA oligonucleotides were annealed to generate a DNA insert fragment, which was then inserted into a lentiviral transfer vector to obtain a lentiviral recombinant vector containing shRNA. The lentiviral recombinant vector was transformed into competent E. coli to obtain an shRNA shuttle plasmid targeting the PRKAA1 gene.

[0017] Step 3: Use shuttle plasmids to transfect target cells to verify the efficiency of AMPKα knockdown and screen out shuttle plasmids with good results;

[0018] Step 4, Lentiviral Packaging:

[0019] Using a three-plasmid system, the shuttle plasmid selected in step three was co-transfected with the packaging plasmid and the envelope plasmid into 293T cells. After culturing for 48 hours, the supernatant was collected to obtain PRKAA1 interference lentivirus.

[0020] Step 5: Verify the lentivirus packaging effect and determine the titer;

[0021] Step 6: Use viral fluid to infect the target cells with the optimal infection MOI (Mean Infection Point) using lentivirus.

[0022] Furthermore, the specific steps for step four are as follows:

[0023] Step 3.1, revival and passage of 293T cells;

[0024] Step 3.2: Mix the shuttle plasmid, packaging plasmid and encapsulation plasmid in a ratio of 4:3:1 and place them in a serum-free culture medium, which is called solution A. Incubate at room temperature for 5 min.

[0025] Step 3.3: Add PEI solution with a concentration of 1 μg / mL to serum-free culture medium, and label it solution B. Let it stand at room temperature for 5 min.

[0026] Step 3.4: Slowly add solution B to solution A, let stand at room temperature for 20 min, mix the two thoroughly, add to a culture dish containing 293T cells, and incubate in an incubator for 6 h;

[0027] Step 3.5: After culturing for 6 hours, discard the original culture medium, add complete culture medium, and culture in a cell culture incubator for 48 hours. Then collect the supernatant, which is the virus solution.

[0028] Furthermore, the lentiviral recombinant vector contains shRNA targeting the gene sequence shown in SEQ ID NO.1, and the viral fluid produced by lentiviral packaging has a viral titer of 10. 8 TU / mL, with an optimal infection MOI of 40.

[0029] (iv) Application of lentiviral recombinant vectors interfering with the PRKAA1 gene in the preparation of products with reduced PRKAA1 gene expression levels. This invention has verified through examples that lentiviral recombinant vectors interfering with the PRKAA1 gene exhibit stable expression, high titer, and excellent infection efficiency, enabling sustained knockdown of PRKAA1 in mammalian cells.

[0030] Beneficial effects:

[0031] (1) The present invention uses RT-qPCR to screen and verify shRNA sequences. The screened sequences have good silencing effect on PRKAA1 gene, accurate targeting, and high knockdown efficiency.

[0032] (2) The lentiviruses of this invention have high packaging efficiency, stable viral titers, and good infection effects. They can stably knock down PRKAA1 in mammalian cells for a long time, making them suitable for long-term experiments and animal studies. Attached Figure Description

[0033] Figure 1 The purpose is to verify the knockdown effect of different target sequences using RT-qPCR.

[0034] Figure 2 It represents the fluorescence intensity of 293T cells 48 hours after transfection.

[0035] Figure 3 This shows the fluorescence expression of lentiviruses at different titers.

[0036] Figure 4 It represents the fluorescence intensity at different MOI values ​​in target cells infected by lentiviruses.

[0037] Figure 5 It involves knocking down the expression of proteins in the AMPK downstream pathway. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. The application principles of this invention will now be described in detail with reference to the accompanying drawings.

[0039] The plasmid extraction method, plasmid transfection method, RNA extraction method, RNA synthesis cDNA and real-time quantitative PCR analysis method, and lentivirus packaging method in Examples 2-6 below are as follows:

[0040] (a) Plasmid extraction method, including the following steps:

[0041] 1) Column equilibration step: Add 500 μL of equilibration solution BL to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0042] 2) Take 1-5 mL of overnight cultured E. coli culture and add it to a centrifuge tube. Centrifuge at 12000 rpm for 1 min and remove as much supernatant as possible (if there is a large amount of bacterial culture, the bacterial precipitate can be collected into a centrifuge tube by multiple centrifugations).

[0043] 3) Add 250 μL of solution P1 (containing RNAase) to the centrifuge tube containing bacterial pellet, and thoroughly suspend the bacterial pellet using a pipette or vortex mixer.

[0044] 4) Add 250 μL of solution P2 to the centrifuge tube and gently invert it 6-8 times to fully lyse the bacteria;

[0045] 5) Add 350 μL of solution P3 to the centrifuge tube, and immediately gently invert it 6-8 times to mix thoroughly. At this time, a white flocculent precipitate will appear. Centrifuge at 12000 rpm for 10 min.

[0046] 6) Transfer the supernatant collected in the previous step to the adsorption column using a pipette, being careful not to aspirate the precipitate. Centrifuge at 12,000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column into the collection tube.

[0047] 7) Add 600 μL of washing buffer PW (with anhydrous ethanol) to the adsorption column, centrifuge at 12000 rpm for 30-60 s, discard the waste liquid in the collection tube, and put the adsorption column into the collection tube.

[0048] 8) Repeat step 7;

[0049] 9) Place the adsorption column into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove the residual washing solution from the adsorption column;

[0050] 10) Place the adsorption column into a clean centrifuge tube, add 50-100 μL of elution buffer EB to the middle of the adsorption membrane, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 2 min, and collect the plasmid solution into the centrifuge tube.

[0051] The above plasmid extraction method uses an alkaline lysis plasmid extraction kit (Tiangen Biopharmaceutical Plasmid Mini-Prep Kit). The Tiangen Biopharmaceutical Plasmid Mini-Prep Kit contains equilibration buffer BL, solution P1, RNase, solution P2, solution P3, and wash buffer PW.

[0052] (II) Plasmid transfection method for bovine mammary epithelial cells (using Thermo Fisher Scientific Lipofectamine® 2000 transfection reagent), including the following steps:

[0053] 1) One day before transfection, use culture medium without antibiotics to plate the cells, so that the cell density reaches 70%-80% before transfection;

[0054] 2) Dilute the plasmid to 100 μL using serum-free medium (Opti-MEM);

[0055] 3) Before using the transfection reagent Lipofectamine 2000, gently mix it, then take 5-12.5 μL of Lipofectamine 2000 and dilute it to 100 μL, and incubate at room temperature for 5 min;

[0056] 4) After incubation, mix the two together, gently stir, and incubate at room temperature for 20 minutes;

[0057] 5) Add the mixture to a 6-well plate of cells, replace with complete culture medium after 6 hours, and extract cell RNA after 48 hours.

[0058] (III) Method for RNA extraction from bovine mammary epithelial cells, wherein the cells are isolated from bovine mammary tissue, including the following steps:

[0059] 1) Aspirate the cell culture medium from the six-well plate and wash the cells with PBS 1-2 times;

[0060] 2) Remove the PBS, then add 1 mL of RNAex (RNA extraction lysis buffer) to each well, and gently shake the culture dish to ensure that the RNAex solution is evenly distributed on the cell surface;

[0061] 3) Use a pipette to repeatedly pipette the cells to detach them, then transfer the lysate containing the cells to a centrifuge tube, and pipette the lysate repeatedly until there is no obvious precipitate in the lysate.

[0062] 4) After standing at room temperature for 5 minutes, add 1 / 5 volume of chloroform to the lysis buffer, mix thoroughly, and let stand at room temperature for 5 minutes.

[0063] 5) Centrifuge at 12000 rpm and 4℃ for 15 min. Carefully remove the centrifuge tube. At this point, the homogenate will be divided into 3 layers: the supernatant (containing RNA), the intermediate protein layer, and the lower organic phase.

[0064] 6) Transfer the supernatant to another new centrifuge tube;

[0065] 7) Add 1 / 2 volume of RNAex isopropanol to the supernatant, mix thoroughly, and let stand at room temperature for 10 minutes;

[0066] 8) Centrifuge at 12000 rpm at 4℃ for 10 min. Discard the supernatant after centrifugation, being careful not to touch the RNA precipitate.

[0067] 9) Add an equal volume of 80% ethanol (pre-cooled to -20℃) to the centrifuge tube to wash the RNA precipitate and the walls of the centrifuge tube. Centrifuge at 7500 rpm at 4℃ for 5 min. Carefully discard the supernatant and do not touch the precipitate.

[0068] 10) Open the centrifuge tube cap and vacuum or dry the precipitate at room temperature for about 5 minutes;

[0069] 11) Add an appropriate amount of RNase-free water to the centrifuge tube to dissolve the RNA, read the RNA concentration and OD value using a UV spectrophotometer, and store the sample RNA at -80℃.

[0070] (iv) RNA synthesis, cDNA, and real-time quantitative PCR analysis methods, including the following steps:

[0071] 1) cDNA was synthesized using the reverse transcription kit Evo M-MLVRT Mix Kit with gDNA Clean for qPCR Ver.2. The gDNA was first removed using a two-step method, and the reaction solution was prepared on ice.

[0072] Table 1. Reaction Solution

[0073]

[0074] Gently mix the mixture using a vortex mixer, briefly centrifuge, and then perform the first step of the PCR reaction at 42°C for 2 min. Add 4 μL of 5×Evo M-MLVRT Reaction Mix Ver.2, briefly centrifuge to mix, and then perform reverse transcription on a PCR instrument at 37°C for 15 min and 85°C for 5 s. Dilute the resulting cDNA with RNase-free water to obtain a uniform concentration. The product can be used for qPCR or stored at -20°C.

[0075] 2) Based on the sequence information of the PRKAA1 gene and the internal reference gene GAPDH of the species bovine (Bos taurus), specific primers were designed and synthesized by Nanjing Qingke Biotechnology Co., Ltd.

[0076] Table 2. Primer sequences

[0077]

[0078] 3) The relative expression level of the target gene was detected using the SYBR Green Premix Pro Taq HS Qpcr Tracking Kit (Rox Plus), and then normalized.

[0079] Table 3. RT-qPCR reaction system

[0080]

[0081] Table 4. RT-qPCR reaction procedure

[0082]

[0083] 3) Statistical analysis

[0084] Using GAPDH as an internal reference gene, The relative expression levels of the target gene were calculated. P-values ​​were determined using one-way ANOVA; the same letter indicated no significant difference (P>0.05), while different letters indicated significant differences (P<0.05). All experimental data were analyzed using one-way ANOVA, and all values ​​are expressed as the mean of three replicates ± SEM. GraphpadPrism 10.0 software was used for plotting.

[0085] (v) Lentiviral packaging method, including the following steps:

[0086] 1) Resuscitation and passage of 293T cells;

[0087] 2) The ratio of shuttle plasmid, packaging plasmid and envelope plasmid used for lentivirus packaging is 4:3:1. Then the mixture of the three is mixed in serum-free culture medium and labeled as solution A. Mix thoroughly and incubate at room temperature for 5 min.

[0088] 3) Take a PEI solution (polyethyleneimine aqueous solution) with a concentration of 1 μg / mL and add it to the serum-free culture medium, which is called solution B. Let it stand at room temperature for 5 min.

[0089] 4) Slowly add solution B to solution A, let stand at room temperature for 20 min, mix the two thoroughly, add to a cell culture dish containing 293T cells, and incubate in an incubator for 6 h;

[0090] 5) After 6 hours, discard the original culture medium, add complete culture medium, and incubate in a cell culture incubator for 48 hours. Then collect the supernatant, which is the original virus solution.

[0091] Example 1: Design of a shuttle plasmid containing shRNA target gene sequence targeting the PRKAA1 gene.

[0092] The design method for shuttle plasmids is as follows:

[0093] (1) Target screening: Based on the gene sequence, bioinformatics software is used to screen 19-21nt target sequences with high specificity and low off-target risk.

[0094] Specifically, based on the mRNA sequence of the PRKAA1 gene (encoding the AMPKα1 subunit), bioinformatics tools were used to predict shRNA target gene sequences with high specificity and low off-target effects, resulting in four candidate target gene sequences, as shown in SEQ ID NO.1-SEQ ID NO.4.

[0095] (2) Hairpin structure design: Design reverse complementary sequences at both ends of the target sequence, insert a stem-loop sequence in the middle, and add an RNA polymerase III terminator at the 3' end.

[0096] (3) Primer synthesis: Add specific restriction endonuclease sites to both ends of the designed shRNA sequence to chemically synthesize two complementary DNA oligonucleotide single strands.

[0097] (4) Two complementary DNA oligonucleotides were annealed to generate a DNA insert fragment, which was then inserted into a lentiviral transfer vector to obtain a lentiviral recombinant vector containing shRNA. The lentiviral recombinant vector was transformed into competent E. coli, and after culturing, single colonies were picked, plasmids were extracted, and PCR sequencing was performed to confirm that the inserted sequence was correct. The recombinant plasmid that has been identified is the shuttle plasmid that can be used for subsequent experiments.

[0098] The selected candidate target gene sequences and shuttle plasmid names are shown in Table 1. The candidate target gene sequences and shRNA vector construction were both completed by Suzhou Gemma Gene Co., Ltd.

[0099] Table 5. Shuttle plasmid names and target gene sequences

[0100]

[0101] Example 2: Verification of AMPKα knockdown efficiency using shuttle plasmid transfection of bovine mammary epithelial cells

[0102] The shuttle plasmids PRKAA1-Bos-1071, PRKAA1-Bos-1275, PRKAA1-Bos-1460, and PRKAA1-Bos-1536, constructed using four target genes, were transfected into the target cells, bovine mammary epithelial cells.

[0103] RNA was extracted from target cells after transfection and subjected to RT-qPCR. The results are as follows: Figure 1 As shown, Figure 1The knockdown efficiency of shRNA-1275, 1536, 1071, and 1460 was significantly different from that of shRNA-NC (no target, used as a negative control) (P<0.05), while the knockdown effect of shRNA-1460 was significantly different from that of the other three groups (P<0.05), indicating that shRNA-1460 had the best knockdown efficiency. PRKAA1-Bos-1460 was used for lentiviral packaging and transfection in subsequent experiments.

[0104] Example 3: Verification of Lentiviral Packaging Effect

[0105] Using a three-plasmid system (PRKAA1-Bos-1460 as the shuttle plasmid, psPAX2 as the packaging plasmid, and PMD2.G as the envelope plasmid), 293T cells were transfected using lentiviral packaging. 48 h later, the fluorescence intensity was as follows: Figure 2 As shown, Figure 2 A in the image is a bright-field image. Figure 2 In the image, B represents the fluorescence image. For example... Figure 2 As shown, when observed under an inverted fluorescence microscope, almost all cells emit green fluorescence.

[0106] Example 4: Investigation of lentivirus titers using viral fluid

[0107] (1) 293T cells were divided into groups of 5 × 10⁻⁶ cells. 3 The cells were densely seeded into 96-well plates, and the lentivirus titer was determined when the cell adhesion rate reached 30%.

[0108] (2) Prepare a mixture of cell culture medium containing 10% FBS and 5 μg / mL polybrene;

[0109] (3) Lentiviral gradient dilution grouping:

[0110] A: Take seven 200μL sterile centrifuge tubes, add 90μL of complete culture medium to each, and label them sequentially;

[0111] B: Add 10 μL of the virus stock solution to tube 1 and mix well;

[0112] C: Take 10 μL of the mixed virus solution from tube 1 and add it to tube 2. Mix well and then take 10 μL and add it to tube 3. Repeat this process for continuous dilution.

[0113] D: Repeat AC for three sets;

[0114] (4) Remove the culture medium from the 96-well plate, add 90 μL of virus from tubes 1-7 in sequence, repeat 3 times, and set up a negative control group at the same time;

[0115] (5) After culturing in the incubator for 8 h, remove the virus dilution, add complete culture medium and continue culturing. After 48 h, observe with an inverted fluorescence microscope and count the GFP positive cells.

[0116] A three-plasmid system (PRKAA1-Bos-1460 as the shuttle plasmid, psPAX2 as the packaging plasmid, and PMD2.G as the envelope plasmid) was used to transfect 293T cells using lentiviral packaging. After 48 hours, the supernatant was collected as the viral stock solution. The viral stock solution was serially diluted to infect 293T cells for viral titer determination. The results are as follows: Figure 3 As shown, 1×10 8 Even after a 10-fold dilution, each well of the virus-infected cells still contained fluorescent cells, indicating a calculated viral titer of 10. 8 TU / mL.

[0117] Figure 3 The pGLVH1-PRKAA1-GFP-Puro corresponding to A in the middle represents the fluorescent expression of viral titer when PRKAA1-Bos-1460 is used as a shuttle plasmid; Figure 3 In the middle B, pGLVH1-NC-GFP-Puro represents the fluorescent expression of viral titer when no target plasmid is present.

[0118] Example 5: Investigation of the optimal MOI value for lentiviral infection of target cells using viral fluid

[0119] (1) Divide the bovine mammary epithelial cells into 10 cells per well. 4 The density is spread into a 24-well plate;

[0120] (2) When the cell density reaches 30%-40%, add lentivirus and polybrene as a co-infectant. Calculate the viral load according to MOI=0, 10, 20, 40, 60, 80, 100. Add the virus to serum-free culture medium and incubate in an incubator for 8-12 hours. Observe the cell status. If there is no effect, continue culturing for 24 hours and then replace with complete culture medium.

[0121] (3) Observe the cell fluorescence expression after 72h-96h to determine the optimal MOI value for lentivirus infection.

[0122] In Example 5, the lentivirus refers to the viral fluid produced after 48 hours of co-transfection of 293T cells using a three-plasmid system (PRKAA1-Bos-1460 as the shuttle plasmid, psPAX2 as the packaging plasmid, and PMD2.G as the envelope plasmid) via lentivirus packaging.

[0123] The results are as follows Figure 4As shown, lentivirus infection of bovine mammary epithelial cell lines was performed. After 72 hours, cell fluorescence expression was observed under an inverted fluorescence microscope, revealing green fluorescence in the cells, indicating an infection rate exceeding 80%. By setting different MOI values, the optimal MOI for infecting target cells was determined. The results showed that the fluorescence expression intensity was highest at MOI=40, while excessively high MOI values ​​led to partial cell death.

[0124] Example 6: Detection of AMPKα pathway-related proteins by knocking down the target gene PRKAA1 in bovine mammary epithelial cells treated with viral fluid.

[0125] (I) Experimental Methods

[0126] 1. Lentiviral infection and construction of stable knockdown strains (puromycin screening)

[0127] 1.1. Seed bovine mammary epithelial cells into 24-well plates and culture for 24 hours. Discard the old culture medium and add 1 / 2 volume of serum-free culture medium. Add lentiviral solution (divided into two groups: shNC lentivirus [control] and shPRKAA1 lentivirus [knockdown]) according to the optimal MOI value determined in Example 4. After 8-12 hours, replenish the culture medium completely (to maintain cell nutrition).

[0128] 1.2 24 hours after infection, discard the virus-containing culture medium and replace it with fresh complete culture medium;

[0129] 1.3. 48 hours after infection, add culture medium containing puromycin (4ug / ml) and screen for 4 days;

[0130] 1.4 Observation control group (cells without virus + puromycin): all cells should die; surviving cells in the infected group are positive clones;

[0131] 1.5. Reduce the concentration of puromycin to "half of the minimum killing concentration" and continue culturing for 3-5 days to amplify a stable knockdown strain (to ensure that PRKAA1 is continuously expressed at low levels).

[0132] 2. Cell plating and LPS treatment (for stable knockdown strains)

[0133] 2.1 One day before LPS treatment, stable knockdown strains (shNC group, shPRKAA1 group) were seeded in 6-well plates with medium without antibiotics and the density was adjusted to 70%-80%.

[0134] 2.2 Add LPS (2 μg / mL) to some wells in the shPRKAA1 group, and label it as the shPRKAA1+LPS group; do not add LPS to the shNC group.

[0135] 2.3 After LPS treatment, cells were collected and total protein was extracted for the detection of AMPKα pathway-related proteins.

[0136] (II) Testing methods:

[0137] Remove the culture medium from the 6-well plate, wash twice with PBS, add 100 μL of protein lysis buffer to each well, and place the 6-well plate on ice for lysis for 5 min. Scrape the cells out of the wells with a cell scraper, transfer to a 1.5 mL centrifuge tube, add 20 μL of SDS protein loading buffer, mix well by pipetting, incubate at 100 °C for 5 min, briefly centrifuge with shaking, and store at -20 °C.

[0138] Remove the sample and protein marker and add them to the wells of the SDS-PAGE precast gel. Add the prepared electrophoresis buffer to the electrophoresis tank, adjust the voltage to 140V, and electrophoresis for 1 hour. Stop electrophoresis when the bromophenol blue band is close to the bottom of the gel. Pre-cut a PVDF membrane to a size similar to the precast gel and pre-activate it by soaking it in methanol. Use the sandwich method for membrane transfer, wet transfer at a constant current of 350mA for 50 minutes. The entire transfer process is carried out in an ice bath to prevent internal overheating and improve transfer efficiency.

[0139] After transfer, the membrane is placed in skim milk powder for blocking. Once blocking is complete, the membrane is removed, and the location of the target protein is determined based on the marker distribution. The protein is then placed in the corresponding primary antibody and incubated overnight at 4°C. After primary antibody recovery, the membrane is washed with prepared TBST. After washing, secondary antibody is added and incubated for 2 hours. The incubation is then stopped, and the membrane is washed again.

[0140] After washing, the protein was incubated with ECL luminescent solution under light-protected conditions, followed by protein imaging in a protein gel imager, and then protein grayscale analysis was performed using ImageJ software. The antibody information used is shown in Table 6.

[0141] Table 6. Antibody Information

[0142]

[0143] The results are as follows Figure 5As shown, after knocking down the target gene PRKAA1 using lentivirus, AMPK protein expression significantly decreased (P<0.05), but the ratio of phosphorylated AMPK to total AMPK increased. SIRT1 and NF-κB, as downstream molecules of the AMPKα signaling pathway, showed significantly decreased SIRT1 protein expression (P<0.05) and significantly increased ratios of phosphorylated NF-κB and acetylated NF-κB (P<0.05) after knockdown. Under LPS stimulation, SIRT1 protein expression was significantly lower than in the control group (P<0.05), and the ratio of phosphorylated AMPK to total AMPK decreased, but the difference was not significant (P>0.05). The ratios of phosphorylated and acetylated NF-κB to total NF-κB significantly increased (P<0.05). After knocking down the target gene PRKAA1 using lentivirus, the expression levels of PFKFB3 and DGAT proteins significantly increased (P<0.05), while the expression level of CPT1 protein significantly decreased (P<0.05). Upon LPS stimulation, the protein expression levels of PFKFB3 and DGAT were significantly higher than those in the blank control group (P<0.05), and the protein expression level of CPT1 was also significantly higher than that in the blank control group (P<0.05).

[0144] In summary:

[0145] (1) The shRNA sequences screened by this invention have high knockdown efficiency: Examples 1 and 2 describe how to design and screen highly efficient and specific shRNA sequences to ensure a good silencing effect on the PRKAA1 gene while avoiding off-target effects.

[0146] (2) Through Examples 3-5, this invention describes how to construct a stable, high-titer, and highly effective shRNA lentiviral vector, thereby achieving continuous knockdown of PRKAA1 in mammalian cells.

[0147] In Example 3 of this invention, after lentivirus packaging (a three-plasmid system, with PRKAA1-Bos-1460 as the shuttle plasmid), it was observed that almost all 293T cells emitted green fluorescence 48 hours after transfection. Figure 2 This verified the "high packaging efficiency". Example 4 involved infecting 293T cells with a serially diluted viral stock solution, and the calculated viral titer was 10. 8 TU / mL ( Figure 3 This demonstrates that "the viral titer is stable." Example 5 investigated the optimal MOI value for lentivirus infection of bovine mammary epithelial cells (the fluorescence expression was strongest at MOI=40, and the infection rate was >80%), demonstrating "good infection effect"; combined with the genomic integration characteristics of lentivirus, "long-term stable knockdown" can be achieved.

[0148] (3) Example 5 describes how to establish a PRKAA1 functional verification process, including molecular detection of PRKAA1 expression downregulation and functional evaluation methods for key phenotypes (such as metabolism, proliferation, inflammation, etc.), which confirms at the molecular level that PRKAA1 knockdown does indeed affect its downstream signaling pathways, thereby revealing the potential function of this gene in metabolism and inflammation.

[0149] (4) The present invention optimizes the experimental operation process, with clear steps, simple operation, stable experimental results, good reproducibility, and can also be used for knockdown studies of other genes, making it easy to promote and use.

[0150] This invention, with its highly efficient and specific shRNA screening strategy, high-titer viral vector construction technology, and systematic functional verification process, successfully overcomes the instability and inefficiency bottlenecks commonly found in existing PRKAA1 gene interference technologies, demonstrating a high potential for technology transfer and commercialization. Given the core research value of the PRKAA1 gene in areas such as metabolic regulation, tumor proliferation, and inflammatory responses, this standardized and highly reproducible experimental system precisely meets the urgent needs of basic medical research and biopharmaceutical companies for high-quality target validation tools. This technology can not only significantly reduce the trial-and-error costs and timelines of downstream drug development and improve drug screening efficiency, but also is expected to generate considerable economic benefits through technology service transfer, reagent kit development, or patent licensing, possessing broad market prospects and profound industrialization value.

[0151] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. A lentiviral recombinant vector that interferes with the PRKAA1 gene, characterized in that, The lentiviral recombinant vector contains shRNA targeting gene sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

2. The method for constructing the lentiviral recombinant vector interfering with the PRKAA1 gene as described in claim 1, characterized in that, Based on the target gene sequence, two complementary oligonucleotide single strands were designed and synthesized. The two complementary DNA oligonucleotides were annealed to generate a DNA insert fragment, which was then inserted into a lentiviral transfer vector to obtain a lentiviral recombinant vector containing shRNA, i.e., a lentiviral recombinant vector that interferes with the PRKAA1 gene.

3. A method for constructing a lentivirus that interferes with the PRKAA1 gene, characterized in that, Includes the following steps: Step 1: shRNA Design and Screening Based on the mRNA sequence of the PRKAA1 gene, bioinformatics tools were used to predict shRNA target gene sequences with high specificity and low off-target effects, resulting in multiple candidate target gene sequences; the candidate target gene sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4. Step 2: Construction of shuttle plasmids: Based on the target gene sequence, two complementary oligonucleotide single strands were designed and synthesized. The two complementary DNA oligonucleotides were annealed to generate a DNA insert fragment, which was then inserted into a lentiviral transfer vector to obtain a lentiviral recombinant vector containing shRNA. The lentiviral recombinant vector was transformed into competent E. coli to obtain an shRNA shuttle plasmid targeting the PRKAA1 gene. Step 3: Use shuttle plasmids to transfect target cells to verify the efficiency of AMPKα knockdown and screen out shuttle plasmids with good results; Step 4, Lentiviral Packaging: Using a three-plasmid system, the shuttle plasmid selected in step three was co-transfected with the packaging plasmid and the envelope plasmid into 293T cells. After culturing for 48 hours, the supernatant was collected to obtain PRKAA1 interference lentivirus. Step 5: Verify the lentivirus packaging effect and determine the titer; Step 6: Use viral fluid to infect the target cells with the optimal infection MOI (Mean Infection Point) using lentivirus.

4. The lentivirus construction method according to claim 3, characterized in that, The specific steps for step four are as follows: Step 3.1, revival and passage of 293T cells; Step 3.2: Mix the shuttle plasmid, packaging plasmid and encapsulation plasmid in a ratio of 4:3:1 and place them in a serum-free culture medium, which is called solution A. Incubate at room temperature for 5 min. Step 3.3: Add PEI solution with a concentration of 1 μg / mL to serum-free culture medium, and label it solution B. Let it stand at room temperature for 5 min. Step 3.4: Slowly add solution B to solution A, let stand at room temperature for 20 min, mix the two thoroughly, add to a culture dish containing 293T cells, and incubate in an incubator for 6 h; Step 3.5: After culturing for 6 hours, discard the original culture medium, add complete culture medium, and culture in a cell culture incubator for 48 hours. Then collect the supernatant, which is the virus solution.

5. The lentivirus construction method according to claim 4, characterized in that, The lentiviral recombinant vector contains shRNA targeting the gene sequence shown in SEQ ID NO.1; The viral fluid used for lentivirus packaging has a viral titer of 10. 8 TU / mL, with an optimal infection MOI of 40.

6. Application of lentiviral recombinant vectors interfering with the PRKAA1 gene in the preparation of products with reduced PRKAA1 gene expression levels.

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