Method for obtaining overexpressed TXNIP macrophage cell strain and application of overexpressed TXNIP macrophage cell strain in antibacterial research
By constructing TXNIP overexpressing macrophage lines, the problem of studying the application of TXNIP gene in Salmonella infection was solved, and an effective experimental model was established, which enhanced its resistance to Salmonella and provided a new target for disease treatment.
Patent Information
- Application Number
- CN202510519330.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-24
- Publication Date
- 2025-07-18
AI Technical Summary
There is a lack of effective methods in the prior art to investigate the role of TXNIP genes in immunomodulation and disease defense, especially in the process of Salmonella infection.
The TXNIP overexpressed macrophages were constructed using eukaryotic expression vectors, and the TXNIP gene was introduced into the RAW264.7 cell line through the lentiviral system, and stable overexpression cell lines were obtained using puromycin screening, and the overexpression effect was verified in combination with RT-qPCR and Western blot.
A stable TXNIP gene overexpression cell line was established, which provided a reliable experimental model, significantly enhanced its resistance to Salmonella infection, and provided a new target for the study and treatment of Salmonella disease.
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Figure CN120330263A_ABST
Abstract
Description
Technical Field
[0001] The present invention discloses a method for obtaining a cell line overexpressing the TXNIP gene and the application of the cell line in antibacterial research, belonging to the field of biotechnology. Background Art
[0002] In recent years, exploring the dynamic interaction between the host and pathogens is of great significance for deeply understanding the disease infection mechanism. The Thioredoxin-interacting protein (TXNIP) gene, as an important part of the thioredoxin system, plays a key role in regulating the cellular redox state and inflammatory response. TXNIP binds to Thioredoxin (TRX) to inhibit its antioxidant function, thereby regulating the intracellular oxidative stress level. The expression of TXNIP is regulated by various factors, including high blood sugar, oxidative stress, and inflammatory signals, etc.
[0003] In the immune response, TXNIP affects the functions of immune cells such as macrophages by regulating the redox balance and the release of inflammatory factors. Studies have shown that TXNIP also plays an important role in the process of coping with pathogen infections. For example, during Brucella infection, TXNIP plays a key role in host immune defense by regulating the NO / ROS antibacterial pathway. In addition, TXNIP is also involved in the regulation of processes such as apoptosis and autophagy, further affecting the host defense mechanism against pathogens.
[0004] In human diseases, the abnormal expression of TXNIP is related to various diseases, including diabetes, cardiovascular diseases, cancers, and inflammatory diseases, etc. The genetic polymorphism of TXNIP has also been found to be related to the susceptibility of certain diseases. For example, the polymorphism of the TXNIP gene is closely related to the incidence risks of type 2 diabetes and inflammatory bowel disease.
[0005] Salmonella ( Salmonella ) as an important zoonotic pathogen modifies the functions of host cells through type III secretion system (T3SS) effector proteins (such as SseK3), interfering with key physiological processes such as endoplasmic reticulum-Golgi transport to promote infection. Recent studies have found that the intracellular redox regulation system of host cells may be involved in the anti-Salmonella immune response. Based on the important role of TXNIP in immune regulation and disease occurrence, on the basis of existing research, the present invention has developed a method for constructing a cell line overexpressing the TXNIP gene and explored the effect of TXNIP overexpression on Salmonella infection. The present invention has significant innovation and application prospects, providing new ideas for studying the role of TXNIP in immune regulation and disease treatment. Summary of the Invention
[0006] The object of the present invention is to provide a method for constructing a macrophage cell line with overexpressed TXNIP gene and its application in the study of Salmonella typhimurium infection. The specific technical solution is as follows: The present invention provides a method for constructing a macrophage cell line with overexpressed TXNIP. A eukaryotic expression vector is used to construct a macrophage cell line with overexpressed TXNIP gene. The construction method includes: searching for the base sequence of the mouse TXNIP gene (NM_001009935.2) in the NCBI database. Using SnapGene software to design a primer pair P1 capable of amplifying the CDS region of the TXNIP gene. After Eco R I and Nhe I enzyme digest the PTRIP-CAGG-Puro plasmid and the target fragment, using T4 ligase to connect the recovered PTRIP-CAGG-Puro vector and the target fragment after enzyme digestion. After transforming the recombinant vector into competent cells, single colonies are picked for colony PCR. After the positive colonies are expanded and cultured, plasmids are extracted. After correct sequencing, it is transfected into tool cells and then the virus supernatant is collected. After centrifugation and filtration, it infects the target cells at a certain ratio with the culture medium, and thus a macrophage cell line with overexpressed TXNIP is obtained.
[0007] Furthermore, the macrophage cell line (RAW264.7), HEK293T cells, and lentiviral expression system (composed of PTRIP-CAGG-Puro, PAX2, and pLP / VSVG) used in this patent are stored and used by the Shaanxi Stem Cell Engineering Technology Research Center; the Salmonella typhimurium used comes from the China Veterinary Culture Collection Center.
[0008] Furthermore, the sequences of the P1 primers are as follows: primer pair P1 Forward primer: 5’-CGGCTAGCGGCTCAATCATGGTGATGTTCA-3’ Reverse primer: 5’-CGGAATTCCTCACTGCACGTTGTTGTTG-3’ Furthermore, the reaction system of the P1 primers: denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min, and preservation at 4°C. Furthermore, all agarose gel electrophoresis uses agarose gel with a mass concentration of 1%. Furthermore, the fast cut enzymes used for enzyme digestion of the PTRIP-CAGG-Puro plasmid and the target fragment are Eco R I and NheI (Takara). Further, the ligation system used is as follows: 100 ng of CAG linearized fragment; 5-fold molar volume of the target fragment; 2 μl of 10× T4 DNA ligase buffer; 1 μl of T4 DNA ligase; ddH2O is added to make up to 20 μL. The ligation procedure is: react at 22°C for 20 - 30 min. Further, the competent cells are DH5α. Further, the tool cells are HEK293T cells. Further, the plasmid transfection method is the liposome transfection method.
[0009] The method for constructing macrophages with overexpressed TXNIP gene is as follows: a) cell transfection and screening; b) verification of the overexpressing cell line. Further, the method in step a) is specifically: co-transfect the plasmid with correct sequencing, PAX2, and VSVG packaging plasmids into HEK293T cells, and collect the virus supernatant after 48 hours. Filter the cell debris using a 0.45 μm filter membrane, leave the virus solution, mix it with the culture medium at a ratio of 1:1, and infect RAW264.7 cells. After 48 hours of infection, the oeTXNIP-Raw264.7 cell line is obtained through puromycin screening.
[0010] Further, the method for verifying the overexpressing cell line in step b) is RT-qPCR and Western blot. The present invention also provides the establishment of a model of Salmonella typhimurium infecting RAW264.7 cells. Infect the cells with a multiplicity of infection (MOI) of 10:1 for a period of time, collect the lysate, coat it on a plate, and observe the colony-forming units (CFU). Further, the Salmonella typhimurium is numbered CVCC3387. Further, the concentration of Salmonella typhimurium infecting the cells is 1.1×10 7 CFU / mL. Further, the number of RAW264.7 cells in each well of the 6-well plate is 1.1×10 6 cells / well. Further, the infection time of Salmonella typhimurium is 4 hours, 6 hours, and 8 hours. Further, the macrophages with overexpressed TXNIP gene may achieve antibacterial effects by interfering with the intracellular survival environment of Salmonella or blocking the functions of its effector proteins.
[0011] The advantage of the present invention is that a stable cell line with overexpressed TXNIP gene is established, providing a reliable experimental model for studying Salmonella infection of cells, providing a new target for the molecular mechanism research and treatment of Salmonella disease, and having important theoretical and application values. Brief Description of the Drawings
[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0013] Figure 1 Electrophoresis result of the product amplified by primer pair P1 for mouse TXNIP gene on agarose gel; Figure 2 For using Eco R I and Nhe I enzyme to digest the PTRIP-CAGG-Puro plasmid and the target fragment, and then the electrophoresis result on agarose gel; Figure 3 Electrophoresis result of the target fragment amplified by primer pair P1 after picking a single colony; Figure 4 For taking the positive colonies for enlarged culture, extracting the plasmid and then Eco RⅠ and Mlu Ⅰ double enzyme digestion, and the electrophoresis result of the obtained product on agarose gel; Figure 5 Picture of the stable transfected cell line after overexpressing TXNIP. Control on the left is the empty vector control, and the right is the overexpressing cell line; Figure 6 Bar graph of quantitative PCR to detect the overexpression efficiency, with β-ACTIN as the internal reference; Figure 7 Western blot was used to detect the protein expression of TXNIP; Figure 8 For infecting cells with a multiplicity of infection (MOI) of 10:1, setting a blank group (no bacterial infection) and an invasion group; Figure 9 For verifying the actual bacterial concentration by gradient dilution method after Salmonella infects cells; The above figure description shows the effect of TXNIP gene overexpression on Salmonella infection, providing an important experimental basis for further research. Embodiment
[0014] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Example 1
[0015] Example 1 of the present invention provides a method for constructing a macrophage cell line with overexpressed TXNIP gene, and the steps include: designing primers for TXNIP gene cloning.
[0016] Find the published mouse TXNIP gene (Gene ID: 56338) from the NCBI database, and use SnapGene to design a primer pair P1 that can amplify the CDS region of the TXNIP gene. Add restriction enzyme sites and their protective bases to the 5' ends of the upstream and downstream primers respectively. The primer sequences are as follows: Primer pair P1 Upstream primer: 5’-CGGCTAGCGGCTCAATCATGGTGATGTTCA-3’ Downstream primer: 5’-CGGAATTCCTCACTGCACGTTGTTGTTG-3’.
[0017] Construction of the CAGG-TXNIP recombinant expression vector.
[0018] Perform PCR amplification using the mouse cDNA sample as a template to amplify a fragment containing the CDS region of the mouse TXNIP gene. The PCR reaction program at the P1 site is: denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min, and preservation at 4°C. The electrophoretic detection diagram of the amplified fragment is shown ( Figure 1 ), M is the Marker. Use the Tiangen universal DNA purification and recovery kit (DP214) for gel cutting and recovery. For the method, please refer to the DP214 instruction manual.
[0019] Digest the PTRIP-CAGG-Puro (CAGG) empty vector and the target fragment with enzymes.
[0020] Use Eco R I and Nhe I fast cut enzymes for double digestion. The double digestion reaction system is 50 μL. Among them, the digestion system of PTRIP-CAGG-Puro (CAG) is QuickCut Eco R I 1 μL, QuickCut Nhe I 1 μL, CAG empty vector with a concentration of 1 μg·μL -1 , 10×QuickCut Buffer 5 μL, and make up to 50 μL with ddH2O; for digesting the target fragment, take 200 ng of the gel recovery product obtained in 2.1, QuickCut Eco R I 1 μL, QuickCut Nhe1 μL of I, 5 μL of 10× QuickCut Buffer, and make up to 50 μL with ddH2O.
[0021] Perform the digestion reaction in a PCR instrument at 37 °C for 25 min. The digestion products (12601 bp and 1210 bp) are shown in the electrophoresis detection diagram ( Figure 2 ), and use a universal DNA purification and recovery kit for system recovery. The method details can be found in its instruction manual.
[0022] T4 ligation: Use Thermo Scientific T4 DNA ligase for T4 ligation. The ligation system is as follows: 100 ng of the CAG linearized vector obtained in 2.2, 5-fold molar volume of the recovered target fragment, 2 μl of 10× T4 DNA ligase buffer, 1 μl of T4 DNA ligase, and make up to 20 μL with ddH2O. React at 22 °C for 20 - 30 min on a PCR instrument to obtain the ligation product.
[0023] Transform the ligation product into DH5α cells. The transformation system is: the volume of the ligation product is one-tenth of the volume of the competent cells. Transformation steps: Take a tube of DH5α competent cells from the -80 °C refrigerator and immediately place it on ice to slowly dissolve. Add the ligation product obtained in the previous step to the competent cells, place it on ice for an ice bath for 30 min, heat shock at 42 °C for 90 s to increase the pores on the cell membrane of the competent cells, which is beneficial for the recombinant plasmid to enter the competent cells. Immediately place it in ice after heat shock and ice bath for 5 min to reduce the pores on the cell membrane. Add 700 μL of LB liquid medium without antibiotics and shake it in a 37 °C constant temperature shaker for 1 h. Centrifuge at 5000 rpm for 3 min with a normal temperature centrifuge, aspirate part of the supernatant in the laminar flow hood, leave about 100 μL of supernatant, pipette and mix it evenly, and then use a disposable spreading rod to spread it on the LB solid medium containing ampicillin resistance and incubate it upside down at 37 °C overnight (about 14 h).
[0024] Pick monoclonal colonies for colony PCR: Prepare a PCR tube, add 30 μl of LB (containing 1000× ampicillin) to the tube, pick monoclonal colonies, pipette and mix them evenly in the LB and then aspirate 1 μl of the bacterial solution and add it to a 20 μl system: 10 μl of 2× Rapid Taq Master Mix, 0.8 μl of each upstream and downstream primer, 1 μl of the bacterial solution, and make up to 20 μl with ddH2O. The PCR reaction system is: 95 °C for 3 min, 95 °C for 15 s, 60 °C for 15 s, 72 °C for 15 s, 72 °C for 5 min. The obtained products are subjected to agarose gel electrophoresis, and then observed and compared in a gel imaging system, and the gel image is photographed and saved, as Figure 3 shown.
[0025] Positive colony expansion culture: Select the colonies identified as positive by colony PCR in the previous step for expansion culture. Prepare a 50 mL centrifuge tube, add 20 mL of LB liquid medium containing ampicillin at a concentration of 1:1000 to the centrifuge tube. Directly add the remaining 29 μL of bacterial liquid to the liquid medium, and incubate it in a constant temperature shaking incubator at 37°C for 14 h.
[0026] Plasmid extraction and identification: Plasmid extraction: Use the Tiangen endotoxin-free plasmid miniprep midiprep kit to extract the plasmid. The method is detailed in the instruction manual. Plasmid identification: Use Eco R Ⅰ and Mlu Ⅰ enzymes for double digestion. The double digestion reaction system is 20 μL, including 0.5 μL of QuickCut Eco RⅠ, 0.5 μL of QuickCut Mlu Ⅰ, 500 ng of plasmid, 2 μL of 10×QuickCutGreen Buffer, and add ddH2O to 20 μL. Perform the digestion reaction in a PCR instrument: Digest at 37°C for 15 min. Electrophorese the obtained product on an agarose gel: Prepare a 1% agarose gel solution by adding 0.3 g of agarose to 30 mL of 1×TAE buffer, heat it in a microwave oven until fully dissolved, add 3 μL of nucleic acid dye Gelview when cooled to about 50°C, pour it into the agarose mold and let it cool naturally to room temperature. Take 10 µL of the double-digested product and add it to the loading well, electrophorese at 120 V for 40 min, place it in a gel imaging system to observe and compare, take a picture and save the gel image, as Figure 4 shown. Send the correctly identified plasmid to Qingke Company for sequencing, and use the plasmid with correct sequencing for the next experiment.
[0027] Lentivirus packaging. Cell preparation: Use 293T cells within passage 4, inoculate them in a 60 mm culture dish, and use DMEM (Corning) complete medium (added with 10% fetal bovine serum) as the culture medium. Culture them in a constant temperature incubator at 37°C and 5% CO2. When the cell growth density reaches about 80%, discard the culture supernatant, replace it with 4 mL of DMEM basal medium, and perform starvation treatment at 37°C for 1 hour.
[0028] Plasmid transfection: Virus packaging system: The ratio of the target plasmid: PAX2: VSVG = 4:3:2. Calculate and prepare 8 μg of plasmid mixture (add in the order of PAX2, VSVG, and the target plasmid). Gently mix 24 μL of PEI (Beyotime) with 250 μL of Opti MEM™ evenly and let it stand for 5 minutes. Subsequently, mix 8 μg of plasmid mixture with 250 μL of Opti MEM™ evenly and let it stand for 5 minutes. Then add it to the PEI-Opti MEM™ mixture, gently mix well and let it stand for 20 minutes. When 20 minutes is almost up, replace the medium of 293T cells with 4 mL of complete DMEM medium. Slowly and evenly drip the plasmid-PEI-Opti MEM™ mixture onto the cell surface, minimizing liquid shaking. Place the cells in a 5% CO2, 37 °C constant temperature incubator for further culture.
[0029] Culture and collection: Replace the medium with complete DMEM medium for continued culture 12 hours after plasmid transfection. Collect the virus solution 48 hours later, centrifuge at 2000 rpm for 5 minutes. The supernatant is the virus solution, which can be directly used to infect RAW264.7 after filtering cell debris through a 0.45 μm filter.
[0030] Lentivirus infection of Raw264.7 and positive cell screening: Cell preparation: Use RAW264.7 cells within 3 passages, inoculate them into a 35 mm culture dish (Thermo), and culture them in a 5% CO2, 37 °C constant temperature incubator. Infect when the density of RAW264.7 cells reaches about 30%.
[0031] Virus infection: Add to RAW264.7 cells according to the volume ratio of "virus solution: DMEM(+) culture medium = 1:1", and add 1 μL of Polybrene (10 mg / mL, Sigma-Aldrich) for every 1 mL volume. Replace the medium with complete DMEM medium 24 hours after lentivirus infection. Observe the green fluorescence expression of the control group CAGG-GFP 48 hours later to judge the transfection efficiency.
[0032] Puromycin screening: Continue to culture the cells after infection. When the cell density reaches 80%, screen the cells with complete DMEM medium containing 1 μg·mL -1 puro (puromycin) for 24 hours, and then replace it with complete DMEM medium. After the cell state recovers, repeat the screening once to obtain a stable transfected cell line ( Figure 5 )
[0033] qPCR was used to detect the overexpression efficiency; RNA extraction: Total RNA was extracted using the TRIzol method. First, the culture medium was discarded, and dead cells were washed away with cold PBS. 1 mL of TRIzol lysis reagent was added to the cell culture dish from which the RNA sample was to be collected, and the lysis reaction was carried out at room temperature for 10 min. Then, the lysate was aspirated into a 1.5 mL centrifuge tube. Chloroform precooled at a ratio of 1:5 was added to the TRIzol lysate, 0.2 mL for every 1 mL of lysate, vortexed for 15 s, and placed on ice for 5 - 10 min. It was placed in a pre-cooled centrifuge at 4°C and centrifuged at 12,000 rpm for 10 min. The upper clear aqueous phase was aspirated and added to a DNase / RNase-free centrifuge tube, approximately 400 μL. An equal volume of isopropanol (400 μL) was added, mixed well by inverting up and down, and placed on ice for 30 min. Then, it was centrifuged at 12,000 rpm at 4°C for 10 min, and the supernatant was discarded. The white precipitate at the bottom was RNA. 1 mL of 75% pre-cooled ethanol was added, inverted up and down, centrifuged at 12,000 rpm at 4°C for 5 min, and the supernatant was discarded, and this was repeated once. Then, it was centrifuged without sample for 3 min to completely dry the ethanol, and dried in a laminar flow hood for 10 min to completely volatilize the ethanol. 20 μL of DEPC water was added, gently pipetted and mixed well, and a micro UV spectrophotometer was used to detect the concentration and purity of the RNA sample. A260 / A280: 1.8 - 2.0 indicated a relatively high purity.
[0034] Reverse transcription into cDNA: The HiScript III RT SuperMix for qPCR(+gDNA wiper) kit was used for reverse transcription. The reverse transcription system was: RNA, 2 μg; 4×gDNA wiper Mix, 4 μL; ddH2O, made up to 16 μL. Reacted at 42°C for 3 min. Subsequently, 4 μL of 5×HiScript Ⅲ qRT SuperMix was added for reverse transcription. The reverse transcription system was: 50°C, 15 min; 80°C, 5 s. The obtained cDNA was stored at -20°C for later use.
[0035] Relative fluorescence quantitative PCR (RT-qPCR) was used to detect the overexpression efficiency: The cDNA was diluted to 20 ng / μL for fluorescence quantitative PCR. The qPCR kit supplied by Tiangen Biochemical Technology Co., Ltd. was used. Care should be taken to avoid mixing the required reaction system under direct light. The reaction system was: cDNA, 1 μL; 2×ChamQ SYBR qPCR Master Mix, 10 μL; 0.5 μL each of the upstream and downstream primers with a concentration of 10 μM; ddH2O, 8 μL. The primers used in this experiment were: q-TXNIP Upstream primer: 5’-TTACCCGAGTCAAAGCCGTC-3’ Downstream primer: 5’-CCATCTCGTTCTCACCTGCT-3’ The quantitative PCR instrument was set for 40 cycles and a three-step reaction program was adopted. After the reaction ended, the experimental results of the qPCR instrument were output and analyzed through the Bio-Rad CFX Manager 3.1 software. Finally, significant analysis was performed through T-test, and the results were obtained ( Figure 6 ). The transcriptome level of TXNIP was upregulated by 9.2 times compared with the control group, indicating successful overexpression.
[0036] Western Blot was used to detect the overexpression efficiency; Cell lysis: The cells were cultured in a 60 mm culture dish. When the cell density reached over 80%, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS and collected by centrifugation. The cells were lysed using RIPA lysis buffer (Beyotime), and protease inhibitor cocktail (Solarbio) and phosphatase inhibitor cocktail (Solarbio) were added. The cells were sonicated at 300 W for 10 seconds, left stationary for 15 seconds, and the cycle was repeated 3 times.
[0037] Protein sample preparation: 5× protein loading buffer (Epizyme) was added to the supernatant, boiled at 100 °C for 10 minutes, and the samples were stored at -80 °C.
[0038] SDS-PAGE and membrane transfer: SDS-PAGE was performed using the Oriscience One-Step PAGE Preparation Kit (10%). Using the AR rapid membrane transfer solution (AccuRef Scientific), the membrane was transferred at a constant current of 400 mA for 30 minutes, and the PVDF membrane was from Sigma.
[0039] Antibody incubation and development: Block with 8% skim milk for 2 hours, incubate the primary antibody overnight at 4 °C, incubate the secondary antibody at room temperature for 1 hour, and develop and expose. The protein marker, antibodies and dilution ratios used are as follows: The protein marker used was GenStar's StarRuler Color Prestained Protein Marker (10 - 180 kDa). The primary antibody used was: Proteintech TXNIP Polyclonal antibody 18243 - 1 - AP, diluted at a ratio of 1:1000. The internal reference used was ABclonal β - Actin Rabbit mAb (High Dilution) AC026, diluted at a ratio of 1:50000. The secondary antibody used was BOSTER HRP Conjugated AffiniPure Goat Anti - rabbit IgG (H + L), diluted at a ratio of 1:50000. Finally, Figure 7 the results were obtained. The protein level of TXNIP was up - regulated by 3 - fold compared with the control group, indicating successful over - expression. Example 2
[0040] Effect of over - expressing macrophage cell line of TXNIP gene on Salmonella typhimurium infection.
[0041] Activation of the strain and preparation for bacterial challenge: After resuscitating the Salmonella strain stored at - 80°C, it was streaked on an LB agar plate for three - zone culture (cultured at 37°C for 18 - 24 hours). A single colony was picked and inoculated into 20 mL of LB liquid medium, placed on a shaker at 37°C and 180 rpm for 12 hours, and passaged twice continuously to ensure that the strain was in the logarithmic growth phase. The OD600 value of the bacterial solution was measured using a spectrophotometer to be 1.099 A. Based on the standard curve (OD600 = 1 A corresponding to 1×10 9 CFU / mL), the concentration of the bacterial solution was estimated to be approximately 1.1×10 9 CFU / mL. Take 1 ml of the bacterial solution, centrifuge at 4000 rpm for 5 min at room temperature, discard the supernatant, wash twice with 1 ml of PBS, and resuspend in antibiotic - free DMEM basal medium.
[0042] Cell seeding: After RAW264.7 was cultured to the logarithmic growth phase, 1.1×10 6 cells were seeded in each well of a 6 - well plate, and a blank control group (no bacterial infection) and a bacterial invasion group (Salmonella) were set up. Infection experiments were carried out according to MOI = 10:1 (1.1×10 7 CFU / mL of Salmonella per well), with 3 replicates as duplicates.
[0043] Salmonella - infected RAW264.7 cells: At 4, 6, and 8 hours after Salmonella invasion, the cells were washed twice with PBS first, and then treated with 200 μg / mL gentamicin for 1 hour to remove the bacteria that had not invaded into the cells. After washing 3 times with PBS, the cells were permeabilized with 0.5% TritonX - 100 solution for 5 minutes, and then the lysate in the wells was collected. After gradient dilution, it was spread on LB agar plates and cultured overnight at 37°C in an inverted position. The results showed that the colony - forming units (CFU) of the TXNIP over - expression group (oeTXNIP) were significantly lower than those of the control group (p < 0.01), as shown in Figure 8 Figure [Figure number corresponding to Figure 8 ], indicating that TXNIP inhibits the intracellular survival of Salmonella through a certain mechanism.
[0044] Actual bacterial concentration detection: Take 1 ml of the bacterial solution and use the serial dilution method (10 6 , 10 7 , 10 8 ) to dilute the bacteria to an appropriate concentration, spread it on the plate. After verification by plate counting, the number of colonies at the 10 7 dilution was 129 and 125 CFU / plate (average 127 CFU / plate), as shown in Figure 9 Figure [Figure number corresponding to Figure 9 ]. Meeting the statistical effective range of 30 - 300 CFU, the actual bacterial concentration was obtained as 1.27×10 9 CFU / mL, with a deviation of less than 10% from the theoretical value estimated based on the standard curve (OD600 = 1A corresponding to 1×10 9 CFU / mL), meeting the experimental accuracy requirements.
Claims
1. A method for constructing a RAW264.7 cell line overexpressing the TXNIP gene, characterized in that, It includes the following steps: a) Obtain a RAW264.7 cell line with overexpressed TXNIP gene by constructing a CAGG-TXNIP recombinant plasmid in combination with a lentiviral vector; b) Verify by RT-qPCR that the mRNA level of TXNIP is upregulated by 9.2 times compared with the control group; c) Verify by Western blot that the protein level of TXNIP is upregulated by about 3 times compared with the control group.
2. A model of TXNIP gene against Salmonella typhimurium infection, characterized in that, It includes the following steps: a) Construct a RAW264.7 cell line with overexpressed TXNIP gene; b) The Salmonella typhimurium used has the number CVCC3387; c) Infect the cell line with an effective amount of Salmonella typhimurium; d) Collect the lysate and coat it on a plate to observe its colony-forming units (CFU).
3. The method according to claim 1, wherein The RAW264.7 cell line with overexpressed TXNIP gene is achieved by transfection with a lentiviral vector.
4. The method according to claim 2, wherein The concentration of Salmonella typhimurium infecting cells is 1.1×10 7 CFU / mL.
5. The method according to claim 2, wherein The infection time of the Salmonella typhimurium on the cells is 4 hours, 6 hours, and 8 hours.
6. The use according to claim 1 and claim 2, characterized in that: Insert the mouse TXNIP gene described in claim 1 into the eukaryotic expression vector PTRIP-CAGG-Puro to construct a high-expression vector containing the mouse TXNIP gene downstream of the CAG promoter, and it has the function of resisting Salmonella infection.