Single plasmid induced expression system as well as preparation method and application thereof

Abstract

The invention provides a single plasmid induced expression system as well as a preparation method and application thereof, and belongs to the technical field of gene engineering. The single plasmid induced expression system comprises a single plasmid, and the nucleotide sequence of the plasmid is shown as SEQ ID No. 1. According to the constructed single plasmid induced expression system, the complex operation of two-time line establishment of traditional induced expression is overcome, an expected transgenic cell line can be obtained through screening only through one-time gene transduction, the operation process is simplified, and the efficiency is improved; compared with a lentivirus system, puromycin can be used for cloning and screening after gene transduction, and tetracycline antibiotic doxycycline is used for inducing target gene expression, so that the operation is simple, the cost is saved, a larger exogenous gene length can be inserted, and the vector capacity is larger; the background expression is lower, the induced expression multiple is higher, and the background leakage is lower while the efficient expression of the gene can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a single plasmid inducible expression system, a preparation method thereof, and an application thereof. Background Art

[0002] Expressing foreign genes intracellularly is the main means to realize gene functions and related research. In mammals, vector systems are usually used to mediate the expression of target genes or achieve the effect of interfering expression. Currently, commonly used vector systems include lentivirus, retrovirus, adenovirus, adeno-associated virus, CRISPR / Cas9-mediated gene editing, etc.

[0003] The lentivirus system can effectively infect proliferating and non-proliferating cells. After infecting cells, it can integrate into the host genome to achieve the expression of foreign genes, and has the advantages of low immunogenicity, tissue-specific expression, and inducible expression. The capacity of the lentiviral vector is as high as 8 kb. Except for promoters, selection genes, and other essential elements, foreign genes less than 2 kb can usually be efficiently packaged. The titer of lentivirus decreases as the inserted foreign gene fragment increases. Different types of cells have different requirements for the lentivirus titer (MOI) when infected with lentivirus. For example, 293T cells and most tumor cell lines can be infected with a lower MOI, while primary cultured cells and stem cells require a higher MOI for lentivirus infection.

[0004] Retroviruses can infect and integrate into the genomes of dividing cells to achieve the expression of foreign genes. The virus titer after packaging is higher than that of lentivirus; its immunogenicity is relatively low, and the capacity can reach 6 kb; it can efficiently infect dividing cells derived from the immune system and hematopoietic system, but there is a phenomenon of gene expression silencing when it is used in stem cells.

[0005] Adenoviruses can infect both dividing and non-dividing cells, have a large capacity for inserting foreign vectors, and have a higher virus titer; however, their immunogenicity is relatively high, and they can only perform transient expression and cannot achieve the integration of foreign genes; they can be used for tissue-specific expression and are not suitable for the inducible expression of foreign genes.

[0006] Adeno-associated virus (AAV) is a single-stranded DNA defective virus, and the replication of AAV requires the participation of a helper virus; the modified recombinant AAV has the characteristics of infecting both dividing and non-dividing cells, low immunogenicity, long-term stable expression, high titer, etc., and is more suitable for the long-term expression of non-dividing tissues and cells; its vector capacity is small, and the length of the foreign gene fragment that can be accommodated is less than 2 kb.

[0007] CRISPR / Cas9-mediated gene editing usually involves introducing corresponding gene expression elements into specific sites within the genome; its insertion site is clear, the number of copies is easy to control, and the genetic stability is good; however, it requires the design of a corresponding donor plasmid for the knock-in site, and the efficiency of gene knock-in is relatively low, the operation cycle is relatively long, and flow sorting is required to obtain successfully transfected clones. A large number of clones need to be identified to obtain gene knock-in clones, which is suitable for experiments with high safety requirements but is not conducive to the rapid acquisition of cell clones.

[0008] In summary, lentivirus has a wider tolerance to cell types and tissues, can be used for long-term studies in vitro and in vivo, and is the preferred method for in vitro gene function studies. Retroviruses are excellent tools for gene delivery in the hematopoietic and immune systems. AAV is the best vector for in vivo animal experiments and cell therapy. Site-directed gene editing technologies such as CRISPR / Cas9 are more suitable for precise editing of specific sites. High load and efficient gene introduction and stable and regulatable exogenous gene expression are still an important issue that needs to be addressed in stem cell research and gene function research.

[0009] At present, two strategies, continuous expression and induced expression, are usually used to study gene function. Continuous expression of differentiation-related genes or interference with the expression of stem cell stemness-related genes may lead to cell differentiation or apoptosis, making it impossible to obtain stable cell lines, which is not conducive to subsequent research on gene function. In the cell differentiation process of stem cells (such as PSCs, NSCs, etc.) and some primary cells (such as PBMCs, etc.), there is a phenomenon of exogenous gene silencing. The commonly used solution is to use strong promoters (such as CAG, EF1a promoter, etc.) to drive exogenous gene expression.

[0010] Stem cells and primary cells usually require higher virus titers for infection. Concentrating the lentiviral cell supernatant suspension can obtain high-titer viruses. In actual operations, lentiviral concentration reagents or ultracentrifugation methods are often used to concentrate the virus. The virus packaging process takes about 7 days from cell preparation to harvesting the virus supernatant, and virus concentration takes about 1 day. The virus packaging and concentration process increases the reagent, labor and time costs in the process of introducing exogenous genes into cells.

[0011] In addition, when using lentivirus to induce the expression of exogenous genes in stem cells, a dual-virus system is required to infect target cells: First, construct a monoclonal cell line stably expressing the modified rtTA element; Second, introduce an exogenous expression gene element driven by the TRE promoter into the above cell line; Finally, select monoclonal cells and verify the effect of induced expression. Monoclonal cell lines need to be picked and established for both virus infections, and the expression of exogenous genes needs to be identified. When using CRISPR-Cas9 for gene knock-in operations with induced expression in cells, the rtTA element with stable and continuous expression and the exogenous expression gene element driven by the TRE promoter are usually knocked into two different gene loci. This cell line establishment scheme requires two cell line establishment operations, while using the single plasmid knock-in scheme will result in a problem of extremely high background expression. Summary of the Invention

[0012] The object of the present invention is to provide a single plasmid inducible expression system, its preparation method and application, so as to solve at least one of the technical problems existing in the above-mentioned background technology.

[0013] To achieve the above object, the present invention adopts the following technical solutions: On the one hand, the present invention provides a single plasmid inducible expression system, which contains a single plasmid, and the nucleic acid sequence of the plasmid is as shown in SEQ ID No. 1.

[0014] On the second hand, the present invention provides a preparation method of the plasmid contained in the single plasmid inducible expression system as described in the first hand, including: Perform single digestion or double digestion using DNA restriction endonucleases EcoRⅠ and BamHⅠ; Obtain the corresponding linearized vector DNA fragment by gel extraction; Obtain the DNA fragment containing the target gene by PCR amplification and gel extraction; Perform cloning by the method mediated by T4 DNA ligase or the recombination method, and identify the positive clone containing the target DNA fragment by PCR; Use the positive clone containing the target DNA fragment for plasmid extraction and preparation.

[0015] As a further limitation of the second aspect of the present invention, in the PCR amplification, the upstream primer sequence of the target gene fragment to be expressed is as shown in SEQ ID No. 3, and the downstream primer sequence is as shown in SEQ ID No. 4.

[0016] As a further limitation of the second aspect of the present invention, construct an overexpression vector of enhanced green fluorescent protein to verify the gene expression effect induced by the single plasmid inducible expression system.

[0017] As a further limitation of the second aspect of the present invention, the gene sequence encoding the fluorescent protein in the enhanced green fluorescent protein expression vector is as shown in SEQ ID No. 5.

[0018] In the third aspect, the present invention provides a biological material containing the plasmid as described in the first aspect, and the biological material is an expression cassette or a host cell.

[0019] In the fourth aspect, the present invention provides an application of the single plasmid inducible expression system as described in the first aspect, and the application is for inducing gene overexpression or inducing knockdown.

[0020] In the fifth aspect, the present invention provides an application of the single plasmid inducible expression system as described in the first aspect, and the application is for the preparation of an expression cassette or a host cell.

[0021] In the sixth aspect, the present invention provides an application of the single plasmid inducible expression system as described in the first aspect, and the application is for transgenic operation in mammalian cells, and the cell types are stem cells, tumor cell lines or primary cells.

[0022] Advantages of the present invention: It overcomes the complex operations of the two-step cell line construction in traditional inducible expression. Only one gene transduction is required to screen and obtain the expected transgenic cell line, simplifies the operation process, and improves the efficiency; it can use puromycin for clone screening after gene transduction. Compared with the use of tetracycline antibiotic doxycycline to induce the expression of target genes and the lentiviral system, the operation is simple and the cost is saved; it can insert a larger exogenous gene length and has a larger vector capacity; it reduces the background expression, increases the induction expression fold, and can achieve high-efficiency gene expression with lower background leakage at the same time.

[0023] Additional aspects and advantages of the present invention will be given in part in the following description, and these will become obvious from the following description, or can be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings without creative efforts based on these drawings.

[0025] Figure 1 It is a structural diagram of the PB-ETPT plasmid (PB-EF1a-Tet-on / 3G-T2A-Puro-MCS-TRE3GV plasmid) described in the embodiments of the present invention.

[0026] Figure 2Structural diagram of the PB-TETP plasmid (PB-MCS-TRE3GV-EF1a-Tet-on / 3G-T2A-Puro plasmid) described in the embodiments of the present invention.

[0027] Figure 3 Schematic diagram of the proportion of EGFP-positive cells in the R1-PB-TETP-EGFP cell line before and after Dox induction in the flow cytometry analysis described in the embodiments of the present invention.

[0028] Figure 4 Schematic diagram of the proportion of EGFP-positive cells in the R1-PB-ETPT-EGFP cell line before and after Dox induction in the flow cytometry analysis described in the embodiments of the present invention.

[0029] Figure 5 Schematic diagram of the statistical results of the average fluorescence intensity (MFI) of EGFP in the R1-PB-ETPT-EGFP and R1-PB-TETP-EGFP cell lines described in the embodiments of the present invention.

[0030] Figure 6 Schematic diagram of the results of the flow cytometry analysis of the uninduced wild-type R1, R1-PB-ETPT-EGFP, and R1-PB-TETP-EGFP cell lines described in the embodiments of the present invention.

[0031] Figure 7 Schematic diagram of the statistical results of the average fluorescence intensity (MFI) of the uninduced wild-type R1, R1-PB-ETPT-EGFP, and R1-PB-TETP-EGFP cell lines described in the embodiments of the present invention.

[0032] Figure 8 Schematic diagram of the proportion of EGFP-positive cells in the H9-PB-TETP-EGFP cell line before and after Dox induction in the flow cytometry analysis described in the embodiments of the present invention.

[0033] Figure 9 Schematic diagram of the proportion of EGFP-positive cells in the H9-PB-ETPT-EGFP cell line before and after Dox induction in the flow cytometry analysis described in the embodiments of the present invention.

[0034] Figure 10 Schematic diagram of the statistical results of the average fluorescence intensity (MFI) of EGFP in the H9-PB-ETPT-EGFP and H9-PB-TETP-EGFP cell lines described in the embodiments of the present invention.

[0035] Figure 11 Schematic diagram of the results of the flow cytometry analysis of the uninduced wild-type H9, H9-PB-ETPT-EGFP, and H9-PB-TETP-EGFP cell lines described in the embodiments of the present invention.

[0036] Figure 12 Schematic diagram of the statistical results of the average fluorescence intensity (MFI) of EGFP in the uninduced wild-type H9, H9-PB-ETPT-EGFP, and H9-PB-TETP-EGFP cell lines described in the embodiments of the present invention.

[0037] Figure 13 Structural diagram of the overexpression plasmid PB-ETPT-ZFP36L2 described in the embodiments of the present invention.

[0038] Figure 14 WB detection result diagram of the induced overexpression of ZFP36L2 in Jurkat cells described in the embodiments of the present invention. Detailed implementation manners

[0039] The embodiments of the present invention provide a single plasmid inducible expression system and its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0040] Specifically, first, in the specific embodiments of the present invention, two single plasmid inducible expression systems are provided. The nucleic acid sequence of the plasmid contained in one single plasmid inducible expression system is shown in SEQ ID No. 1 (PB-ETPT plasmid); the nucleic acid sequence of the plasmid contained in the other single plasmid inducible expression system is shown in SEQ ID No. 2 (PB-TETP plasmid).

[0041] Such as Figure 1 、 Figure 2As shown, the two plasmids are composed of the same DNA elements, except that the positions of the DNA elements in the two plasmids are different. The difference in the induction effect reflected during application is mainly achieved by optimizing the positions of the DNA elements, and there is no difference in subsequent preparation and use. The same DNA restriction enzyme can be used for digestion to obtain linearized DNA fragments with the same sticky ends, and the same exogenous gene amplification products, the same gene cloning preparation method, and cell transfection method can be inserted. In the figure, TR represents the terminal repeat sequence; ins represents the HS4 insulator sequence derived from chicken; EF1α promoter represents the promoter derived from human elongation factor 1 alpha; Tet-on / 3G represents the optimized rtTA (reverse tetracycline-controlled transactivator) protein; TRE3GV represents the third-generation tetracycline-responsive promoter; T2A represents the 2A peptide derived from Thosea asigna virus; PuroR represents the puromycin N-acetyltransferase gene, the puromycin resistance gene; bGH poly(A) signal represents the bovine growth hormone polyribonucleotide tailing signal; SV40 poly(A) signal represents the simian virus 40 polyribonucleotide tailing signal; MCS (multiple cloning sites) represents the multiple cloning site; ori represents the replication origin; AmpR promoter represents the AmpR promoter of the ampicillin resistance gene; AmpR represents the ampicillin resistance gene.

[0042] The components in the above two vector systems can be replaced with other components, and the expression effect of the essential plasmid system is not affected after replacement: The Puro resistance gene can be replaced with resistance genes such as BSD (Blasticindin resistance gene), Hygro (hygromycin resistance gene), Neo (neomycin resistance gene), or Zeo (zeocin resistance gene), etc., and corresponding drugs can be used for screening. T2A can be replaced with self-cleaving 2A peptides such as P2A, E2A, F2A, etc., or can be replaced with an IRES sequence for expressing two proteins driven by one promoter; there should be no stop codon before the 2A peptide sequence, while there needs to be a stop codon before the IRES sequence. The EF1α promoter can be replaced with the CAG promoter or the CMV promoter, and can only be replaced with the CAG promoter in stem cells, and the CMV promoter can be used for replacement in other cell lines. The TRE3GV promoter can be replaced with other tetracycline-responsive promoters. The Tet-on / 3G protein-coding gene can be replaced with other types of rtTA protein-coding genes. bGHpolyA and SV40 polyA can be replaced with other polyA sequences, such as hGH polyA, rbGlob polyA, etc.

[0043] In this implementation, experimental methods for preparing the above two plasmids were provided, and verification experiments on the performance of the two plasmids were carried out, including the following experimental procedure steps: Experiment 1 Plasmid transformation: Transform the PB-ETPT plasmid or the PB-TETP plasmid into Escherichia coli competent cells, spread them on an LB plate with ampicillin resistance, and pick monoclonal colonies for plasmid extraction after overnight culture. The specific steps are as follows: 1) Take about 20 μL of a tube of competent cells from the -80 °C refrigerator, place it on ice for about 5 min to thaw. 2) Add 1 - 10 ng of plasmid to the competent cells, gently pipette and mix well, and then incubate on ice for 30 min. The types of Escherichia coli competent cells that can be used include DH5α, TOP10, STBL3, etc. 3) Place the competent cell-DNA mixture in a 42 °C water bath for heat shock for 75 s, and then place it on ice for 2 min. 4) Add 1 mL of liquid LB without ampicillin to the transformed product, mix well, and take 100 μL of the medium to spread on an LB plate with ampicillin resistance. There is no need for a resuscitation step when transforming the plasmid, otherwise there will be too many clones to pick monoclonal colonies.

[0044] Experiment 2 Plasmid Extraction: A plasmid miniprep kit was used for the small-scale preparation of plasmids. In this example, the Tiangen Plasmid Mini Midiprep Kit (DP118) was used for plasmid extraction. There is no requirement to remove endotoxin for the plasmid extraction kit used for plasmid cloning, but the plasmid extraction for cell transfection should require endotoxin removal. The specific steps are as follows: 1) Preparation of bacterial solution: Pick a single colony and inoculate it into 15 mL of liquid LB medium with ampicillin resistance (Amp), and culture it overnight at 37 °C in a shaker at a rotation speed of 220 rpm for 14 - 16 h. The next day, centrifuge at 5000 rpm for 10 min, discard the supernatant, and collect the bacterial cells. 2) Perform the column equilibration step according to the instructions: Add 500 μL of equilibration buffer BL to the adsorption column CP4, centrifuge at 12000 rpm for 1 min, pour out the filtered equilibration buffer, and place the adsorption column back into the collection tube. 3) Add 500 μL of solution P1 to the bacterial cell pellet, and resuspend the bacterial solution thoroughly by pipetting or vortexing. 4) Add 500 μL of solution P2 to the centrifuge tube, gently invert it up and down 8 times to fully lyse the bacterial cells. The lysed bacterial solution should be clear and viscous to prevent incomplete lysis due to excessive bacterial cells. 5) Add 500 μL of solution P4 to the centrifuge tube, immediately gently invert it up and down 8 times to neutralize the lysis process, and white flocculent precipitates will appear in the tube at this time. Let it stand at room temperature for 10 min, and centrifuge at 12000 rpm for 10 min. After centrifugation, the white precipitate aggregates at the bottom of the tube. 6) Add the supernatant from the previous step to the filtration column CS in portions, centrifuge at 12000 rpm for 1 min, and collect the filtrate and place it in a new 2 mL centrifuge tube. 7) Add 0.3 times the volume of isopropanol to the collected filtrate, mix it by inverting up and down, and transfer it to the pretreated and activated adsorption column CP4, centrifuge at 12000 rpm for 1 min, discard the filtrate, and sequentially pass the filtrate with isopropanol added to the collection tube through the adsorption column CP4 and centrifuge to discard the filtrate. 8) Add 500 μL of protein removal solution PD to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate. 9) Add 600 μL of wash buffer PW to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate. 10) Place the adsorption column CP4 back into the collection tube and repeat step 9 once. 11) Place the adsorption column CP4 back into the collection tube and centrifuge at 12000 rpm for 2 min. 12) Place the adsorption column CP4 in a clean centrifuge tube, add an appropriate amount of elution buffer TB dropwise onto the membrane in the middle part of the adsorption column, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min to collect the plasmid solution. 13) Measure the concentration of the plasmid using a nucleic acid and protein analyzer and store it at -20 °C.

[0045] Experiment 3 Plasmid Digestion: Select appropriate restriction sites to digest the plasmid according to the method adopted in the subsequent cloning experiment. In this example, two common restriction sites, BamHⅠ and EcoRⅠ, were reserved in the two plasmids for subsequent cloning operations, and the DNA restriction enzymes from NEB were used for the digestion experiment.

[0046] The restriction enzyme reaction system is as follows: rCutSmart™ Buffer, 5 μL; EcoRⅠ-HF, 2 μL; BamHⅠ-H, 2 μL; plasmid DNA, 6 μg; ddH2O, made up to a total volume of 50 μL; digest with restriction enzymes at 37 °C for 2 h.

[0047] Experiment 4: Amplification of the target gene: The target gene fragment with an insert was prepared by PCR amplification. In this example, the primer sequences designed when constructing the EGFP inducible expression plasmid by the recombination method are as follows: the upstream primer sequence is shown in SEQ ID No. 3, and the downstream primer sequence is shown in SEQ ID No. 4. They were synthesized by a DNA synthesis company and diluted to a concentration of 10 μM for use. The source of the amplification template can be plasmid DNA or cDNA template obtained by reverse transcription of RNA extracted from cells or tissues with high expression of the target gene. High-fidelity enzymes were used to amplify the target gene to reduce the probability of base mutations during the amplification process. In this example, the amplification enzyme used was Phanta Max Super-Fidelity DNA Polymerase (product number: PD505-d3) from Nanjing Novoprotein Scientific Inc. The following PCR reaction system was prepared in a 200 μL centrifuge tube: 2 × Phanta Max Buffer, 25 μL; dNTP Mix (10 mM each), 1 μL; Primer F, 2 μL; Primer R, 2 μL; Phanta Max Super-Fidelity DNA Polymerase, 1 μL; DNA template or cDNA, 1 ng or 1 μL; ddH2O, to 50 μL.

[0048] After the above system was prepared and centrifuged briefly, amplification was carried out in a PCR instrument. The PCR instrument used in this example was the T100 type from Bio-Rad. The PCR amplification conditions were set as follows: (1) Pre-denaturation: 95 °C for 3 min; (2) Denaturation: 95 °C for 15 s; (3) Annealing: 55 °C for 15 s; (4) Extension: 72 °C for 30 s; (5) Final extension: 72 °C for 5 min. Among them, one cycle was performed for the denaturation, annealing, and extension steps, and the number of cycles was set to 30. The remaining steps were performed once.

[0049] Experiment 5 Agarose Gel Electrophoresis and DNA Recovery: Use an agarose DNA recovery kit to recover the target DNA fragment after PCR amplification and the vector fragment after plasmid digestion. In this example, the DNA recovery kit from Tiangen Biochemical (Beijing) Co., Ltd. (product number DP219) is used. The specific experimental steps are as follows: 1) Prepare a 1% agarose gel using 1×TAE buffer. 2) Add 1×DNA loading buffer to the PCR product and the digestion product to a final concentration, and then load the samples for electrophoresis. The electrophoresis is carried out at a constant voltage of 5 V / cm for 30 min. 3) Cut the gel after electrophoresis with a Tanon 1600 gel imaging system to obtain the gel strip containing the target DNA fragment. After cutting, weigh it and place it in a clean 1.5 mL centrifuge tube. 4) Add solubilization solution PE to the gel strip at a ratio of 300 μL per 100 mg of the gel strip, and solubilize the gel at 50 °C for 5 - 10 min, during which invert and mix 2 - 3 times to fully solubilize the gel. 5) Add the solubilized solution to adsorption column CA, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, and discard the filtrate. 6) Add washing solution PW to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate. 7) Repeat step 6 for rinsing and centrifuging. 8) Place adsorption column CA back into the collection tube and centrifuge at 12,000 rpm for 2 min, then let it stand at room temperature for 2 - 5 min to completely dry and remove the washing solution. 9) Place adsorption column CA in a clean centrifuge tube, add an appropriate amount of elution buffer TB dropwise to the middle of the adsorption membrane. Let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min to collect the eluted DNA solution, and measure the concentration using a nucleic acid and protein analyzer.

[0050] Experiment 6 Ligation and Transformation of the Target Gene Fragment: 1) Ligation: The construction of the recombinant plasmid containing the target gene can be carried out using T4 DNA ligase or Gibson assembly method. In this example, T4 DNA ligase from NEB (product number: M0202S) is used. The ligation steps using T4 DNA ligase are briefly described as follows: Prepare the following reaction system on ice: 10×T4 DNA Ligase Reaction Buffer, 1 μL; Linealized vector, 50 ng; Insert DNA fregment, 10 - 50 ng; T4 DNA Ligase, 1 μL; ddH2O, to 10 μL; Ligate at room temperature for 10 - 30 min or at 16 °C for 30 min - 1 h. Add all the ligation products to Escherichia coli competent cells for transformation. The transformation steps are the same as those described in Experiment 1 for plasmid transformation.

[0051] 2) Cloning by recombination method: The plasmids in this example can be cloned using the Gibson recombination principle. The recombination kit used is the One Step Cloning Kit (Cat. No.: C117-02) from Nanjing Novoprotein Scientific Inc. The specific operation method is as follows: Prepare the following reaction on ice: Linealized vector, 50 ng; Insert DNA fragment, 10 - 50 ng; 2 × CE Mix V3, 5 μL; ddH2O, to 10 μL; 50 °C, 5 - 15 min, store at 4 °C or on ice.

[0052] Add all the recombinant products to the competent cells for transformation. The transformation steps are the same as those described for plasmid transformation in Experiment 1. After transformation, directly add an appropriate amount of LB liquid medium without ampicillin (Amp-) to resuspend the bacterial solution and then spread it on an LB plate (Amp+). After overnight incubation for 14 - 16 h, pick colonies for PCR identification.

[0053] Experiment 7 Identification of positive clones: Clone identification is carried out by colony PCR and DNA sequencing. Colony PCR is amplified using the 2×Hieff® PCR Master Mix (With Dye, Cat. No.: 10102ES09) from Yeasen Biotech Co., Ltd. Prepare the following reaction system on ice: 2×Hieff ® PCR Master Mix, 5 μL; Primer F (10 μM), 0.5 μL; Primer R (10 μM), 0.5 μL; ddH2O, 4 μL.

[0054] Prepare the corresponding volume of PCR mixture according to the number of clones to be identified, and aliquot it into 200 μL centrifuge tubes at a volume of 10 μL. Use a 10 μL white pipette tip to pick a single colony and inoculate it at the marked position on the Amp+ LB plate. After picking the colonies, the plate is placed at 37 °C for continued incubation for 3 - 6 h. The identified positive clones can be used for subsequent shaking culture and plasmid extraction; repeatedly pipette the 10 μL pipette tip in the PCR mixture, briefly centrifuge, and then amplify in a Bio-Rad T100 thermal cycler. The PCR amplification conditions are set as follows: (1) Pre-denaturation: 94 °C for 3 min; (2) Denaturation: 94 °C for 15 s; (3) Annealing: 55 °C for 15 s; (4) Extension: 72 °C for 30 s; (5) Final extension: 72 °C for 5 min. Among them, one cycle is executed for the denaturation, annealing, and extension steps, and the number of cycles is set to 30. The remaining steps are executed once.

[0055] After the PCR reaction was completed, 2.5 μL of the reaction product was taken for agarose gel electrophoresis, and the electrophoresis conditions were carried out as described in Experiment 5. The electrophoresis results were observed using a Tanon gel imaging system (Tanon 1600). Whether the target band of the expected size appeared in each lane was used to determine whether the clone corresponding to each lane was a positive clone. The positive clones identified by PCR were selected for shaking culture and amplification. For each plasmid, two positive clones were picked and sent for bacterial liquid sequencing or the plasmid was extracted and then sent to a sequencing company for sequencing. The plasmid with the correct sequencing result was selected for the subsequent cell transfection experiment.

[0056] Experiment 8: Mouse embryonic stem cell culture: R1 mouse embryonic stem cells were purchased from ATCC and cultured under feeder-free conditions. The components of the mESM medium used included DMEM, 15% FBS, 1% GlutaMax, 1% NEAA, 1% penicillin-streptomycin, 0.1 mM β-mercaptoethanol, and 1000 U / mL mLIF. The culture dish was coated with 0.1% gelatin 1 h in advance, and the cells were inoculated at a density of 2×10 5 / cm 2 . Trypsin at 0.25% was used for digestion and passage, and passage was carried out every two days.

[0057] Experiment 9: Human embryonic stem cell culture: H9 human embryonic stem cells were cultured in a Matrigel-coated culture plate, and the medium used was mTeSR1 medium (product number 85850) from STEMCELL. When passaging, 0.5 mM EDTA was used for digestion and passage, and fresh medium was changed every day. For single-cell passage, Accutase (STEMCELL, product number 07920) was used for digestion and passage, and 10 μM Y27632 was added to promote cell survival. The passage time was determined according to the cell growth situation, and passage was carried out approximately once every 5 days.

[0058] Experiment 10: Transfect cells to construct a cell line that can induce the expression of the target gene: Both human and mouse embryonic stem cells can be used to establish overexpressing cell lines by transfection. Lipofectamine 2000 (ThermoFisher, product number 11668019) or Lipofectamine 3000 (ThermoFisher, product number L3000008) and other transfection reagents were used for cell transfection. The plasmids for transfection included PB inducible expression plasmids (PB-TETP-EGFP or PB-ETPT-EGFP) and PB transposase (CMV-hyPBase) plasmids, which were mixed at a mass ratio of 1:1 and then transfected. The cells were passaged one day before transfection, and the cells were inoculated at a density of 1×10 5 / cm 2Inoculate cells at a certain cell density. Taking the transfection of a 24-well plate with Lipofectamine 2000 as an example, the transfection process is briefly described as follows: Take a 1.5 mL EP tube and add 50 μL of Opti-MEM, then add 1 - 2 μL of Lipofectamine 2000, mix well and set aside; Take a new 1.5 mL EP tube and add 500 ng of plasmid, mix well; After incubating at room temperature for 5 min, slowly add the plasmid suspension into the first EP tube, mix well and incubate at room temperature for 10 min, mix again and continue incubating for 10 min. Add the transfection mixture to the cell-seeded culture plate and gently mix evenly. After overnight incubation for 14 - 16 h, replace with a new medium. When transfecting human embryonic stem cells, 10 μM of Y27632 needs to be added, and the fresh medium is replaced the next day while removing Y27632.

[0059] After 48 h of transfection, add 2 μg / mL of puromycin for screening for 2 - 4 days. To compare the protein expression levels of the target gene in polyclones with and without the inducer doxycycline (Dox), appropriate monoclonal clones can be further selected for analysis and cell cryopreservation.

[0060] Experiment 11 Flow cytometry analysis of induced expression level: Treat mouse or human embryonic stem cell EGFP-induced expression cell lines with 1 μg / mL Dox for 48 h, digest them with 0.25% trypsin or Accutase respectively to prepare single-cell suspensions, terminate digestion with a medium containing 10% FBS, centrifuge at 300 g for 5 min to collect cell pellets; Wash once with DPBS, centrifuge at 300 g for 5 min to collect cell pellets, resuspend the cells with an appropriate volume of DPBS, filter through a 70 μm cell strainer, and then perform flow cytometry analysis. For flow cytometry analysis, select the main cell population, select single cells for further analysis, adjust the voltage and then analyze the expression of cells expressing EGFP in the FITC channel. Analyze the mean fluorescence intensity (MFI) of EGFP before and after Dox induction to compare the differences in induced expression effects among different cell lines. This experiment is independently repeated three times and then statistically analyzed. To avoid the subjective influence during the process of selecting clones, the mean fluorescence intensity (MFI) of EGFP in polyclones was directly analyzed in this experiment.

[0061] In a specific application embodiment, the plasmid constructed by the above method can be used for gene function research, including induced overexpression and induced knockdown.

[0062] In another specific application embodiment, the plasmid constructed by the above method can be used for transgenic operations in mammalian cells, and the cell types include but are not limited to various stem cells, tumor cell lines, primary cells and other various cell lines.

[0063] The above two single plasmids established in the embodiments of the present invention can be used as transposon vectors for inducible gene expression in mouse embryonic stem cells (mESCs). Both vectors can efficiently induce gene expression, but there are differences in the expression effects of the two vectors. Among them, the PB-ETPT vector has a higher induction efficiency and a lower background leakage; the PB-TETP vector can achieve a higher expression intensity after induction, but has a higher background expression. Therefore, the PB-ETPT vector is an excellent vector for inducing gene expression in mESCs.

[0064] In the embodiments of the present invention, verification tests were carried out to verify that the above two vectors have high expression efficiency, induction efficiency and expression intensity, as well as the differences in expression efficiency, induction efficiency and expression intensity between the two vectors. To verify the gene expression effect, two enhanced green fluorescent protein overexpression vectors were constructed, namely PB-TETP-EGFP and PB-ETPT-EGFP. The gene sequences of the two enhanced green fluorescent protein overexpression vectors are the same, as shown in SEQ ID No. 5.

[0065] In the embodiments of the present invention, the experimental materials and equipment required for the above verification tests include: CMV-hyPBase plasmid, PB-TETP plasmid, PB-ETPT plasmid, PB-TETP-EGFP plasmid, PB-ETPT-EGFP plasmid, plasmid extraction kit, restriction endonuclease, gel recovery kit, LB solid and liquid media, bacterial culture flasks, bacterial culture dishes, cell culture plates, human and mouse embryonic stem cell media, transfection reagent or electroporator.

[0066] Experimental results: As Figure 3 shown, the flow cytometry analysis results show that the proportion of EGFP-positive cells in the R1-PB-TETP-EGFP polyclonal cell line after induction with doxycycline (Dox) for 48 h is 99.6%, indicating that the cell line can successfully induce the expression of EGFP.

[0067] As Figure 4 shown, the flow cytometry analysis results show that the proportion of EGFP-positive cells in the R1-PB-ETPT-EGFP polyclonal cell line after induction with doxycycline (Dox) for 48 h is 99.7%, and the proportion of positive cells is also very high, indicating that the cell line can successfully induce the expression of EGFP.

[0068] As Figure 5As shown, the mean fluorescence intensity (MFI) of EGFP in the R1-PB-ETPT-EGFP and R1-PB-TETP-EGFP cell lines after induction with doxycycline (Dox) for the same time (48 h) was analyzed. The results showed that the EGFP MFI in R1-PB-ETPT-EGFP cells increased by approximately 487-fold compared with that before induction, and the EGFP MFI in R1-PB-TETP-EGFP cells increased by approximately 435-fold compared with that before induction. The statistical analysis results of MFI showed that the induction effect of EGFP in R1-PB-ETPT-EGFP cells was significantly better than that in R1-PB-TETP-EGFP cells, with a highly significant difference (** p<0.01). In the figure, plasmid 1 is the PB-ETPT plasmid and plasmid 2 is the PB-TETP plasmid.

[0069] As Figure 6 and Figure 7 shown, the background expression of EGFP in uninduced wild-type R1 cells and the R1-PB-ETPT-EGFP and R1-PB-TETP-EGFP cell lines was analyzed. The results showed that the EGFP MFI in R1-PB-ETPT-EGFP cells was approximately 4 times that of wild-type control cells, the EGFP MFI in R1-PB-TETP-EGFP cells was approximately 17.9 times that of wild-type control cells, and the EGFP MFI in R1-PB-TETP-EGFP cells was approximately 4.4 times that of R1-PB-ETPT-EGFP cells. The above results indicate that the EGFP expression in R1-PB-ETPT-EGFP cells was lower than that in R1-PB-TETP-EGFP cells before induction, with a highly significant difference (**** p<0.0001). Figure 7 In the figure, plasmid 1 is the PB-ETPT plasmid and plasmid 2 is the PB-TETP plasmid.

[0070] In summary, the above two transposon vectors capable of inducing gene expression in mESCs (mouse embryonic stem cells) were established in this example. Both vectors can efficiently induce gene expression, but there are differences in the expression effects of the two vectors. Among them, the PB-ETPT vector has a higher induction efficiency and a lower background leakage; the PB-TETP vector can achieve a higher expression intensity after induction, but has a higher background expression. Therefore, the PB-ETPT vector is an excellent vector for inducing gene expression in mESCs.

[0071] In this example, the expression effects of the two vectors in the hESCs cell line H9 were tested. Two polyclonal H9 cell lines capable of inducing the expression of EGFP were established by transfection. And the induction expression effects of the two vectors were analyzed by flow cytometry, and the results are as follows.

[0072] AsFigure 8 As shown, the flow cytometry analysis results showed that the proportion of EGFP-positive cells in the H9-PB-TETP-EGFP polyclonal cell line was 97.8% after induction with doxycycline (Dox) for 48 h. The very high proportion of positive cells indicated that the cell line establishment could successfully induce EGFP expression.

[0073] As Figure 9 shown, the flow cytometry analysis results showed that the proportion of EGFP-positive cells in the H9-PB-ETPT-EGFP polyclonal cell line was 97.9% after induction with doxycycline (Dox) for 48 h, which also indicated that the cell line was established and could induce EGFP expression.

[0074] As Figure 10 shown, the mean fluorescence intensity (MFI) of EGFP in the H9-PB-ETPT-EGFP and H9-PB-TETP-EGFP cell lines after induction with doxycycline (Dox) for the same time (48 h) was analyzed. The results showed that the EGFP MFI in the H9-PB-ETPT-EGFP cells increased by approximately 4096-fold after induction compared with that before induction, and the EGFP MFI in the H9-PB-TETP-EGFP cells increased by approximately 633-fold compared with that before induction. The results showed that the induction fold increase of EGFP in the H9-PB-ETPT-EGFP cells was approximately 6.5 times that in the H9-PB-TETP-EGFP cells, and the difference was extremely significant (**** p<0.0001). In the figure, plasmid 1 is the PB-ETPT plasmid and plasmid 2 is the PB-TETP plasmid.

[0075] Combined Figure 11 with Figure 12 shown, the background expression of EGFP in uninduced wild-type H9 cells and the H9-PB-ETPT-EGFP and H9-PB-TETP-EGFP cell lines was analyzed. The results showed that the EGFP MFI in the H9-PB-ETPT-EGFP cells was approximately 1.9 times that of the wild-type control cells, the EGFP MFI in the H9-PB-TETP-EGFP cells was approximately 13.3 times that of the wild-type control cells, and the EGFP MFI in the H9-PB-TETP-EGFP cells was approximately 7.1 times that in the H9-PB-ETPT-EGFP cells when not induced. The results indicated that the background expression of EGFP in the H9-PB-ETPT-EGFP cells was lower than that in the H9-PB-TETP-EGFP cells when not induced, and the difference was extremely significant (**** p<0.0001). Figure 12 In

[0076] From the above experimental comparison, it can be seen that when the two gene inducible expression vectors constructed in the embodiments of the present invention are used for human embryonic stem cells (hESCs), the PB-ETPT vector has a higher inducible expression efficiency, manifested as a higher up-regulation multiple of the induced gene expression and a lower background of gene expression when not induced. Therefore, the PB-ETPT vector is an excellent vector for transgenic inducible expression in hESCs.

[0077] In a specific embodiment, the overexpression plasmid PB-ETPT-ZFP36L2 of the target gene ZFP36L2 was successfully constructed using the plasmid preparation method described in this embodiment. The structure of the constructed plasmid is as Figure 13 shown. And Lipofectamine 2000 was used to co-transfect Jurkat cells with PB-ETPT-ZFP36L2 and CMV-hyPBase plasmids and screened with puromycin to successfully obtain an inducible overexpression cell line. After adding 1 μg / mL of doxycycline and inducing for 12 h, the inducible overexpression effect was detected by Western blot. The results are as Figure 14 shown. Hemagglutinin tag and ZFP36L2 protein were not expressed in uninduced Jurkat overexpression cells, while the expression of hemagglutinin tag and ZFP36L2 protein was significantly up-regulated in induced Jurkat overexpression cells. The above results show that the background expression of the overexpression cells is extremely low without the inducer, and the overexpression effect after induction is very obvious, which confirms that good gene inducible expression effect can be achieved by using PB-ETPT plasmid in Jurkat cells. As shown in the following Experiments 12 and 13.

[0078] Experiment 12 Establishment of Jurkat inducible overexpression line and verification of overexpression effect: Overexpression of the target gene ZFP36L2 (the gene coding sequence length including the tag sequence is 1527 bp) was carried out in the human T lymphocyte leukemia cell line Jurkat cells. The sequence of the target gene ZFP36L2 used in this embodiment is shown in SEQ ID No. 6, which is the sequence after adding the HA tag.

[0079] The sequence of the constructed overexpression plasmid PB-ETPT-ZFP36L2 is shown in SEQ ID No. 7. The primer sequences used for gene cloning in the construction of this overexpression plasmid are as follows: the upstream primer sequence is shown in SEQ ID No. 8, and the downstream primer is shown in SEQ ID No. 9. Adding a hemagglutinin tag (HA-tag) to the primer sequences facilitates the verification of the overexpression effect, and the sequencing method is used to confirm that the overexpression clone sequence is correct. Subsequently, cell transfection is carried out using Lipofectamine 2000 transfection reagent according to the aforementioned method. After 48 hours of transfection, 1 μg / mL puromycin is added for screening, and the cells are allowed to grow to a sufficient number for effect verification.

[0080] Experiment 13 Western blot detection: First, collect the cultured cells for protein extraction. Collect the cultured cells, centrifuge at 300g for 5 min at room temperature, and discard the supernatant; add DPBS and wash twice, centrifuge at 300g for 5 min at room temperature, and discard the supernatant. Take 1 - 2×10 6 cells and add 200 μL of RIPA lysis buffer (Beyotime, catalog number P0013C) for lysis. Add protease and phosphatase inhibitors (Beyotime, catalog number P1046) to the lysis buffer in a ratio of 1:50 in advance to prevent protein degradation. Vigorously pipette and mix well, then place on ice for 5 min for lysis, centrifuge at 12000 rmp at 4℃ for 10 min, and take the supernatant for BCA protein quantification (YaEnzyme, catalog number ZJ101L). Then take 50 μg of protein after high-temperature denaturation for PAGE gel electrophoresis. Use a 10% PAGE gel (YaEnzyme, catalog number PG112) for electrophoresis, transfer the membrane by wet transfer method, with a constant current of 300 mA for 2 hours, block with a blocking solution (TBST containing 5% skim milk), add the primary antibody and incubate overnight on a horizontal shaker at 4℃ (ZFP36L2 antibody from Santa Cruz, catalog number sc-365908, dilution ratio 1:200, HA antibody from CST, catalog number 3724S, dilution ratio 1:2000), wash 3 times with 1×TBST for 10 minutes each time, add the secondary antibody and incubate at room temperature for 1 h. Wash 3 times with 1×TBST for 10 minutes each time. According to the kit instructions, mix equal amounts of A and B solutions and drop them onto the transfer membrane for chemiluminescence (Tanon, catalog number 180-5001), and use a chemiluminescence imaging system (Bio-Rad, ChemiDoc TM MP) for image acquisition.

[0081] In summary, the present invention constructs a single plasmid inducible expression system, which overcomes the complex operations of the two-step system construction in traditional inducible expression. Only one gene transduction experiment is required to screen and obtain the expected transgenic cell line, simplifying the experimental process and improving the experimental efficiency. Puromycin can be used for clone screening after gene transduction, and the tetracycline antibiotic doxycycline is used to induce the expression of the target gene. Compared with the lentiviral system, the inducible expression system constructed by the present invention is simpler in operation, saves costs, can achieve a larger vector capacity, has a lower background expression, a higher induction expression multiple, and can achieve high-efficiency gene expression while having a lower background leakage.

[0082] Although the specific implementation manners of the present invention have been described above in conjunction with the accompanying drawings, it is not a limitation to the protection scope of the present invention. Those skilled in the art should understand that based on the technical solutions disclosed in the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts should be covered within the protection scope of the present invention.

Claims

1. A single plasmid inducible expression system, characterized in that, It contains a single plasmid, and the nucleic acid sequence of the plasmid is as shown in SEQ ID No.

1.

2. A method for preparing a plasmid contained in the single plasmid inducible expression system according to claim 1, characterized in that, Including: Performing single enzyme digestion or double enzyme digestion using restriction endonucleases EcoRⅠ and BamHⅠ; Obtaining the corresponding linearized vector DNA fragment by gel recovery; Obtaining the DNA fragment containing the target gene by PCR amplification and gel recovery; Performing cloning by the method mediated by T4 DNA ligase or the recombination method, and obtaining positive clones containing the target DNA fragment by PCR identification; Performing plasmid extraction and preparation using the positive clones containing the target DNA fragment.

3. The method for preparing the plasmid according to claim 2, wherein The PCR primer sequence of the target gene fragment to be expressed designed in the PCR amplification is as shown in SEQ ID No.

3.

4. The method for preparing the plasmid according to claim 2, characterized in that, Constructing an enhanced green fluorescent protein overexpression vector to verify the gene expression effect induced by the single plasmid induction expression system.

5. The method for preparing the plasmid according to claim 2, wherein The gene sequence encoding the fluorescent protein in the enhanced green fluorescent protein overexpression vector is as shown in SEQ ID No.

4.

6. A biological material containing the plasmid as described in claim 1, characterized in that, The biological material is an expression cassette or a host cell.

7. Use of a single plasmid inducible expression system as described in claim 1, characterized in that, The application is for inducing gene overexpression or inducing knockdown.

8. Use of a single plasmid inducible expression system as described in claim 1, characterized in that, The application is for the preparation of an expression cassette or a host cell.

9. Use of a single plasmid inducible expression system as described in claim 1, characterized in that, The application is for transgenic operation in mammalian cells.

10. The application according to claim 9, wherein The mammalian cell types for transgenic operation are stem cells, tumor cell lines or primary cells.

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