Self-activated micromolecule induction system, cell and application and method in gene expression induction

Abstract

The invention provides a self-activated micromolecule induction system, a cell and application and a method in inducing gene expression, and belongs to the technical field of gene engineering. According to the self-activated small molecule induction system, element composition optimization is carried out on the basis of a green light response system, the induction efficiency of a target gene is improved through cascade reaction, the background leakage level can be reduced, the self-activated small molecule induction system can play an important role in target gene induction, and the application prospect is wide. The method has a good application prospect in the field of protein expression and virus vector production and preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a self-activating small molecule induction system, cells and applications and methods in inducing gene expression. Background Art

[0002] The mammalian cell inducible expression system is an important tool for studying gene function and protein production. The most widely used inducible gene expression technology is the Tet system, also known as the tetracycline regulatory system. The Tet system uses the regulatory effect of tetracycline antibiotics to control gene expression. The system is usually divided into two parts: the first part is the TetR protein, which is a transcriptional repressor that can bind to the TetO sequence on the promoter to inhibit the expression of the target gene. The other part is the induction condition. In the presence of tetracycline (or its derivative polycycline, such as doxycycline), TetR binds to tetracycline, thereby releasing the inhibition of TetO and activating the expression of the target gene. Because this system requires the use of antibiotic molecules such as tetracycline for induced expression regulation, it has certain limitations for clinical applications.

[0003] The green light-responsive system is another system that can induce the expression of target proteins in mammalian cells. Based on a light-sensitive bacterial transcription factor CarH and its corresponding DNA operating sequence CarO, it is engineered to control gene expression in mammalian cells. Its inducing molecule is vitamin B12 (AdoB12), which is drug-free and does not require the use of antibiotics (such as tetracycline), thus avoiding the potential effects of drugs on cell physiology and cytotoxicity. This system has a shorter response time and can quickly start or inhibit gene expression. Changes can usually be observed within a few minutes, while the Tet system may take longer to reach a stable expression level. It can be widely used in mammalian cells and plant cells (Chatelle C, Ochoa-Fernandez R, Engesser R, et al. A Green-Light-Responsive System for the Control of Transgene Expression in Mammalian and Plant Cells. ACS Synth Biol. 2018;7(5):1349-1358. doi:10.1021 / acssynbio.7b00450).

[0004] However, there is still room for further development of existing technologies for the expression level of target genes. Improving the expression level of target genes is the key direction for further development of current technologies. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention constructs a self-activating small molecule induction system based on the green light response system to induce the target gene expression vector, significantly enhances the expression of the target protein through positive feedback regulation, and replaces the promoter to reduce background leakage. The relevant sequence is constructed into a plasmid vector, which is easy to operate.

[0006] The present invention first provides a self-activating small molecule induction system for expressing a target gene. The self-activating small molecule induction system includes the following elements on the same vector: Expression element 1 for transcriptional activation, expression element 2 for operator regulation; operator and promoter; expression element 3 of target gene; The promoter includes promoter 1 for promoting expression element 1 and expression element 2 and promoter 2 for promoting expression element 3; promoter 1 and promoter 2 are the same or different; The operon regulates promoter 1 and promoter 2 simultaneously.

[0007] The source of the expression element 1 for transcriptional activation includes but is not limited to any one or more of VP16, VP64, P65, HSF1, CBP, P300, FHL1, FHL2, FHL3, FHL4, and ACT, preferably the transcriptional activation domain of VP16.

[0008] Further preferably, the expression element 1 has a nucleotide sequence as shown in SEQ ID NO.4.

[0009] The expression element 2 for operon regulation includes but is not limited to: any one or more of CarH, LacI, trpG, galR, Gal4, and TetR, preferably CarH.

[0010] In the present invention, the expression element 2 can also be understood as a regulatory gene of an operon, and the regulatory gene regulates the corresponding operon.

[0011] Preferably, the expression element 2 has a nucleotide sequence as shown in SEQ ID NO.5.

[0012] Preferably, the operon is CarO8, and its nucleotide sequence is SEQ ID NO.1.

[0013] The promoter 1 and promoter 2 may be the same or different: As an example, when the promoter 1 and promoter 2 are the same, they can both be hCMVmin, and the nucleotide sequence thereof is SEQ ID NO.2; As an example, when the promoter 1 and the promoter 2 are different, the promoter 1 is hCMVmin, and the promoter 2 is CMV-T6, and CMV-T6 has a nucleotide sequence as shown in SEQ ID NO.3.

[0014] In some specific embodiments, the self-activating small molecule inducible system includes, in sequence on the same vector: expression element 1-expression element 2-promoter 1-operator-promoter 2-expression element 3.

[0015] The self-activating small molecule induction system of the present invention can be used for the induced expression of various proteins, that is, the expression element 3 can be adaptively adjusted as needed, and the expression element 3 can express any one or more target proteins. As those skilled in the art understand, the expression element 3 can include any one or more expression genes.

[0016] Optionally, the expression element 3 also includes a reporter gene for detecting the expression level of the target gene.

[0017] As an example, the present invention provides several feasible types of expression genes, including but not limited to: any one or more of reporter genes, virus packaging genes and foreign protein expression genes.

[0018] Preferably, the reporter gene includes but is not limited to: GFP, Luciferase, mCherry and the like.

[0019] Preferably, the viral packaging gene includes but is not limited to: lentiviral packaging gene, AAV packaging gene, etc.

[0020] Preferably, the reporter gene is a fluorescent protein gene; the fluorescent protein is EGFP.

[0021] Specifically, the vector is selected from a prokaryotic expression vector or a eukaryotic expression vector, including but not limited to a lentiviral expression vector, an AAV expression vector, and a transposon expression vector.

[0022] Preferably, the vector is a mammalian cell expression vector; more preferably, it is pBSK.

[0023] The present invention further provides a cell comprising the aforementioned self-activating small molecule inducible system.

[0024] Preferably, the cells are mammalian cells.

[0025] The present invention also provides the use of the aforementioned self-activating small molecule induction system or cell in inducing gene expression.

[0026] Specifically, the application is to increase the expression level of the target gene.

[0027] The increasing the expression level of the target gene includes but is not limited to any one or more of the following aspects: increasing gene expression efficiency, increasing the level of expression products, increasing the transcription level, increasing the translation level, and increasing the gene copy number.

[0028] The present invention also provides a method for constructing the aforementioned self-activating small molecule induction system, wherein the construction method at least comprises amplifying the corresponding elements and then inserting them into a vector backbone.

[0029] Specifically, the amplification is to amplify each element separately, or, the amplification is to amplify any one or more of the elements simultaneously.

[0030] Based on the above content, the present invention also provides a method for expressing a target gene, which at least includes using the aforementioned self-activating small molecule induction system or cell to induce expression of the target gene.

[0031] According to the general understanding of those skilled in the art, the method may also include adding inducing molecules to cause a cascade reaction and increase the expression level of the target gene.

[0032] The principle of the self-activating small molecule induction system of the present invention: 1) Principle of green light response system: In existing literature, constitutive promoters are used to drive the expression of activation elements. Monomers cannot activate the expression of target genes in large quantities and are limited to the background level. However, by adding small molecules AdoB12, CarH and VP16 are tetramerized and combined with the operator (8-tandem DNA sequence, CarO8) in the promoter region of the induced gene, which has the effect of gene induction expression.

[0033] 2) Principle of the self-activating small molecule induction system: Based on the green light response system, the operator sequence of CarO8 is also added before CarH and VP16, and the same operator sequence is used to control the activation element and the reporter gene. In this way, the small molecule AdoB12 binds to CarH and VP16, and after tetramerization, it will positively regulate and activate the expression of CarH and VP16, cascade amplify the activation signal, and thus induce the expression of the target gene to a greater extent. This system realizes positive feedback regulation based on the literature, using the same operator to first induce the expression of the activation element and then activate the expression of the target gene, thereby improving the induction efficiency of the target gene, and constructing the relevant sequences into a plasmid vector, which is easier to operate.

[0034] 3) Reduce the background leakage level Principle: This system expects a higher level of target protein expression after induction, and the background level before induction is as low as possible. The core promoter sequence in the current vector structure affects the background level, which has a certain level of driving target protein expression before induction. Therefore, replacing the core promoter sequence can reduce the background leakage expression (the sequence comes from the tet system literature: Loew R, Heinz N, Hampf M, Bujard H, Gossen M. ImprovedTet-responsive promoters with minimized background expression. BMCBiotechnol. 2010 Nov 24;10:81. doi: 10.1186 / 1472-6750-10-81. PMID: 21106052;PMCID: PMC3002914.).

[0035] See the example of the invention technical principle diagram for Figure 1 .

[0036] The beneficial effects of the present invention include the following two aspects: 1. First, a self-activating small molecule induction system was established to enhance the expression of the target gene through positive regulatory feedback.

[0037] 2. Secondly, replace the core promoter sequence to reduce background leakage expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 An example of a schematic diagram of the technical principle of the present invention.

[0039] Figure 2 This is the plasmid map of piggybac-CMV-CarH-VP16 in Example 1.

[0040] Figure 3 This is the plasmid map of PEGFP-CarO8-hCMVmin in Example 1.

[0041] Figure 4 This is the plasmid map of pBSK-CarO8hCMVmin-CarH-VP16 in Example 1.

[0042] Figure 5 This is the plasmid map of pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP in Example 1.

[0043] Figure 6 This is a comparison of cell morphology and fluorescence 72 hours after transfection in Example 1.

[0044] Figure 7-1 and Figure 7-2 The fluorescence ratio comparison results of Example 1 72 hours after transfection.

[0045] Figure 8 The statistical results of the average fluorescence intensity 72 hours after transfection in Example 1 are shown.

[0046] Fig. 9 This is a technical schematic diagram of replacing the core promoter in Example 2.

[0047] Fig.10 This is the plasmid map of pBSK-VP16-CarH-hCMVmin-CarO8-T6-EGFP in Example 2.

[0048] Fig.11 This is a comparison of cell morphology and fluorescence 72 hours after transfection in Example 2.

[0049] Figure 12-1 and Figure 12-2 The fluorescence ratio comparison results of Example 2 72 hours after transfection.

[0050] Fig.13 The statistical results of the average fluorescence intensity 72 hours after transfection in Example 2 are shown.

[0051] Fig.14 This is the plasmid map of pCCL-VP16-CarH-hCMVmin-CarO8-T6-EGFP in Example 3.

[0052] Fig.15 The statistical results of the average fluorescence intensity 96 hours after lentiviral transduction in Example 3 are shown. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0054] The information of some experimental materials used in the examples is shown in Table 1: Table 1

[0055] Those skilled in the art know that the selection of specific experimental materials can be conventionally replaced according to the disclosure of the prior art, so the implementation of the technical solution of the present invention is not limited to the above types. The relevant operations involved in the embodiments of the present invention, unless otherwise specified, are all completed using the kit in Table 1, including but not limited to: when it comes to the gel recovery step, the gel recovery kit in Table 1 is used to refer to the instructions; when it comes to plasmid extraction, the plasmid mini-extraction kit in Table 1 is used to refer to the instructions.

[0056] In the present invention, "plasmid vector", "plasmid" and "vector" have the same or similar meanings.

[0057] In the present invention, "LB" refers to the name of a culture medium used to culture bacteria.

[0058] In the present invention, "DMEM (dulbecco's modified eagle medium)" is a culture medium containing various amino acids and glucose, which is developed on the basis of MEM culture medium and is a common culture medium type in the art.

[0059] In the present invention, "FBS" refers to fetal bovine serum, which is an important product widely used by researchers in scientific research and industry as a cell culture medium. It is an important basic growth medium and rich in nutrients. It is a rich source of proteins and growth factors that support cell growth.

[0060] In the present invention, "DPBS" is the abbreviation of Dulbecco's Phosphate-Buffered Saline, which is Dulbecco's Phosphate Buffered Saline in Chinese. It is one of the commonly used balanced salt solutions in biochemical research, and its main components include NaCl, KCl, KH2PO4, Na2HPO4, etc.

[0061] In the present invention, mean fluorescence intensity is also called MFI (Mean Fluorescence Intensity), which is a unit of measurement used to describe the average fluorescence intensity of fluorescent dyes on the surface of cells or particles. The MFI mean fluorescence intensity unit is usually used in experiments such as flow cytometry and fluorescence microscopy.

[0062] In the present invention, XhoI enzyme, NotI enzyme, NheI enzyme, NcoI enzyme, are sometimes abbreviated as XhoI, NotI, NheI, NcoI, and those skilled in the art can understand that they represent corresponding enzymes or restriction sites.

[0063] In the present invention, "LTR" refers to long terminal repeats, which are regulatory elements at both ends of the retroviral genome.

[0064] In the present invention, "activation element" also refers to "Activator", which includes CarH and VP16.

[0065] In the present invention, "Core Insulator" refers to a core insulator, which is used to prevent adjacent control elements from interfering with each other.

[0066] In the present invention, "promoter" refers to a promoter that initiates the transcription of a DNA sequence.

[0067] In the present invention, "Enhancer" refers to an enhancer that enhances the transcription of a DNA sequence.

[0068] In the present invention, "SV40 poly(A) signal" refers to the simian virus 40 polyadenylation signal, which increases the stability of mRNA.

[0069] In the present invention, "AmpR" refers to the ampicillin resistance gene, which is used for the selection of plasmids in Escherichia coli.

[0070] In the present invention, "KanR" refers to the kanamycin resistance gene, which is used for the screening of plasmids in Escherichia coli.

[0071] In the present invention, "ori" refers to the E. coli replication origin, which regulates the rolling circle replication of the entire plasmid in E. coli.

[0072] In the present invention, "f1 ori" refers to the f1 phage replication initiation site, which controls the replication of single-stranded DNA.

[0073] In the present invention, "Core promoter" refers to the core promoter, which initiates the transcription of a DNA sequence.

[0074] In the present invention, "Operator" refers to an operon, which regulates the transcription and expression of a gene.

[0075] In the present invention, "EGFP" is the abbreviation of Enhanced Green Fluorescent Protein, which refers to enhanced green fluorescent protein, and is generally used as a reporter gene.

[0076] In the present invention, "poly A" refers to polyadenylic acid, which has the function of terminating mRNA transcription and protecting mRNA.

[0077] In the present invention, "overlap extension polymerase chain reaction" is also referred to as "overlap extension PCR", which is generally referred to as "overlap PCR" in the art.

[0078] Some plasmid maps in the present invention are produced by software, and the output format is retained. Some of the retained English contents are all contents that can be understood by those skilled in the art in combination with the content of the invention and the definitions of related terms.

[0079] As is well known to those skilled in the art, the process of constructing a vector involves operations such as enzyme cleavage, primer amplification, and enzyme coupling. Therefore, some of the fragments involved in the construction process may retain some non-functional fragments (including but not limited to fragments related to enzyme cleavage sites, primer related fragments, etc.) in most cases. Therefore, in the embodiments, some of the fragments are included in the target fragment, rather than being completely consistent with the target fragment, which can be understood by those skilled in the art.

[0080] Example 1 A self-activating small molecule induction system and its use in inducing target gene expression In this embodiment, different plasmid vectors are constructed to load the self-activating small molecule induction system. After the relevant plasmid vectors are transfected into 293T cells, the induction effect on the expression of the target gene is determined by detecting the fluorescence intensity. Specifically, the following steps are included: 1. Vector Construction The relevant information of the vector constructed in this example is shown in Table 1: Table 1 Carrier information

[0081] The plasmid maps of each vector in Table 1 are shown in Figure 2-Figure 5 , the construction method is as follows: 1. Construction of plasmid piggybac-CMV-CarH-VP16 (sequence number 1): (1) The purchased mammalian cell transposition plasmid (Ubao Bio, VT1663) was double-digested with SpeI and HpaI, and the 4789 bp fragment was recovered from the gel to obtain a linear vector.

[0082] (2) Synthesize the target gene: CarH has the nucleotide sequence shown in SEQ ID NO.6; VP16 has the nucleotide sequence shown in SEQ ID NO.7.

[0083] (3) Three fragments were obtained by PCR amplification using plasmid pCDNA3.1 and the synthetic target genes CarH and VP16 as templates.

[0084] The sequence of fragment 1-1 is SEQ ID NO.8; the sequence of fragment 1-2 is SEQ ID NO.9; and the sequence of fragment 1-3 is SEQ ID NO.10.

[0085] (4) The three fragments were then subjected to overlap extension PCR using CMV-SpeI-F (SEQ ID NO. 11) and VP16-PCR-R (SEQ ID NO. 12) to obtain the final fragment and a fragment with a length of 1920 bp was recovered from the gel.

[0086] (5) The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, and reacted at 50°C for 15 min.

[0087] (6) Transform the ligation product into competent E. coli TransStbl3 and mix gently; place on ice for 20 min, heat shock at 42°C for 45 s, and immediately place on ice for 2 min; add liquid LB without antibiotics and culture at 37°C with shaking for 60 min; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on an LB agar plate containing ampicillin; and culture inverted at 37°C for 16 h.

[0088] (7) Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37°C with shaking for 16 hours; use a plasmid mini-extraction kit (TIANGEN Rapid Plasmid Mini-Extraction Kit) to extract the plasmid. After double restriction digestion with SpeI and HpaI, sequencing is performed to obtain the piggybac-CMV-CarH-VP16 plasmid.

[0089] 2. Construction of plasmid pEGFP-CarO8-hCMVmin (sequence number 2): (1) The purchased plasmid vector pEGFP-C1 (Ubao Bio, VT1118) was double-digested with PciI and AgeI, and the linear vector with a length of 4073 bp was obtained by gel recovery; (2) Synthesizing the target sequence CarO8-hCMVmin (SEQ ID NO.13); (3) Using the synthesized target sequence CarO8-hCMVmin as a template, PCR amplification was performed to obtain fragment 2-1, and the fragment was recovered by gel extraction (SEQ ID NO. 14).

[0090] (4) The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, and reacted at 50°C for 15 min.

[0091] (5) Transform the ligation product into competent E. coli TransStbl3 and mix gently; place on ice for 20 min, heat shock at 42°C for 45 s, and immediately place on ice for 2 min; add liquid LB without antibiotics and culture at 37°C with shaking for 60 min; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on an LB agar plate containing ampicillin; and culture inverted at 37°C for 16 h.

[0092] (6) Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37°C with shaking for 16 hours; extract the plasmid using a plasmid extraction kit. Perform double restriction digestion with PciI and AgeI, and then perform sequencing to obtain the PEGFP-CarO8-hCMVmin-GFP plasmid.

[0093] 3. Construction of plasmid pBSK-CarO8hCMVmin-CarH-VP16 (sequence number 3): (1) The plasmid pBSK (BioWind, 18447) was double-digested with XhoI and NotI, and recovered with a gel recovery kit to obtain a linear vector with a length of 2888 bp: (2) Three fragments were obtained by PCR amplification using piggybac-CMV-CarH-VP16 and PEGFP-CarO8-hCMVmin-GFP as templates, respectively.

[0094] The sequence of fragment 3-1 is SEQ ID NO.15; the sequence of fragment 3-2 is SEQ ID NO.16; and the sequence of fragment 3-3 is SEQ ID NO.17.

[0095] The above three fragments were then subjected to overlap extension PCR to obtain the final fragment and the gel-recovered sequence was SEQ ID NO.18.

[0096] (3) The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, and reacted at 50°C for 15 min.

[0097] (4) Transform the ligation product into competent E. coli TransStbl3 and mix gently; place on ice for 20 min, heat shock at 42°C for 45 s, and immediately place on ice for 2 min; add liquid LB without antibiotics and culture at 37°C with shaking for 60 min; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on an LB agar plate containing ampicillin; and culture inverted at 37°C for 16 h.

[0098] (5) Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37℃ with shaking for 16 hours; use a plasmid extraction kit to extract the plasmid. After double restriction digestion with XhoI and NotI, sequencing is performed to obtain the pBSK-CarO8hCMVmin-CarH-VP16 plasmid.

[0099] 4. Construction of plasmid pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (sequence number 4): (1) Double digest the plasmid pBSK with XhoI and NotI, and recover the plasmid by gel extraction to obtain the vector (refer to step 3 (1)).

[0100] (2) PCR amplification was performed using pCDNA3.1, piggybac-CMV-CarH-VP16 (sequence number 1), and PEGFP-CarO8-hCMVmin-GFP (sequence number 2) plasmids as templates to obtain four fragments, namely: The sequence of fragment 4-1 is SEQ ID NO.19; the sequence of fragment 4-2 is SEQ ID NO.20; the sequence of fragment 4-3 is SEQ ID NO.21; the sequence of fragment 4-4 is SEQ ID NO.22; The above four fragments were then subjected to overlap extension PCR to obtain the final fragment, which was recovered from the gel and has the sequence of SEQ ID NO.23.

[0101] Subsequently, refer to (3)-(5) in step 1 to obtain the pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP plasmid.

[0102] 2. Plasmid Transfection The experimental groups were set up as follows: Control group: piggybac-CMV-CarH-VP16 and pEGFP-CarO8-hCMVmin, the activation region of the vector is not regulated by CarO8, as the control, with the inducer as group 1, and without the inducer as group 2.

[0103] Two experimental groups: pBSK-CarO8hCMVmin-CarH-VP16 and PEGFP-CarO8-hCMVmin, the activation region is regulated by CarO8, the activation vector and the reporter gene vector are two vectors, the inducer is added as group 3, and the inducer is not added as group 4; pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP, the activation region and the reporter gene were constructed on one vector and regulated by the operator sequence CarO8. Group 5 was added with inducer, and group 6 was not added with inducer.

[0104] Leakage expression control group: PEGFP-CarO8-hCMVmin, not induced as Group 7.

[0105] Negative control group: Adherent 293T cells without plasmid transfection and without inducer were used as negative control as group 8.

[0106] The amount of transfection plasmids: 3.34μg / well for dual plasmids (1:1), 1.67μg / well for single plasmids. The transfection reagent was PEI, the amount was 2μL / well, the solution was changed 24h after transfection and AdoB12 coenzyme was added for induction, the amount was 20μM at the final concentration. Flow cytometry was performed 72h after transfection, and the fluorescence ratio and fluorescence intensity of each group were compared.

[0107] The specific steps are as follows: a) Adherent 293T cells were seeded in a 6-well plate one day in advance, with approximately 6×10 cells per well. 5 10 cells, add 2 mL DMEM (containing 10% FBS), and place in a 37°C, 5% CO2 incubator for static culture.

[0108] b) After the cells have grown for 18-24 hours, observe their confluence under a microscope. Transfection is optimal when the confluence is 60%-90%.

[0109] c) Thaw the plasmid and transfection reagent needed for transfection at room temperature for later use.

[0110] d) Prepare a sterile cryotube, add 100 μL DMEM (without FBS), add the calculated plasmid, mix well and let stand for 5 minutes, and mark it as solution A.

[0111] e) Prepare a sterile centrifuge tube, add 100 μL DMEM (without FBS) and 2 μL PEI transfection reagent, mix well and let stand for 5 min. Label it as solution B.

[0112] f) Add solution B to solution A, mix well and let stand for 10-15 minutes. The transfection complex is now ready.

[0113] g) The transfection complex was slowly added dropwise into the 6-well plate and placed in a 37°C, 5% CO2 incubator for static culture.

[0114] h) After culturing for 24 hours, discard the culture medium containing the transfection complex in the 6-well plate, add new transfection medium DMEM (containing 10% FBS), add AdoB12 coenzyme induction, protect from light, and place in a carbon dioxide incubator at 37°C and 5% CO2 concentration for static culture.

[0115] i) 72 h after transfection, cells were harvested and analyzed by flow cytometry.

[0116] 3. GFP fluorescence detection and analysis The 293T cells in each well were digested and neutralized, centrifuged to remove the supernatant, and resuspended in DPBS for flow cytometry detection. Figure 6 , Figure 7-1 , Figure 7-2 and Figure 8 shown.

[0117] The results showed that the mean fluorescence intensity (MFI) of each group after induction was significantly higher than that before induction. The MFI of group 5 was 3.6 times that of group 1. The construction of this self-activation induction system increased the expression of the target protein after induction.

[0118] Example 2 Replacing the core promoter to reduce background expression Considering the complexity of the transfection system with two plasmids, the high leakage expression of the plasmid itself and the low induction level, a promoter with reduced leakage expression was selected to construct the vector and perform a second verification. The technical principle diagram is shown in the figure. Fig. 9 As shown. The following steps are included: 1. Vector Construction Based on the pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (sequence number 4) vector constructed in Example 1, the core promoter of the reporter gene was changed to CMV-T6, and the constructed vector was named pBSK-VP16-CarH-hCMVmin-Car08-T6-EGFP. The plasmid map is shown in Fig.10 The details are as follows: 1. Double digest the plasmid pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (sequence number 4) with NheI and NcoI, and recover the fragment of 5930 bp on gel to obtain the vector; 2. Design primers: T6-NheI-F (SEQ ID NO. 24); T6-NcoI-R (SEQ ID NO. 25); PCR amplification without template and gel recovery were performed to obtain a fragment with the sequence of SEQ ID NO.26.

[0119] 3. Connect the recovered vector and fragment using a seamless cloning kit, 10 μL system, and react at 50°C for 15 min.

[0120] 4. Transform the ligation product into the competent E. coli TransStbl3 and mix gently; place on ice for 20 minutes, heat shock at 42°C for 45 seconds, and immediately place on ice for 2 minutes; add liquid LB without antibiotics and culture at 37°C with shaking for 60 minutes; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on the LB agar plate containing ampicillin; and culture at 37°C inverted for 16 hours.

[0121] 5. Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37℃ with shaking for 16h; use a plasmid extraction kit to extract the plasmid. After double restriction digestion with NheI and NcoI, sequencing and identification were performed to obtain the pBSK-VP16-CarH-hCMVmin-CarO8-T6-EGFP plasmid.

[0122] 2. Plasmid Transfection The experiment was set up as follows: Control group: piggybac-CMV-CarH-VP16 and pEGFP-CarO8-hCMVmin, the activation region of which is not regulated by CarO8, served as controls, with the addition of inducer as Group 1 and without the addition of inducer as Group 2.

[0123] Three experimental groups: pBSK-CarO8hCMVmin-CarH-VP16 and PEGFP-CarO8-hCMVmin, the activation region is regulated by CarO8, the activation vector and the reporter gene vector are two vectors, the inducer is added as group 3, and the inducer is not added as group 4; pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP, the activation region and the reporter gene were constructed on one vector and regulated by the operator sequence CarO8. Group 5 was added with inducer, and group 6 was not added with inducer.

[0124] pBSK-VP16-CarH-hCMVmin-Car08-T6-EGFP, the activation region and the reporter gene were constructed into one vector and regulated by the operator sequence CarO8. The reporter gene promoter was changed to CMV-T6, and the inducer was added to group 7, and the inducer was not added to group 8.

[0125] Negative control group: Adherent 293T cells without plasmid transfection and without inducer were used as negative control as group 9.

[0126] The amount of transfection plasmids: 3.34μg / well for dual plasmids (1:1), 1.67μg / well for single plasmids. The transfection reagent was PEI, the amount was 2μL / well, the solution was changed 24h after transfection and AdoB12 coenzyme was added for induction, the amount was 20μM at the final concentration. Flow cytometry was performed 72h after transfection, and the fluorescence ratio and fluorescence intensity of each group were compared.

[0127] For specific operation steps, please refer to a)-i) in Example 1.

[0128] 3. GFP fluorescence detection and analysis The 293T cells in each well were digested and neutralized, centrifuged to remove the supernatant, and resuspended in DPBS for flow cytometry detection. Fig.11 , Figure 12-1 , Figure 12-2 and Fig.13 shown.

[0129] The results showed that the mean fluorescence intensity (MFI) of each group after induction was significantly higher than that before induction. The MFI of group 5 was 3.4 times that of group 1, and the MFI of group 7 was 2.7 times that of group 1. The self-activation induction system was constructed to improve the expression of the target protein after induction. The MFI of group 8 was 43% of that of group 6, and the background leakage level was reduced by 57%. The expression intensity of group 7 after induction was 18.5 times higher than that of group 8, and the expression intensity of group 5 after induction was 9.8 times higher than that of group 6. After replacing the core promoter CMV-T6, the protein expression multiple after induction was increased compared with the background expression level.

[0130] Example 3 Preparation of lentivirus using a self-activating small molecule induction system The purpose of this example is to prepare a lentiviral plasmid carrying GFP through a self-activating small molecule induction system and package the lentivirus. After the lentivirus is infected and integrated into the target cells, it is induced by small molecules to detect the expression of the target protein.

[0131] 1. Vector Construction Based on the pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (sequence number 4) vector constructed in Example 2, the vector backbone was changed to a lentiviral backbone vector sequence, and the constructed vector was named pCCL-VP16-CarH-hCMVmin-Car08-T6-EGFP. The plasmid map is shown in Fig.14 The details are as follows: 1. Double digest the plasmid pCCL-SIN-cPPT-MCS-RbPA (refer to patent application number CN202210373842.6) with XhoI and KpnI, and recover the fragment of 6253 bp on gel to obtain the vector; 2. Use the template pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (sequence number 4) for PCR amplification and gel recovery to obtain a fragment with the sequence of SEQ ID NO.27.

[0132] 3. Connect the recovered vector and fragment using a seamless cloning kit, 10 μL system, and react at 50°C for 15 min.

[0133] 4. Transform the ligation product into the competent E. coli TransStbl3 and mix gently; place on ice for 20 minutes, heat shock at 42°C for 45 seconds, and immediately place on ice for 2 minutes; add liquid LB without antibiotics and culture at 37°C with shaking for 60 minutes; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on the LB agar plate containing ampicillin; and culture at 37°C inverted for 16 hours.

[0134] 5. Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37℃ with shaking for 16h; use a plasmid extraction kit to extract the plasmid. After double restriction digestion with NheI and NcoI, sequencing and identification were performed to obtain the pCCL-VP16-CarH-hCMVmin-CarO8-T6-EGFP plasmid.

[0135] 2. Virus Packaging WayneLVPro™ HEK293T cells were passaged into corresponding cell shake flasks 48h in advance, and the cell density was 2.0×10 6 -3.0×10 6 The total amount of plasmid was 0.5 μg / million cells, the shuttle plasmid pCCL-VP16-CarH-hCMVmin-CarO8-T6-EGFP of the third-generation lentiviral packaging system, and the auxiliary plasmid LentiHelper TM Gagpol (GenScript Biopharmaceuticals, NF-03), helper plasmid LentiHelper TM Rev (GenScript Biopharmaceuticals, NF-04), helper plasmid LentiHelper TM VSV-G (GenScript Biopharmaceuticals, NF-01) was co-transfected into 293T cells at a mass ratio of 4:2:1:1. The ratio of transfection reagent PEIpro®-HQ (Polyplus, Catalog No.: 301-01L) to plasmid was 1:1 (volume μL: mass μg). The glucose concentration was measured before transfection and adjusted to 6 g / L, and then adjusted every 20-24 hours until the virus was harvested. 20-24 hours after cell transfection, an anti-agglomeration agent (Thermo, Catalog No.: 0010057DG) was added at 1 / 10000 of the transfection volume. 44-52 hours after cell transfection, the supernatant was harvested by centrifugation at 4-8°C and 10,000g for 20 minutes. The prepared virus was named LV-EF1α-VP16-CarH-hCMVmin-CarO8-T6-EGFP.

[0136] 3. Virus infection of 293T cells LV-EF1α-VP16-CarH-hCMVmin-CarO8-T6-EGFP virus infected 293T cells adherent to 6-well plates, mixed and placed in a carbon dioxide incubator for 24 hours. 24 hours after infection, the medium was changed and the cells were evenly divided into two groups for plating, one group without inducer and one group with 20μM AdoB12 coenzyme for induction. GFP protein expression was detected by flow cytometry 72 hours after induction.

[0137] IV. GFP fluorescence detection and analysis The 293T cells in each well were digested and neutralized, centrifuged to remove the supernatant, and resuspended in DPBS for flow cytometry detection. Fig.15 shown.

[0138] The results showed that after lentivirus infection and integration into 293T cells, protein expression could be induced by AdoB12 coenzyme with an induction multiple of 53.68 times.

[0139] The above embodiments are only used to illustrate the present invention, not to limit the present invention. Adjustments and improvements of conventional technical means made by those skilled in the art without departing from the technical concept of the present invention are all within the scope of protection of the present invention.

Claims

1. A self-activating small molecule induction system, characterized in that: For expressing the target gene, the self-activating small molecule induction system includes the following elements on the same vector: Expression element 1 for transcriptional activation, expression element 2 for operator regulation; operator and promoter; expression element 3 of target gene; The promoter includes promoter 1 for promoting expression element 1 and expression element 2 and promoter 2 for promoting expression element 3; promoter 1 and promoter 2 are the same or different; The operon regulates promoter 1 and promoter 2 simultaneously.

2. The self-activating small molecule induction system according to claim 1, characterized in that: The sequence on the same vector includes: expression element 1-expression element 2-promoter 1-operator-promoter 2-expression element 3.

3. The self-activating small molecule induction system according to claim 1, characterized in that: The operon is CarO8, and its nucleotide sequence is SEQ ID NO.

1.

4. The self-activating small molecule induction system according to claim 1, characterized in that: The promoter 1 and promoter 2 are the same, both are hCMVmin, and the nucleotide sequence thereof is SEQ ID NO.2; Alternatively, the promoter 1 and the promoter 2 are different, the promoter 1 is hCMVmin, the promoter 2 is CMV-T6, and CMV-T6 has a nucleotide sequence as shown in SEQ ID NO.

3.

5. The self-activating small molecule induction system according to claim 1, characterized in that: The expression element 1 is derived from the transcriptional activation domain of VP16.

6. The self-activating small molecule induction system according to claim 5, characterized in that: The expression element 1 has a nucleotide sequence as shown in SEQ ID NO.

4.

7. The self-activating small molecule induction system according to claim 1, characterized in that: The expression element 2 is the expression element of CarH.

8. The self-activating small molecule induction system according to claim 7, characterized in that: The expression element 2 has a nucleotide sequence as shown in SEQ ID NO.

5.

9. The self-activating small molecule induction system according to claim 1, characterized in that: The target gene is selected from any one or more of a reporter gene, a virus packaging gene and a foreign protein expression gene.

10. The self-activating small molecule induction system according to claim 9, characterized in that: The expression element 3 also includes a reporter gene for detecting the expression level of the target gene.

11. The self-activating small molecule induction system according to claim 10, characterized in that: The reporter gene includes a fluorescent protein gene; the fluorescent protein includes EGFP.

12. The self-activating small molecule induction system according to claim 1, characterized in that: The vector is selected from a prokaryotic expression vector or a eukaryotic expression vector.

13. The self-activating small molecule induction system according to claim 12, characterized in that: The vector is a mammalian cell expression vector.

14. The self-activating small molecule induction system according to claim 13, characterized in that: The vector is pBSK.

15. A cell comprising the self-activating small molecule inducible system according to any one of claims 1 to 14.

16. The cell according to claim 15, characterized in that The cells are mammalian cells.

17. Use of the self-activating small molecule induction system according to any one of claims 1 to 14 or the cell according to any one of claims 15 to 16 in inducing gene expression.

18. The use according to claim 17, characterized in that The application is to increase the expression level of the target gene.

19. The method for constructing a self-activating small molecule inducible system according to any one of claims 1 to 14, characterized in that: It includes amplifying the corresponding elements and then inserting them into the vector backbone.

20. The construction method according to claim 19, characterized in that: The amplification is to amplify each element separately, or the amplification is to amplify any one or more of the elements simultaneously.

21. A method for expressing a target gene, characterized in that: The method comprises inducing the expression of the target gene using the self-activating small molecule induction system described in any one of claims 1 to 14 or the cell described in any one of claims 15 to 16.

22. The method according to claim 21, characterized in that The method also includes adding inducing molecules to cause a cascade reaction and increase the expression level of the target gene.

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