A self-activating small molecule induction system, cells, and applications and methods in inducing gene expression

Abstract

The present invention provides a self-activating small molecule induction system, cells, and applications and methods in inducing gene expression, belonging to the technical field of genetic engineering. The self-activating small molecule induction system optimizes the component composition based on the green light response system, improves the induction efficiency of the target gene through a cascade reaction, and can reduce the background leakage level, playing an important role in the induction of the target gene and having good application prospects in the field of protein expression and the production and preparation of viral vectors.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly 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 utilizes the regulatory effect of tetracycline antibiotics to control gene expression. This system is generally divided into two parts: The first part is the TetR protein. TetR is a transcriptional repressor that can bind to the TetO sequence on the promoter and inhibit the expression of the target gene. The other part is the induction condition. In the presence of tetracycline (or its derivative doxycycline), TetR binds to tetracycline, thereby releasing the inhibition of TetO and activating the expression of the target gene. Since this system requires the use of antibiotic molecules such as tetracycline for inducible 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 operator sequence CarO, through engineering transformation, it can control gene expression in mammalian cells. Its inducer molecule is vitamin B12 (AdoB12), which is drug-independent and does not require the use of antibiotic drugs (such as tetracycline), avoiding the potential impact of drugs on cell physiology and cytotoxicity. This system has a shorter response time and can quickly initiate or inhibit gene expression. Usually, changes can be observed within a few minutes, while the Tet system may take a longer time to reach a stable expression level. It can be widely applied to 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 in the expression level of the target gene in the prior art. Improving the expression level of the target gene is a key direction for further development based on the current technology. Summary of the Invention

[0005] To solve the problems existing in the prior art, based on the green light response system, the present invention constructs a self-activating small molecule induction system to induce a 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 related sequences are 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, and the self-activating small molecule induction system includes the following elements on the same vector:

[0007] Expression element 1 for transcriptional activation, expression element 2 for operon regulation; operon and promoter; expression element 3 of the target gene;

[0008] The promoter includes promoter 1 for initiating expression element 1 and expression element 2 and promoter 2 for initiating expression element 3; promoter 1 and promoter 2 are the same or different;

[0009] The operon regulates both promoter 1 and promoter 2 simultaneously.

[0010] 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, ACT, and preferably the transcriptional activation domain of VP16.

[0011] Further preferably, the expression element 1 has the nucleotide sequence shown in SEQ ID NO.4.

[0012] The expression element 2 for operon regulation includes but is not limited to any one or more of CarH, LacI, trpG, galR, Gal4, TetR, and preferably CarH.

[0013] In the present invention, the expression element 2 can also be understood as the regulatory gene of the operon, and the regulatory gene regulates the corresponding operon.

[0014] Preferably, the expression element 2 has the nucleotide sequence shown in SEQ ID NO.5.

[0015] Preferably, the operon is CarO8, and its nucleotide sequence is SEQ ID NO.1.

[0016] The promoter 1 and promoter 2 can be the same or different:

[0017] As an example, when the promoter 1 and the promoter 2 are the same, they can both be hCMVmin, and its nucleotide sequence is SEQ ID NO.2;

[0018] 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 the nucleotide sequence shown in SEQ ID NO.3.

[0019] In some specific embodiments, the self-activating small molecule induction system sequentially includes on the same vector: expression element 1 - expression element 2 - promoter 1 - operator - promoter 2 - expression element 3.

[0020] The self-activating small molecule induction system of the present invention can be used for the inducible expression of various proteins, that is, the expression element 3 can be adaptively adjusted according to needs, and the expression element 3 can express any one or more target proteins. As understood by those skilled in the art, the expression element 3 can include any one or more expression genes.

[0021] Optionally, the expression element 3 further includes a reporter gene for detecting the expression level of the target gene.

[0022] 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.

[0023] Preferably, the reporter gene includes but is not limited to: GFP, Luciferase, mCherry, etc.

[0024] Preferably, the virus packaging gene includes but is not limited to: lentivirus packaging gene, AAV packaging gene, etc.

[0025] Preferably, the reporter gene is a fluorescent protein gene; the fluorescent protein is EGFP.

[0026] Specifically, the vector is selected from prokaryotic expression vectors or eukaryotic expression vectors, including but not limited to lentiviral expression vectors, AAV expression vectors, and transposon expression vectors.

[0027] Preferably, the vector is a mammalian cell expression vector; more preferably, it is pBSK.

[0028] The present invention further provides a cell comprising the aforementioned self-activating small molecule induction system.

[0029] Preferably, the cell is a mammalian cell.

[0030] The present invention also provides the use of the aforementioned self-activating small molecule induction system or cells in the induction of gene expression.

[0031] Specifically, the use is to increase the expression level of a target gene.

[0032] The increase in the expression level of the target gene includes, but is not limited to, any one or more of the following aspects: improving gene expression efficiency, increasing the level of the expression product, enhancing the transcription level, raising the translation level, and increasing the gene copy number.

[0033] The present invention also provides a method for constructing the aforementioned self-activating small molecule induction system, which at least includes amplifying the corresponding elements and then inserting them into the vector backbone.

[0034] Specifically, the amplification is to amplify each element separately, or the amplification is to simultaneously amplify any one or more of the elements.

[0035] Based on the foregoing, the present invention also provides a method for expressing a target gene, which at least includes inducing the expression of the target gene by using the aforementioned self-activating small molecule induction system or cells.

[0036] According to the general understanding of those skilled in the art, the method may further include adding an inducer molecule to cause a cascade reaction to increase the expression level of the target gene.

[0037] Principle of the self-activating small molecule induction system of the present invention:

[0038] 1) Principle of the green light-responsive system: In existing literature, a constitutive promoter is used to drive the expression of the activation element. The monomer alone cannot activate the high expression of the target gene and is limited to the background level. However, by adding the small molecule AdoB12, CarH and VP16 are tetramerized, and bind to the operator (8 tandem DNA sequences, CarO8) in the promoter region of the induced gene, achieving the effect of inducing gene expression.

[0039] 2) Principle of the self-activating small molecule induction system: On the basis of the green light-responsive system, the operator sequence of CarO8 is also added in front of CarH and VP16, and the same operator sequence is used to control the activation element and the reporter gene. Thus, when the small molecule AdoB12 binds to CarH and VP16 and achieves tetramerization, it will positively regulate the expression of CarH and VP16, cascading and amplifying the activation signal, and thus inducing the expression of the target gene to a greater extent. This system realizes positive feedback regulation on the basis of the literature. Using the same operator, it first induces the expression of the activation element and then activates the expression of the target gene, improving the induction efficiency of the target gene, and constructing the related sequences into a plasmid vector, making the operation more convenient.

[0040] 3) Principle of reducing background leakage level: The system expects a higher level of target protein expression after induction and as low a background level as possible before induction. Currently, the core promoter sequence in the vector structure affects the background level and drives the expression of the target protein at a certain level before induction. Therefore, replacing the sequence of the core promoter can reduce background leakage expression (the source of this sequence is the tet system literature: Loew R, Heinz N, Hampf M, Bujard H, Gossen M. Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol. 2010 Nov 24;10:81. doi: 10.1186 / 1472-6750-10-81. PMID: 21106052; PMCID: PMC3002914.).

[0041] For an example of the schematic diagram of the invention technology principle, see Figure 1 .

[0042] The beneficial effects of the present invention include the following two aspects:

[0043] 1. First, a self-activating small molecule induction system was established, and the expression of the target gene was improved through positive regulation feedback.

[0044] 2. Second, the sequence of the core promoter was replaced to reduce background leakage expression. Description of the Drawings

[0045] Figure 1 Schematic diagram of the invention technology principle of the present invention.

[0046] Figure 2 Plasmid map of piggybac-CMV-CarH-VP16 in Example 1.

[0047] Figure 3 Plasmid map of PEGFP-CarO8-hCMVmin in Example 1.

[0048] Figure 4 Plasmid map of pBSK-CarO8hCMVmin-CarH-VP16 in Example 1.

[0049] Figure 5 Plasmid map of pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP in Example 1.

[0050] Figure 6 Cell morphology and fluorescence comparison diagram 72 h after transfection in Example 1.

[0051] Figure 7-1 and Figure 7-2 The comparison result of fluorescence ratio 72 h after transfection in Example 1.

[0052] Figure 8 The statistical result of average fluorescence intensity 72 h after transfection in Example 1.

[0053] Figure 9 The schematic diagram of the technology for replacing the core promoter in Example 2.

[0054] Figure 10 The plasmid map of pBSK-VP16-CarH-hCMVmin-CarO8-T6-EGFP in Example 2.

[0055] Figure 11 The cell morphology and fluorescence comparison diagram 72 h after transfection in Example 2.

[0056] Figure 12-1 and Figure 12-2 The comparison result of fluorescence ratio 72 h after transfection in Example 2.

[0057] Figure 13 The statistical result of average fluorescence intensity 72 h after transfection in Example 2.

[0058] Figure 14 The plasmid map of pCCL-VP16-CarH-hCMVmin-CarO8-T6-EGFP in Example 3.

[0059] Figure 15 The statistical result of average fluorescence intensity 96 h after lentiviral transduction in Example 3. Detailed implementation manners

[0060] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are usually carried out under conventional conditions if not otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels if not otherwise specified.

[0061] Some of the experimental material information used in the examples is shown in Table 1 below:

[0062] Table 1

[0063]

[0064] Those skilled in the art are aware that for the selection of specific experimental materials, conventional substitutions can be made according to the disclosures of the prior art. Therefore, the implementation of the technical solution of the present invention is not limited to the above types. In the embodiments of the present invention, for related operations, unless otherwise specified, they are all completed using the kits in Table 1, including but not limited to: when it comes to the gel extraction step, the gel extraction kit in Table 1 is used with reference to the instructions; when it comes to plasmid extraction, the plasmid mini-prep kit in Table 1 is used with reference to the instructions.

[0065] In the present invention, "plasmid vector", "plasmid", and "vector" have the same or similar meanings.

[0066] In the present invention, "LB" refers to the name of a medium used to culture bacterial strains.

[0067] In the present invention, "DMEM (dulbecco's modified eagle medium)" is a medium containing various amino acids and glucose, which is developed on the basis of MEM medium and is a common medium type in this field.

[0068] In the present invention, "FBS" refers to fetal bovine serum, which is widely used as an important product in cell culture by researchers in the scientific research community and the industrial community. It is an important basic growth medium and is rich in nutrients. It is a rich source of proteins and growth factors that support cell growth.

[0069] In the present invention, "DPBS" is the abbreviation of Dulbecco's Phosphate-Buffered Saline, and its Chinese name is Dulbecco's phosphate buffered saline solution. It is one of the commonly used balanced salt solutions in biochemical research, and its main components include NaCl, KCl, KH2PO4, Na2HPO4, etc.

[0070] In the present invention, the mean fluorescence intensity, also known as MFI (Mean Fluorescence Intensity), is a unit of measurement used to describe the average fluorescence intensity of a fluorescent dye 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.

[0071] In the present invention, XhoI enzyme, NotI enzyme, NheI enzyme, NcoI enzyme, sometimes abbreviated as XhoI, NotI, NheI, NcoI, and those skilled in the art can understand that they represent the corresponding enzymes or enzyme cleavage sites.

[0072] In the present invention, "LTR" refers to long terminal repeat, which is a regulatory element at both ends of the retroviral genome.

[0073] In the present invention, the "Activator" also represents "Activator", and includes CarH and VP16.

[0074] In the present invention, "Core Insulator" refers to the core insulator, which is used to block the mutual interference of adjacent regulatory elements.

[0075] In the present invention, "promoter" refers to the promoter, which initiates the transcription of the DNA sequence.

[0076] In the present invention, "Enhancer" refers to the enhancer, which enhances the transcription of the DNA sequence.

[0077] In the present invention, "SV40 poly(A) signal" refers to the simian vacuolating virus 40 polyadenylation signal, which increases the stability of mRNA.

[0078] In the present invention, "AmpR" refers to the ampicillin resistance gene, which is used for the screening of plasmids in Escherichia coli.

[0079] In the present invention, "KanR" refers to the kanamycin resistance gene, which is used for the screening of plasmids in Escherichia coli.

[0080] In the present invention, "ori" refers to the Escherichia coli replication origin, which regulates the rolling circle replication of the entire plasmid in Escherichia coli.

[0081] In the present invention, "f1 ori" refers to the f1 phage replication origin, which controls the replication of single-stranded DNA.

[0082] In the present invention, "Core promoter" refers to the core promoter, which initiates the transcription of the DNA sequence.

[0083] In the present invention, "Operator" refers to the operon, which regulates the transcription and expression of genes.

[0084] In the present invention, "EGFP" is the abbreviation of Enhanced Green Fluorescent Protein, which refers to the enhanced green fluorescent protein and is generally used as a reporter gene.

[0085] In the present invention, "polyA" refers to polyadenylic acid, which has the functions of terminating mRNA transcription and protecting mRNA.

[0086] In the present invention, the "overlap extension polymerase chain reaction", also known as "overlap extension PCR", is generally referred to as "overlap PCR" in the art.

[0087] Some of the plasmid maps in this invention were made using software, and the output format was retained. All the partially retained English content can be understood by those skilled in the art in combination with the invention content and relevant vocabulary interpretations.

[0088] As is well known to those skilled in the art, operations such as restriction enzyme digestion, primer amplification, and ligation are involved in the process of constructing vectors. Therefore, in most cases, some fragments involved in the construction process may retain some non-functional fragments (including but not limited to fragments related to restriction enzyme sites, primer-related fragments, etc.). Therefore, some of the fragments in the examples are in an inclusion relationship with the target fragment, rather than being exactly the same as the target fragment, which can be understood by those skilled in the art.

[0089] Example 1 A self-activating small molecule induction system and for inducing the expression of target genes

[0090] In this example, different plasmid vectors were constructed to load the self-activating small molecule induction system. After the relevant plasmid vectors were transfected into 293T cells, the induction effect on the expression of target genes was determined by detecting the fluorescence intensity. The specific steps are as follows:

[0091] I. Vector construction

[0092] The relevant information of the vectors constructed in this example is shown in Table 2:

[0093] Table 2 Vector information

[0094]

[0095] The plasmid maps of each vector in Table 2 are shown in Figures 2 - 5 , and the construction method is as follows:

[0096] 1. Construction of plasmid piggybac-CMV-CarH-VP16 (Serial number 1):

[0097] (1) The purchased mammalian cell transposon plasmid (Youbao Biotechnology, VT1663) was digested with SpeI and HpaI double restriction enzymes, and a 4789 bp length fragment was recovered by gel electrophoresis to obtain a linear vector.

[0098] (2) Synthesis of the target gene:

[0099] CarH has the nucleotide sequence shown in SEQ ID NO.6; VP16 has the nucleotide sequence shown in SEQ ID NO.7.

[0100] (3) PCR amplification was performed using plasmid pCDNA3.1 and the synthesized target genes CarH and VP16 as templates to obtain 3 fragments.

[0101] The sequence of fragment 1-1 is SEQ ID NO.8; the sequence of fragment 1-2 is SEQ ID NO.9; the sequence of fragment 1-3 is SEQ ID NO.10.

[0102] (4) Then, overlap extension PCR was performed on the 3 fragments using CMV-SpeI-F (SEQ ID NO.11) and VP16-PCR-R (SEQ ID NO.12) to obtain the final fragment, and the fragment with a length of 1920 bp was recovered by gel extraction.

[0103] (5) The recovered vector and fragment were ligated using a seamless cloning kit in a 10 μL system and reacted at 50 °C for 15 min.

[0104] (6) The ligation product was transformed into Escherichia coli competent TransStbl3, gently mixed; incubated on ice for 20 min, heat shocked at 42 °C for 45 s, immediately incubated on ice for 2 min; added with liquid LB without antibiotics, cultured with shaking at 37 °C for 60 min; after discarding part of the supernatant, the bacterial solution was evenly spread on an LB agar plate containing ampicillin using a sterile spreading rod; cultured upside down at 37 °C for 16 h.

[0105] (7) Single colonies were picked and inoculated into liquid LB containing ampicillin, cultured with shaking at 37 °C for 16 h; the plasmid was extracted using a plasmid miniprep kit (TIANGEN rapid plasmid miniprep kit). After double digestion identification with SpeI and HpaI, sequencing identification was carried out to obtain the piggybac-CMV-CarH-VP16 plasmid.

[0106] 2. Construction of plasmid pEGFP-CarO8-hCMVmin (Serial number 2):

[0107] (1) The purchased plasmid vector pEGFP-C1 (Youbao Biotechnology, VT1118) was digested with PciI and AgeI, and the linear vector with a length of 4073 bp was recovered by gel extraction;

[0108] (2) The target sequence CarO8-hCMVmin (SEQ ID NO.13) was synthesized;

[0109] (3) Using the synthesized target sequence CarO8-hCMVmin as a template, PCR amplification was carried out to obtain fragment 2-1 and the fragment was recovered by gel extraction (SEQ ID NO.14).

[0110] (4) The recovered vector and fragment were ligated using a seamless cloning kit in a 10 μL system and reacted at 50 °C for 15 min.

[0111] (5) Transform the ligation product into Escherichia coli competent TransStbl3, mix gently; incubate on ice for 20 min, heat shock at 42 °C for 45 s, and immediately incubate on ice for 2 min; add antibiotic-free liquid LB, shake culture at 37 °C for 60 min; discard part of the supernatant and spread the bacterial solution evenly onto an LB agar plate containing ampicillin with a sterile spreading rod; incubate upside down at 37 °C for 16 h.

[0112] (6) Pick a single colony and inoculate it into liquid LB containing ampicillin, shake culture at 37 °C for 16 h; extract the plasmid using a plasmid mini-prep kit. After double digestion identification with PciI and AgeI, and then sequencing identification, the plasmid PEGF - CarO8 - hCMVmin - GFP is obtained.

[0113] 3. Construction of plasmid pBSK - CarO8hCMVmin - CarH - VP16 (Serial number 3):

[0114] (1) Digest plasmid pBSK (Biofeng, 18447) with XhoI and NotI, and recover it using a gel extraction kit to obtain a linear vector with a length of 2888 bp:

[0115] (2) Perform PCR amplification using piggybac - CMV - CarH - VP16 and PEGF - CarO8 - hCMVmin - GFP as templates respectively to obtain 3 fragments.

[0116] The sequence of fragment 3 - 1 is SEQ ID NO.15; the sequence of fragment 3 - 2 is SEQ ID NO.16; the sequence of fragment 3 - 3 is SEQ ID NO.17.

[0117] Then perform overlap extension PCR on the above 3 fragments to obtain the final fragment and recover the sequence as SEQ ID NO.18 by gel extraction.

[0118] (3) Ligate the recovered vector and the fragment using a seamless cloning kit, in a 10 μL system, react at 50 °C for 15 min.

[0119] (4) Transform the ligation product into Escherichia coli competent TransStbl3, mix gently; incubate on ice for 20 min, heat shock at 42 °C for 45 s, and immediately incubate on ice for 2 min; add antibiotic-free liquid LB, shake culture at 37 °C for 60 min; discard part of the supernatant and spread the bacterial solution evenly onto an LB agar plate containing ampicillin with a sterile spreading rod; incubate upside down at 37 °C for 16 h.

[0120] (5) Pick a monoclonal colony and inoculate it into liquid LB containing ampicillin, and culture it with shaking at 37 °C for 16 h; extract the plasmid using a plasmid mini-prep kit. After double digestion with XhoI and NotI for identification and then sequencing identification, the plasmid pBSK-CarO8hCMVmin-CarH-VP16 was obtained.

[0121] 4. Construction of plasmid pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (No. 4):

[0122] (1) Perform double digestion of plasmid pBSK with XhoI and NotI, and recover the gel to obtain the vector (refer to step (1) of step 3).

[0123] (2) Use plasmids pCDNA3.1, piggybac-CMV-CarH-VP16 (No. 1), and PEGFP-CarO8-hCMVmin-GFP (No. 2) as templates for PCR amplification to obtain 4 fragments, which are respectively:

[0124] 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;

[0125] Then perform overlap extension PCR on the above 4 fragments, obtain the final fragment and recover the gel, and the sequence is SEQ ID NO.23.

[0126] Subsequently, refer to (3)-(5) in step 1 to obtain the plasmid pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP.

[0127] II. Plasmid transfection

[0128] The experimental groups were set as follows:

[0129] Control group:

[0130] piggybac-CMV-CarH-VP16 and pEGFP-CarO8-hCMVmin, where the vector activation region is not regulated by CarO8, are used as controls. The group with the inducer added is group 1, and the group without the inducer added is group 2.

[0131] Two experimental groups:

[0132] 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 group with inducer added is group 3, and the group without inducer added is group 4;

[0133] pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP, the activation region and the reporter gene are constructed on one vector and are jointly regulated by the operator sequence CarO8. The group with inducer added is group 5, and the group without inducer added is group 6.

[0134] Leakage expression control group:

[0135] PEGFP-CarO8-hCMVmin, without induction as group 7.

[0136] Negative control group:

[0137] Adherent 293T cells, without plasmid transfection and without inducer added as the negative control, which is group 8.

[0138] Dosage of transfected plasmids: 3.34 μg / well (1:1) for double plasmids, 1.67 μg / well for single plasmid. The transfection reagent is PEI, with a dosage of 2 μL / well. Replace the medium 24 h after transfection and add AdoB12 coenzyme for induction, with a final concentration of 20 μM. Flow cytometry detection is performed 72 h after transfection, and the fluorescence ratio and fluorescence intensity of each group are compared.

[0139] The specific operation steps are as follows:

[0140] a) Seed adherent 293T cells in a 6-well plate 1 day in advance, with about 6×10 5 cells seeded in each well, add 2 mL of DMEM (containing 10% FBS), and place it in a secondary incubator at 37°C and 5% CO2 for static culture.

[0141] b) Observe the confluence under a microscope 18 - 24 h after cell growth. The best transfection effect occurs when the confluence is between 60% and 90%.

[0142] c) Thaw the plasmids and transfection reagents required for transfection at room temperature for standby.

[0143] d) Prepare 1 sterile cryopreservation tube, add 100 μL of DMEM (without FBS), add the calculated plasmid, mix well and let it stand for 5 min, and label it as solution A.

[0144] e) Prepare 1 sterile centrifuge tube, add 100 μL of DMEM (without FBS) and 2 μL of PEI transfection reagent, mix well and let it stand for 5 min, and label it as solution B.

[0145] f) Add solution B to solution A, mix well and let it stand for 10 - 15 min. The transfection complex is then prepared.

[0146] g) Slowly add the transfection complex drop by drop into a 6 - well plate and place it in an incubator at 37°C with 5% CO₂ for static culture.

[0147] h) After culturing for 24 h, discard the medium containing the transfection complex in the 6 - well plate, add fresh transfection medium DMEM (containing 10% FBS), induce with AdoB12 coenzyme, protect from light, and place it in an incubator at 37°C with 5% CO₂ for static culture.

[0148] i) Harvest the cells after transfection for 72 h for flow cytometry detection.

[0149] III. GFP fluorescence detection and analysis

[0150] Digest and neutralize the 293T cells in each well, centrifuge to remove the supernatant, resuspend with DPBS and then perform flow cytometry detection. The results are as Figure 6 、 Figure 7-1 、 Figure 7-2 and Figure 8 shown.

[0151] The results show that: the mean fluorescence intensity (MFI) after induction in each group is significantly higher than the MFI before induction. The MFI of group 5 is 3.6 times that of group 1. The self - activating induction system is constructed, which improves the expression of the target protein after induction.

[0152] Example 2 Replace the core promoter to reduce the background level of expression

[0153] Considering that the transfection system of two plasmids is complex, the leakage expression of the plasmid itself is high, and the induction level is low, a promoter with reduced leakage expression is selected to construct a vector and perform a second verification. The technical principle diagram is as Figure 9 shown. The steps are as follows:

[0154] I. Vector construction

[0155] Based on the pBSK - VP16 - CarH - hCMVmin - CarO8 - hCMVmin - EGFP (serial number 4) vector constructed in Example 1, change the core promoter of the reporter gene part to CMV - T6, and the constructed vector is named pBSK - VP16 - CarH - hCMVmin - Car08 - T6 - EGFP. The plasmid map is shown in Figure 10 . Specifically as follows:

[0156] 1. The plasmid pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (SEQ ID NO.4) was digested with NheI and NcoI, and the fragment with a length of 5930 bp was recovered by gel extraction to obtain the vector.

[0157] 2. Primers were designed:

[0158] T6-NheI-F (SEQ ID NO.24);

[0159] T6-NcoI-R (SEQ ID NO.25);

[0160] PCR amplification was carried out without a template and the product was recovered by gel extraction to obtain a fragment with the sequence of SEQ ID NO.26.

[0161] 3. The recovered vector and the fragment were ligated using a seamless cloning kit in a 10 μL system at 50 °C for 15 min.

[0162] 4. The ligation product was transformed into Escherichia coli competent cells TransStbl3, gently mixed; incubated on ice for 20 min, heat shocked at 42 °C for 45 s, immediately incubated on ice for 2 min; added liquid LB without antibiotics, cultured with shaking at 37 °C for 60 min; after discarding part of the supernatant, the bacterial solution was evenly spread onto an LB agar plate containing ampicillin using a sterile spreading rod; cultured inverted at 37 °C for 16 h.

[0163] 5. Single colonies were picked and inoculated into liquid LB containing ampicillin, cultured with shaking at 37 °C for 16 h; plasmids were extracted using a plasmid miniprep kit. After digestion with NheI and NcoI for identification and then sequencing identification, the plasmid pBSK-VP16-CarH-hCMVmin-CarO8-T6-EGFP was obtained.

[0164] II. Plasmid Transfection

[0165] The following settings were made for this experiment:

[0166] Control group:

[0167] piggybac-CMV-CarH-VP16 and pEGFP-CarO8-hCMVmin. The activation region of this vector is not regulated by CarO8. As a control, the group with an inducer was set as group 1, and the group without an inducer was set as group 2.

[0168] Three experimental groups:

[0169] 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. Group 3 is with inducer added, and group 4 is without inducer added.

[0170] pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP, the activation region and the reporter gene are constructed on one vector and are jointly regulated by the operator sequence CarO8. Group 5 is with inducer added, and group 6 is without inducer added.

[0171] pBSK-VP16-CarH-hCMVmin-Car08-T6-EGFP, the activation region and the reporter gene are constructed on one vector and are jointly regulated by the operator sequence CarO8. The promoter of the reporter gene is changed to CMV-T6. Group 7 is with inducer added, and group 8 is without inducer added.

[0172] Negative control group:

[0173] Adherent 293T cells are not transfected with plasmids and no inducer is added as the negative control, which is group 9.

[0174] Dosage of transfected plasmids: 3.34 μg / well (1:1) for double plasmids, 1.67 μg / well for single plasmid. The transfection reagent is PEI with a dosage of 2 μL / well. The culture medium is changed 24 h after transfection and coenzyme AdoB12 is added for induction with a final concentration of 20 μM. Flow cytometry detection is performed 72 h after transfection to compare the fluorescence ratio and fluorescence intensity of each group.

[0175] The specific operation steps refer to a)-i) in Example 1.

[0176] III. GFP fluorescence detection and analysis

[0177] After digesting and neutralizing the 293T cells in each well, the supernatant is removed by centrifugation, and the cells are resuspended with DPBS for flow cytometry detection. The results are as Figure 11 、 Figure 12-1 、 Figure 12-2 and Figure 13 shown.

[0178] 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-activating induction system was constructed, which improved 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 expression multiple of the protein after induction was increased compared with the background expression level.

[0179] Example 3 Preparation of Lentivirus Using a Self-Activating Small Molecule Induction System

[0180] The purpose of this example is to prepare a lentiviral plasmid carrying GFP using a self-activating small molecule induction system and package lentivirus. Verify that after the lentivirus infects and integrates into the target cells, it is induced by small molecules, and detect the expression level of the target protein.

[0181] I. Vector Construction

[0182] Based on the vector pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (sequence number 4) constructed in Example 2, the vector backbone was changed to the lentiviral backbone vector sequence, and the constructed vector was named pCCL-VP16-CarH-hCMVmin-Car08-T6-EGFP. The plasmid map is shown in Figure 14 . Specifically as follows:

[0183] 1. The plasmid pCCL-SIN-cPPT-MCS-RbPA (refer to the patent with application number CN202210373842.6) was digested with XhoI and KpnI double enzymes, and a 6253 bp length fragment was recovered by gel electrophoresis to obtain the vector;

[0184] 2. Using the template pBSK-VP16-CarH-hCMVmin-CarO8-hCMVmin-EGFP (sequence number 4) for PCR amplification and gel recovery, a fragment was obtained, and the sequence was SEQ ID NO.27.

[0185] 3. The recovered vector and fragment were ligated using a seamless cloning kit, with a 10 μL system and a reaction at 50 °C for 15 min.

[0186] 4. Transform the ligation product into Escherichia coli competent TransStbl3, gently mix; incubate on ice for 20 min, heat shock at 42 °C for 45 s, immediately incubate on ice for 2 min; add liquid LB without antibiotics, culture with shaking at 37 °C for 60 min; discard part of the supernatant and then evenly spread the bacterial liquid onto an LB agar plate containing ampicillin using a sterile spreading rod; incubate upside down at 37 °C for 16 h.

[0187] 5. Pick a single colony and inoculate it into liquid LB containing ampicillin, culture with shaking at 37 °C for 16 h; use a plasmid miniprep kit to extract the plasmid. After double digestion identification with NheI and NcoI, then perform sequencing identification to obtain the plasmid pCCL-VP16-CarH-hCMVmin-CarO8-T6-EGFP.

[0188] II. Virus packaging

[0189] WayneLVPro™ HEK293T cells are passaged into the corresponding cell culture flask 48 h in advance, and transfection is carried out when the cell density reaches 2.0×10 6 -3.0×10 6 cells / mL. The total amount of plasmid is 0.5 μg per million cells. The shuttle plasmid pCCL-VP16-CarH-hCMVmin-CarO8-T6-EGFP of the third-generation lentiviral packaging system, the helper plasmid LentiHelper TM Gagpol (GenScript ProBio, NF-03), the helper plasmid LentiHelper TM Rev (GenScript ProBio, NF-04), the helper plasmid LentiHelper TM VSV-G (GenScript ProBio, NF-01) are co-transfected into 293T cells according to the mass ratio of 4:2:1:1. The ratio of the transfection reagent PEIpro®-HQ (Polyplus, product number: 301-01L) to the plasmid is 1:1 (volume μL: mass μg). Measure the glucose concentration before transfection and adjust it to 6 g / L, and then adjust it every 20 - 24 h until virus harvesting. Add an anti-clumping agent (Thermo, product number: 0010057DG) at one-thousandth of the transfection volume 20 - 24 h after cell transfection. Centrifuge at 4 - 8 °C and 10000 g for 20 min to harvest the supernatant 44 - 52 h after cell transfection. The prepared virus is named LV-EF1α-VP16-CarH-hCMVmin-CarO8-T6-EGFP.

[0190] III. Virus infection of 293T cells

[0191] The 6-well plate adherent 293T cells were infected with LV-EF1α-VP16-CarH-hCMVmin-CarO8-T6-EGFP virus. After mixing, they were placed in a carbon dioxide incubator for 24 h. The medium was changed 24 h after infection. The cells were evenly divided into two groups for plating. One group was without inducer, and the other group was induced with 20 μM AdoB12 coenzyme. The expression of GFP protein was detected by flow cytometry 72 h after induction.

[0192] IV. GFP fluorescence detection and analysis

[0193] After digesting and neutralizing the 293T cells in each well, the supernatant was removed by centrifugation, and the cells were resuspended with DPBS for flow cytometry detection. The results are as Figure 15 shown.

[0194] The results showed that after the lentivirus infection and integration into 293T cells, the protein expression could be induced by AdoB12 coenzyme, and the induction fold was 53.68 times.

[0195] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Any adjustment and improvement of conventional technical means made by those skilled in the art without departing from the technical concept of the present invention are within the scope of protection of the present invention.

Claims

1. A self-activating small molecule induction system, characterized in that, For expression of a target gene, the self-activating small molecule induction system includes the following elements on the same vector, in the following order: Expression element 1 - Expression element 2 - Promoter 1 - Operator - Promoter 2 - Expression element 3; The expression element 1 is derived from the transcriptional activation domain of VP16, with a nucleotide sequence of SEQ ID NO.4, and is used for transcriptional activation; The expression element 2 is the expression element of CarH, with a nucleotide sequence of SEQ ID NO.5, and is used to regulate the operator CarO8; The expression element 3 is used to express the target gene; The promoter 1 is used to initiate expression element 1 and expression element 2; The promoter 2 is used to initiate expression element 3; The operator is CarO8, with a nucleotide sequence of SEQ ID NO.1, and regulates both promoter 1 and promoter 2 simultaneously; The promoter 1 and promoter 2 are the same or different; When the promoter 1 and promoter 2 are the same, both are hCMVmin, with a nucleotide sequence of SEQ ID NO.2; Alternatively, when the promoter 1 and promoter 2 are different, the promoter 1 is hCMVmin, and the promoter 2 is CMV-T6, and the nucleotide sequence of CMV-T6 is SEQ ID NO.

3.

2. The self-activating small molecule induction system according to claim 1, wherein The target gene is selected from any one or more of a reporter gene, a viral packaging gene, and an exogenous protein expression gene.

3. The self-activating small molecule induction system according to claim 2, wherein The expression element 3 further includes a reporter gene for detecting the expression level of the target gene.

4. The self-activating small molecule induction system according to claim 3, wherein The reporter gene includes a fluorescent protein gene; the fluorescent protein includes EGFP.

5. The self-activating small molecule induction system according to claim 1, wherein The vector is a eukaryotic expression vector.

6. The self-activating small molecule induction system according to claim 5, wherein The vector is a mammalian cell expression vector.

7. The self-activating small molecule induction system according to claim 6, characterized in that, The vector is pBSK.

8. A cell comprising the self-activating small molecule induction system according to any one of claims 1-7.

9. The cell according to claim 8, characterized in that, The cell is a mammalian cell.

10. Use of the self-activating small molecule induction system according to any one of claims 1-7 or the cell according to any one of claims 8-9 in inducing gene expression, characterized in that, The application is for non-disease diagnosis and treatment.

11. The application according to claim 10, wherein The application is to increase the expression level of the target gene.

12. Method for constructing a self-activating small molecule induction system according to any one of claims 1-7, characterized in that, Including inserting into the vector backbone after amplifying the corresponding elements.

13. The construction method according to claim 12, wherein The amplification is to amplify each element separately, or, the amplification is to amplify any one or more of the elements simultaneously.

14. A method for expressing a target gene, characterized in that, The method includes inducing the expression of the target gene using the self-activating small molecule induction system according to any one of claims 1-7 or the cell according to any one of claims 8-9, and the method is not a disease diagnosis and treatment method.

15. The method according to claim 14, wherein The method further includes adding an inducer molecule to cause a cascade reaction and increase the expression level of the target gene.

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