Improved expression-inducing system and use thereof

Abstract

A transcription activating protein TraR of a TraR / 3-oxo-C8-HSL expression-inducing system. The transcription activating protein TraR has a transcription activating domain, and the transcription activating domain contains an NF-κB p65 sequence and a heat shock factor 1 sequence. Further provided are a nucleic acid encoding the transcription activating protein TraR, a cell and an expression-inducing system containing the transcription activating protein TraR, and a method for using the expression-inducing system to regulate the expression of a target nucleic acid.

Description

Improved inducible expression system and its application

[0001] Priority information

[0002] This application claims priority to Chinese patent application number 2024102055962, filed with the China Patent Office on February 23, 2024, entitled “Improved Inducible Expression System and Its Application,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to an improved inducible expression system, in particular, to an improved transcriptional activator protein, a nucleic acid encoding the same, a cell or an inducible expression system comprising the same, and a method for regulating the expression of a target nucleic acid using the inducible expression system. Background Art

[0004] Regulation of gene expression can be achieved by controlling several steps, including transcription, RNA splicing, translation, and post-translational modification. Regulating specific genes or target nucleic acid fragments during the transcription step is the most widespread and effective method of expression regulation. Inducible expression systems, which control the on / off transcription and expression of specific genes and nucleic acid fragments within cells through external factors, play a key role in biopharmaceuticals, gene therapy, cell therapy, and other fields. These inducible expression systems typically consist of two components: 1) a specific nucleic acid sequence upstream of the 5' end of the target nucleic acid fragment, which can bind to a transcriptional activator or repressor to regulate transcription of the target nucleic acid fragment, such as a nucleic acid sequence containing an operator (hereinafter referred to as a "response element"); and 2) a regulatory protein that can bind to or dissociate from the specific nucleic acid sequence under external control and has transcriptional activation or repression functions. These external factors can be environmental factors or substances involved in biological processes, such as metabolites or artificial compounds. The transcriptional activity of the target nucleic acid fragment under induced conditions and the leaked transcriptional activity under non-induced conditions are two important indicators for evaluating the quality of inducible expression systems. A good inducible expression system needs to have high induced expression activity and low leaky expression activity.

[0005] The TraR / N-(3-oxo-octanoyl)-L-homoserine lactone (hereinafter referred to as "3-oxo-C8-HSL") inducible expression system (hereinafter referred to as the "TraR / 3-oxo-C8-HSL inducible expression system" or "TraR inducible expression system") is derived from the quorum sensing system of Agrobacterium tumefaciens (see Fuqua, WC et al. "Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators." Journal of bacteriology vol. 176, 2 (1994): 269-75, which is incorporated herein by reference). TraR is a transcriptional regulatory protein belonging to the LuxR family. Its property of binding to 3-oxo-C8-HSL has been used to develop a novel inducible expression system (see Neddermann, Petra et al. "A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR." EMBO reports vol. 4, 2 (2003): 159-65, which is incorporated herein by reference). The system comprises: a regulatory sequence comprising a response element (hereinafter referred to as TraRRE) containing a tra box sequence (e.g., 1-7 copies) and a minimal promoter sequence containing a TATA box; and a transcriptional activator protein TraR that can bind to the response element in the presence of the inducer 3-oxo-C8-HSL or a derivative or functional analog thereof, wherein TraR comprises three domains: a DNA binding domain, a ligand binding domain, and a transcriptional activation domain, wherein the transcriptional activation domain adopts the sequence of the mammalian NF-κB family p65 protein (amino acids 287-551). The transcription of the target nucleic acid fragment connected to the response element TraRRE containing the tra box sequence is regulated by adding 3-oxo-C8-HSL or its derivatives or functional analogs.Theoretically, in the absence of 3-oxo-C8-HSL or its derivatives or functional analogs, the transcriptional regulatory protein TraR will not bind to the response element TraRRE, thereby inhibiting the transcription of the downstream regulated target nucleic acid fragment; while in the presence of 3-oxo-C8-HSL or its derivatives or functional analogs, the transcriptional regulatory protein TraR can bind to the response element TraRRE, thereby allowing the transcription of the downstream regulated target nucleic acid fragment.

[0006] Currently, methods exist for regulating target gene expression using the TraR inducible expression system. However, when used in mammalian cells, the induced expression activity under the control of this system is significantly lower than that under the control of common constitutively active promoters. Typically, in an inducible expression system, enhancing the expression activity of the regulated target nucleic acid fragment under induced conditions also correspondingly increases its leaky expression activity under non-induced conditions. Common strategies for controlling leaky expression activity under non-induced conditions, such as reducing the affinity of the transactivator for the operator under inducible conditions through point mutations or reducing the basal activity of a minimal promoter downstream of the operator, can affect the maximum expression activity after induction. This results in most inducible expression systems reducing the expression activity under induced conditions in order to limit leaky expression activity under non-induced conditions. Therefore, there is a need for an improved TraR / 3-oxo-C8-HSL inducible expression system that can achieve significantly improved induced expression activity without significantly increasing leaky expression activity. Summary of the Invention

[0007] The present invention discovered that by optimizing the transcriptional activator TraR in the TraR / 3-oxo-C8-HSL inducible expression system, the induced expression activity under the control of this system can be greatly improved while limiting leaky expression. The optimization includes adding the heat shock factor 1 (HSF1) sequence to the original transcriptional activation domain (p65) of TraR to obtain a new transcriptional activation domain (p65-HSF1).

[0008] The present invention also found that by constructing a composite inducible expression system based on the TraR / 3-oxo-C8-HSL inducible expression system, the ratio of the induced expression amount to the leaked expression amount of the regulated nucleic acid sequence can be further improved.

[0009] In a first aspect, the present disclosure provides a transcriptional activator protein TraR of a TraR / 3-oxo-C8-HSL inducible expression system, wherein the transcriptional activator protein TraR has a transcriptional activation domain, and the transcriptional activation domain comprises an NF-κB p65 sequence and a heat shock factor 1 sequence. In one example, the NF-κB p65 sequence is a mammalian NF-κB p65 sequence. In one example, the NF-κB p65 sequence is a human NF-κB p65 sequence. In one example, the NF-κB p65 sequence is as shown in 1-798 bp of SEQ ID NO:36. In one example, the HSF1 sequence is a mammalian HSF1 sequence. In one example, the HSF1 sequence is a human HSF1 sequence. In one example, the HSF1 sequence is as shown in 571-942 bp of SEQ ID NO:39. In one example, the transcriptional activation domain comprises the sequence of SEQ ID NO: 40, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In one example, the transcriptional activation domain comprises the sequence of SEQ ID NO: 40. In one example, the transcriptional activator protein TraR has the sequence of SEQ ID NO: 38, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In one example, the transcriptional activator protein TraR has the sequence of SEQ ID NO: 38.

[0010] In a second aspect, the present disclosure provides a nucleic acid encoding the transcriptional activator protein TraR according to the first aspect above. In one example, the sequence of the nucleic acid is a codon-optimized sequence. In one example, the nucleic acid has a sequence as shown in SEQ ID NO: 37 or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In one example, the nucleic acid has a sequence as shown in SEQ ID NO: 37.

[0011] It should be noted that, for nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0012] In a third aspect, the present disclosure provides a cell comprising the transcriptional activator protein TraR according to the first aspect or the nucleic acid according to the second aspect. In one example, the cell is a eukaryotic cell, comprising at least one of a fungus, an insect cell, a plant cell, and a mammalian cell. In one example, the cell is a mammalian cell, including a 293T cell, a BHK cell, a CHO cell, a NSO cell, or a COS cell. In one example, the cell is a human cell, for example, a 293T cell.

[0013] In a fourth aspect, the present disclosure provides an inducible expression system, comprising the transcriptional activator protein TraR according to the first aspect, the nucleic acid according to the second aspect, or the cell according to the third aspect. In one example, the inducible expression system is a TraR / 3-oxo-C8-HSL inducible expression system, further comprising a regulatory sequence comprising a response element comprising (e.g., 2, 3, 4, 5, 6, 7, or more copies of) a tra box sequence, in one example, the response element having the sequence shown in SEQ ID NO: 25.

[0014] In a fifth aspect, the present disclosure provides a composite inducible expression system, comprising: a regulatory sequence comprising a first response element comprising a tra box sequence, a minimal promoter sequence comprising a TATA box, and a second response element located downstream of the TATA box capable of binding to a transcriptional repressor protein; and the transcriptional activator protein TraR according to the first aspect above, and the transcriptional repressor protein. In one example, the first response element comprises 2, 3, 4, 5, 6, 7, or more copies of the tra box sequence. In one example, the first response element has the sequence set forth in SEQ ID NO: 25. In one example, the second response element capable of binding to a transcriptional repressor protein is: a CuO operator capable of binding to a transcriptional repressor protein CymR, a LacO operator capable of binding to a transcriptional repressor protein LacI, or a PadRO (RC) operator capable of binding to a transcriptional repressor protein PadR. In one example, the transcriptional repressor protein is a transcriptional repressor protein CymR, a transcriptional repressor protein LacI, or a transcriptional repressor protein PadR. In one example, the CuO operator has the sequence of SEQ ID NO:31. In one example, the LacO operator has the sequence of SEQ ID NO:29. In one example, the PadRO (RC) operator has the sequence of SEQ ID NO:30. In one example, the second response element is located 10 to 100 bp downstream of the TATA box sequence. In one example, the second response element is located 30 to 50 bp downstream of the TATA box sequence. In one example, the second response element is located 50 bp downstream of the TATA box sequence. In one example, the regulatory sequence of the composite inducible expression system has the sequence set forth in SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In one example, the regulatory sequence of the complex inducible expression system has a sequence shown in SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28. In one example, the regulatory sequence further comprises a spliceable intron sequence located between the downstream of the second response element and the upstream of the regulated nucleic acid sequence.In one example, the spliceable intron sequence is any intron sequence capable of RNA splicing in mammalian cells, including but not limited to: rabbit β-globin intron, hybrid intron derived from human β-globulin and immunoglobulin heavy chain intron, EF-1α intron A, SV40 intron, hybrid intron derived from adenovirus and immunoglobulin heavy chain intron, modified human cytomegalovirus intron, hybrid intron derived from chicken β-actin (CBA) and minute virus of mice (MMV) intron, chimera derived from chicken β-actin and rabbit β-globulin intron, and mP1 intron; or any intron of any gene of any eukaryote; or an artificial intron designed based on intron splicing rules. In one example, the intron is human β-globin intron (BGI).

[0015] In a sixth aspect, the present disclosure provides a method for regulating nucleic acid expression in eukaryotic cells, the method comprising using the transcription activator protein TraR according to the first aspect above, the nucleic acid according to the second aspect above, the cell according to the third aspect above, the inducible expression system according to the fourth aspect above, or the composite inducible expression system according to the fifth aspect above.

[0016] It should be noted that the eukaryotic cells described herein are not particularly limited. The eukaryotic cells can include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as armyworms, plant cells such as tobacco, and mammalian cells such as 293T cells, BHK cells, CHO cells, COS cells, and myeloma cells. In one embodiment, the eukaryotic cells described herein are preferably mammalian cells, including 293T cells, BHK cells, CHO cells, NSO cells, or COS cells, and more preferably human cells, such as 293T cells.

[0017] In a seventh aspect, the present disclosure provides a method for regulating the expression of a nucleic acid sequence at at least four expression levels, the method comprising using a composite inducible expression system, the composite inducible expression system comprising a regulatory sequence, the regulatory sequence comprising at least a first response element and a second response element, and a minimal promoter sequence comprising a TATA box located between the two response elements, the first response element being capable of binding to its transcriptional activator protein in the presence of a first inducer, and the second response element being capable of dissociating from its transcriptional repressor protein in the presence of a second inducer, wherein a first expression level is achieved in the absence of any inducer, a second expression level is achieved in the absence of the second inducer without the addition of the first inducer, a third expression level is achieved in the absence of the first inducer without the addition of the second inducer, and a fourth expression level is achieved in the presence of both the first inducer and the second inducer. In one example, the first response element is such a response element that, in the absence of the first inducer, the transcriptional activator does not bind to the first response element, thereby inhibiting transcription of the downstream regulated target nucleic acid fragment, and in the presence of the first inducer, the transcriptional activator is capable of binding to the first response element, thereby allowing transcription of the downstream regulated target nucleic acid fragment. In one example, the first response element is a response element containing a tra box sequence. In one example, the transcriptional activator protein capable of binding to the first response element is the transcriptional activator protein TraR according to the first aspect above. In one example, the second response element is such a response element that, in the absence of a second inducer, the transcriptional repressor protein binds to the second response element, thereby inhibiting the transcription of the downstream regulated target nucleic acid fragment, and in the presence of the second inducer, the transcriptional repressor protein dissociates from the second response element, thereby allowing the transcription of the downstream regulated target nucleic acid fragment. In one example, the second response element is a CuO operator capable of binding to the transcriptional repressor protein CymR, a LacO operator capable of binding to the transcriptional repressor protein LacI, or a PadRO (RC) operator capable of binding to the transcriptional repressor protein PadR.

[0018] In an eighth aspect, the present disclosure provides a nucleic acid sequence comprising a TraR response element sequence containing a tra box sequence, a minimal promoter sequence comprising a TATA box, and a CuO operator sequence downstream of the TATA box that is capable of binding to the transcriptional repressor protein CymR. In one example, the CuO operator sequence is spaced 40 to 60 bp from the TATA box. In another example, the CuO operator sequence is spaced 50 bp from the TATA box.

[0019] In a ninth aspect, the present disclosure provides a vector, a cell or an inducible expression system, wherein the vector, the cell or the inducible expression system comprises the nucleic acid sequence described in the eighth aspect.

[0020] By using the transcriptional activator TraR of the present invention and the TraR / 3-oxo-C8-HSL inducible expression system or the composite inducible expression system comprising the same, the regulated target nucleic acid can greatly improve the inducible expression activity under the control of the system without significantly increasing the leaky expression.

[0021] In the tenth aspect, the present disclosure provides a pharmaceutical composition, which includes the transcription activator protein TraR described in the first aspect, the nucleic acid described in the second aspect, the cell described in the third aspect, the inducible expression system described in the fourth aspect, the composite inducible expression system described in the fifth aspect, the nucleic acid sequence described in the eighth aspect, or the vector, cell or inducible expression system described in the ninth aspect, and the target gene. It should be noted that the "target gene" described in this application refers to the exogenous gene that the expression system and / or vector needs to carry, and the exogenous gene enters the target cell after transfection and / or transformation of the cell, and realizes the expression of the target gene in the target cell. When the expression of the target gene in the target cell can achieve a direct or indirect therapeutic effect, the pharmaceutical composition formed by the above-mentioned substances achieves the effect of targeting the target gene into the target cell, thereby realizing targeted treatment of the disease.

[0022] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 shows the effects of the transcriptional activator of the optimized TraR inducible expression system on the induced expression level ( FIG1A ) and the non-induced leaky expression level ( FIG1B ) of the target nucleic acid fragment according to one embodiment of the present disclosure.

[0024] FIG2 shows the effects of a single inducible expression system and a composite inducible expression system on the induced expression amount and the non-induced leakage expression amount of a target nucleic acid fragment according to one embodiment of the present disclosure.

[0025] FIG3 shows the graded expression activities of the composite inducible expression system under different induction combinations according to one embodiment of the present disclosure. DETAILED DESCRIPTION

[0026] The following examples are provided to illustrate the technical solutions of the present invention, and should not be considered as limiting the scope and spirit of the present invention.

[0027] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0028] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0029] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0030] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0031] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0032] As used herein, the term "at least 70% identity" refers to a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to its corresponding reference sequence, including amino acid sequences, nucleic acid sequences, fusion sequences of DNA and RNA, or fusion sequences of amino acids and nucleic acids.

[0033] As used herein, the term "nucleotide" refers to ribonucleotides, deoxynucleotides or modified forms of either type of nucleotide, and combinations thereof.

[0034] As used herein, the term "transformation" or "transfection" refers to the introduction of a nucleic acid (eg, a vector) into a cell by various techniques known in the art.

[0035] The transcription repressor protein CymR (cysteine ​​metabolism repressor protein) and Cumate operator (CuO) involved in the present disclosure are derived from the Cumate Inducible System. The system is derived from the p-cym operon of Pseudomonas putida, including the operator CuO downstream of the TATA box, the inducer Cumate or its derivative or functional analogue, and the repressor protein CymR protein capable of binding to the operator CuO. In the absence of Cumate, the CymR protein binds to the operator CuO to inhibit the transcription of the downstream regulated target nucleic acid fragment; when the CymR protein binds to Cumate, its affinity to the operator CuO decreases and separates, and the transcription of the downstream regulated target nucleic acid fragment is no longer inhibited. For example, the operator CuO is connected between the constitutively active promoter sequence and the regulated target nucleic acid fragment, and the CymR repressor protein is constitutively expressed in the cell. When the inducer Cumate or its derivatives are absent in the culture medium, the expressed CymR protein binds to the operator CuO, thereby inhibiting the transcription of the regulated target nucleic acid fragment; when the above-mentioned inducer is added to the culture medium, the CymR protein bound to Cumate or its derivatives separates from the operator CuO, and the transcription of the regulated target nucleic acid fragment is no longer inhibited (see Mullick, A. et al. (2006). "The cumate gene-switch: a system for regulated expression in mammalian cells." BMC Biotechnol 6: 43; and WO02088346A2 and WO2006037215A1, which are incorporated herein by reference). Currently, the widely used Cumate induction system includes, for example, SBI System Biosciences' SparQ TM Cumate Switch system.

[0036] The transcription repressor protein LacI (lactose operon repressor protein) and operator LacO (lactose operon) involved in the present disclosure are derived from the IPTG-induced expression system, which includes the operator LacO downstream of the TATA box, the inducer IPTG or its derivatives or functional analogs, and the repressor protein LacI protein capable of binding to the operator LacO. In the absence of IPTG, the LacI protein binds to the operator LacO, inhibiting the transcription of the downstream regulated target nucleic acid fragment; when the LacI protein binds to IPTG, its affinity with the operator LacO decreases and separates, so that the transcription of the downstream regulated target nucleic acid fragment is no longer inhibited. For example, the operator LacO is connected between the constitutively active promoter sequence and the regulated target nucleic acid fragment, and the LacI repressor protein is constitutively expressed in the cell. When the inducer IPTG or its derivatives are not present in the culture medium, the expressed LacI protein binds to the operator LacO, thereby inhibiting the transcription of the regulated target nucleic acid fragment; when the above-mentioned inducer is added to the culture medium, the LacI protein bound to IPTG or its derivatives separates from the operator LacO, thereby making the transcription of the regulated target nucleic acid fragment no longer inhibited (see U.S. Patent No. 4,833,080A, which is incorporated herein by reference).

[0037] The present disclosure relates to a transcriptional repressor protein PadR (phenolic acid decarboxylase repressor) and an operator PadRO (RC) derived from a sodium ferulate (SF) inducible expression system. The system comprises an operator PadRO (RC) downstream of a TATA box, an inducer SF or a derivative or functional analog thereof, and a repressor protein PadR capable of binding to the operator PadRO (RC) (see Wang, Yidan et al., "Aversatile genetic control system in mammalian cells and mice responsive to clinically licensed sodium ferulate." Science Advances vol. 6, 32eabb9484.7Aug. 2020, incorporated herein by reference). In the absence of SF, the PadR protein binds to the operator PadRO (RC) and inhibits transcription of a downstream regulated target nucleic acid fragment. Upon binding to SF, the PadR protein loses its affinity for the operator PadRO (RC) and dissociates, and transcription of the downstream regulated target nucleic acid fragment is no longer inhibited. For example, the operator PadRO(RC) is linked between a constitutively active promoter sequence and the regulated target nucleic acid fragment, while the PadR protein is constitutively expressed in the cell. When the inducer SF or its derivatives are absent from the culture medium, the expressed PadR protein binds to the operator PadRO(RC), thereby inhibiting the transcription of the regulated target nucleic acid fragment. However, when the inducer is added to the culture medium, the PadR protein bound to SF or its derivatives dissociates from the operator PadRO(RC), thereby uninhibiting the transcription of the regulated target nucleic acid fragment.

[0038] Example 1: Plasmid construction

[0039] The molecular cloning techniques used in the following examples, such as PCR amplification of DNA fragments, restriction endonuclease digestion of DNA fragments, gel recovery of DNA fragments, T4 DNA ligase ligation of two or more DNA fragments, transformation of competent cells for ligation products, plasmid miniprep and identification, etc., are all well known in the art. The following reagents are involved in the following examples: PCR enzyme (Thermo, F-530S); restriction endonuclease (NEB); T4 DNA ligase (Invitrogen, 15224041); DNA fragment gel recovery kit (Omega, D2500-02); plasmid miniprep kit (TIANGEN, DP105-03); competent cells (XL-10Gold, Hunan Fenghui Biotechnology Co., Ltd., JZ011); the primer sequences shown in SEQ ID NO: 1 to SEQ ID NO: 13 were synthesized by Jinweizhi; the nucleic acid sequences shown in SEQ ID NO: 14 to SEQ ID NO: 23 and SEQ ID NO: 42 to SEQ ID NO: 46 were synthesized by GenScript. Plasmid sequencing and identification were completed by Beijing Qingke Biotechnology Co., Ltd. Table 1 is the primer information for constructing the plasmid. Table 2 is a description of the sequence element composition. Table 3 is a description of each functional element in the plasmid. Table 4 is the plasmid number and corresponding name constructed by the present invention. The element sequence information used by each plasmid involved in the following examples is an example for realizing the present invention. Those skilled in the art can expect that the effects described in the present invention can be achieved by replacing each element sequence on the plasmid used in the following examples with other element sequences with similar biological functions, including but not limited to the backbone sequence of the plasmid (such as replication origin, resistance gene, etc.), restriction site sequence, induction system response element sequence, insulator sequence, promoter sequence, intron sequence, polyadenylation signal (PolyA) sequence, gene sequences optimized by different codons, mutants of the above functional element sequences and gene sequences, and cloning position, cloning order and cloning direction of each functional element sequence and gene sequence. The specific plasmid construction method is as follows:

[0040] 1. Construction of pCMV-BGI-Fluc-Rluc plasmid: The 18BF011 plasmid (the same as the 18BF011 plasmid in Chinese patent application CN202010366440.4, which is incorporated herein by reference in its entirety) was double-digested with restriction endonucleases MluI and SphI, and the digested fragment was ligated with the 18BF004 plasmid (the same as the 18BF004 plasmid in Chinese patent application CN202010366440.4) that had also been double-digested with MluI and SphI to obtain the pCMV-BGI-MCS plasmid. Using the synthesized sequence SEQ ID NO: 14 as a template, PCR was performed using primers 220524-C-EF1a-F (AgeI) (SEQ ID NO: 1) and 220524-FP-EF1aRluc-R (SEQ ID NO: 2) to amplify the EF1α gene fragment (235 bp). Using the pGL4.70[hRluc] plasmid (Promega, E6881) as a template, PCR was performed using primers 220524-FP-EF1aRluc-F (SEQ ID NO: 3) and 220505-FP-T2ARluc-R1 (SEQ ID NO: 4) to amplify the hRluc gene fragment (988 bp). NO: 13) as primers to amplify the hRlucT2A gene fragment (1008 bp); using the 19BF080 plasmid (the same as the 19BF080 plasmid in Chinese patent application CN202010366440.4) as a template, PCR was performed using 220505-FP-T2AoptiPuroR-F (SEQ ID NO: 5) and SV40pA-R-C190925-R6 (SEQ ID NO: 6) as primers to amplify the optiPuroR gene fragment (765 bp); the amplified EF1α gene fragment, hRlucT2A gene fragment and optiPuroR gene fragment were connected by fusion PCR as a template and 220524-C-EF1a-F (AgeI) (SEQ ID NO: 1) and SV40pA-R-C190925-R6 (SEQ ID NO: 6) were used as primers. NO:6) was used as primers for PCR amplification to obtain the pCMV-BGI-MCS-Rluc-FuPCR (1962 bp) gene fragment; the fragment was then double-digested with restriction endonucleases AgeI and AvrII and ligated to the pCMV-BGI-MCS plasmid that had been treated with the same double enzymes, thereby constructing the pCMV-BGI-MCS-Rluc plasmid.Using 18YYH26 plasmid (same as 18YYH26 plasmid in Chinese patent application CN202010366440.4) as template, 220506-C-Fluc-F (BamHI) (SEQ ID NO: 7) and 220506-FP-Fluc-R1 (SEQ ID NO: 8); 220506-FP-Fluc-F1 (SEQ ID NO: 9) and 220506-FP-Fluc-R2 (SEQ ID NO: 10) and 220506-FP-Fluc-F2 (SEQ ID NO: 11) and 220506-C-Fluc-R (XhoI) (SEQ ID NO: 12) were cloned into 18YYH26 plasmid. NO: 12) as primers for PCR amplification of three DNA fragments of 204 bp, 670 bp and 866 bp (to remove the BstBI and SpaI restriction sites); the three amplified DNA fragments were ligated using a fusion PCR method as templates and PCR amplified using primers 220506-C-Fluc-F (BamHI) (SEQ ID NO: 7) and 220506-C-Fluc-R (XhoI) (SEQ ID NO: 12) to obtain a Fluc (v2) (1678 bp) gene fragment; the fragment was then double-digested with restriction endonucleases BamHI and XhoI and ligated to a pCMV-BGI-MCS-Rluc plasmid that had been treated with the same double enzyme digestion, thereby obtaining a pCMV-BGI-Fluc-Rluc plasmid.

[0041] 2. Construction of pCMV-Fluc-Rluc plasmid: The pCMV-BGI-Fluc-Rluc plasmid was digested with restriction endonuclease BstBI, the DNA fragment (7634 bp) was recovered from gel, and the recovered fragments were ligated with T4 ligase to obtain the pCMV-Fluc-Rluc plasmid.

[0042] 3. Construction of pTRE3G-Fluc-Rluc plasmid and pTraRRE-Fluc-Rluc plasmid: The synthesized sequences SEQ ID NO: 15 (394 bp) and SEQ ID NO: 16 (434 bp) were double-digested with restriction endonucleases MluI and ClaI, respectively, and ligated into the pCMV-BGI-Fluc-Rluc plasmid that had been treated with the same double enzyme digestion, thereby obtaining the pTRE3G-Fluc-Rluc plasmid and the pTraRRE-Fluc-Rluc plasmid.

[0043] 4. Construction of pTraRRELacO50-Fluc-Rluc plasmid, pTraRREPadRO(RC)50-Fluc-Rluc plasmid, pTraRRECuO100-Fluc-Rluc plasmid, pTraRRECuO60-Fluc-Rluc plasmid, pTraRRECuO50-Fluc-Rluc plasmid, pTraRRECuO40-Fluc-Rluc plasmid, pTraRRECuO30-Fluc-Rluc plasmid and pTraRRECuO14-Fluc-Rluc plasmid: The synthetic sequences SEQ ID NO: 17 (481 bp), SEQ ID NO: 18 (503 bp), SEQ ID NO: 42 (538 bp), SEQ ID NO: 43 (498 bp), SEQ ID NO: 19 (490 bp), SEQ ID NO: 44 (478 bp), SEQ ID NO: 45 (470 bp) and SEQ ID NO: NO:46 (470bp) was double-digested with restriction endonucleases MluI and ClaI and ligated to the pCMV-BGI-Fluc-Rluc plasmid that had undergone the same double-digestion treatment, thereby obtaining pTraRRELacO50-Fluc-Rluc plasmid, pTraRREPadRO(RC)50-Fluc-Rluc plasmid, pTraRRECuO100-Fluc-Rluc plasmid, pTraRRECuO60-Fluc-Rluc plasmid, pTraRRECuO50-Fluc-Rluc plasmid, pTraRRECuO40-Fluc-Rluc plasmid, pTraRRECuO30-Fluc-Rluc plasmid and pTraRRECuO14-Fluc-Rluc plasmid.

[0044] 5. Construction of pTraRRELacO50-BGI-Fluc-Rluc plasmid, pTraRREPadRO(RC)50-BGI-Fluc-Rluc plasmid and pTraRRECuO50-BGI-Fluc-Rluc plasmid: The pCMV-BGI-MCS plasmid was digested with the restriction endonuclease BstBI and ligated to the BstBI restriction sites of the pTraRRELacO50-Fluc-Rluc plasmid, pTraRREPadRO(RC)50-Fluc-Rluc plasmid and pTraRRECuO50-Fluc-Rluc plasmid, respectively, to obtain pTraRRELacO50-BGI-Fluc-Rluc, pTraRREPadRO(RC)50-BGI-Fluc-Rluc and pTraRRECuO50-BGI-Fluc-Rluc plasmids.

[0045] 6. Construction of pCAGGS-rtTA(Adv) / optiCymR plasmid: This plasmid is identical to the 19BF074 plasmid in Chinese patent application CN202010366440.4.

[0046] 7. Construction of pCAGGS-rtTA(Adv) / optiLacI plasmid and pCAGGS-rtTA(Adv) / optiPadR plasmid: The synthesized sequences SEQ ID NO:20 (1148 bp) and SEQ ID NO:21 (1040 bp) were double-digested with restriction endonucleases ClaI and XhoI, respectively, and ligated into the pCAGGS-rtTA(Adv) / optiCymR plasmid that had been treated with the same double enzyme digestion, thereby obtaining the pCAGGS-rtTA(Adv) / optiLacI plasmid and the pCAGGS-rtTA(Adv) / optiPadR plasmid.

[0047] 8. Construction of pCAGGS-optiTraR (p65) plasmid and pCAGGS-optiTraR (p65-HSF1) plasmid: The synthesized sequences SEQ ID NO: 22 (1604 bp) and SEQ ID NO: 23 (1754 bp) were double-digested with restriction endonucleases ClaI and XhoI, respectively, and ligated to the 18BF008 plasmid (the same as the 18BF008 plasmid in Chinese patent application CN202010366440.4) that had undergone the same double enzyme digestion treatment, thereby obtaining the pCAGGS-optiTraR (p65) plasmid and the pCAGGS-optiTraR (p65-HSF1) plasmid.

[0048] Table 1. Primer information

[0049] Table 2. Description of sequence element composition

[0050] Table 3. Functional elements in plasmids

[0051] Example 2: Effect of optimizing the activation domain of regulatory proteins on induced expression and non-induced leakage expression

[0052] In order to study the effect of optimizing the activation domain of the regulatory protein on the induced expression amount and the non-induced expression amount of the TraR inducible expression system, the TraR regulatory protein with p65 as the activation domain (referred to as TraR (p65) in this article) in the existing TraR inducible expression system was replaced with a TraR regulatory protein with p65-HSF1 as the activation domain (referred to as TraR (p65-HSF1) in this article). Plasmids carrying the coding sequences for different regulatory proteins (pCAGGS-optiTraR(p65) plasmid carrying the TraR(p65) coding sequence, and pCAGGS-optiTraR(p65-HSF1) plasmid carrying the TraR(p65-HSF1) coding sequence) were transiently transfected into cells along with a plasmid carrying a response element and a regulated target nucleic acid sequence (luciferase coding sequence) located downstream of the response element. The expression levels of the target nucleic acid (luciferase fluorescence) were measured with and without the inducer 3-oxo-C8-HSL, and compared with the results of the Tet-On inducible expression system (TRE3G: response element in the Tet-On(3G) inducible expression system; rtTA(adv): tetracycline-dependent transactivator in the Tet-On Advanced inducible expression system). The specific experimental methods are as follows:

[0053] 293T cells (ATCC) were seeded in 96-well plates (black) (Corning Life Sciences, Inc., 3916) at a density of 1.0E+04 cells / well in 200 μl of DMEM complete medium. After 24 hours of culture, the plasmid combinations shown in Table 4 were transfected according to the PEI transfection method. 20 μl of transfection reagent was added to each well during transfection, and the total plasmid amount was 0.3 μg, of which the amount of the plasmid carrying the response element and the regulated target nucleic acid was 0.05 μg. The molar ratio of the plasmid carrying the response element and the regulated target nucleic acid to the plasmid carrying the regulatory protein coding sequence was 2: 1, and the remaining plasmid amount was supplemented by 18BF003 (see SEQ ID NO: 1 in Chinese patent application CN202010442534.5, which is incorporated herein by reference in its entirety). Empty plasmid. The mass ratio of total plasmid to PEI MAX (Polysciences, 24765-1) was 1:4, and four wells were transfected with each plasmid combination. Three hours after transfection, complete DMEM medium was replaced, and inducers were added to two wells: 20 μM 3-oxo-C8-HSL (Aladdin, N350215) for the TraR-inducible expression system and the CMV control, and 1 μg / ml DOX (Sangon, A600889) for the TRE3G-regulatable expression system. No inducers were added to the other two wells. Twenty-four hours after transfection, relative fluorescence units (RLU) were measured in each well using a fluorescence microplate reader (Perkin Elmer Victor V) using the Dual Luciferase Reporter Assay Kit (Norvozymes, DL101-01) according to the manufacturer's instructions.

[0054] Table 4. Transfection plasmid combinations

[0055] The results are shown in Figure 1A and Table 5. Replacing the activation domain of the TraR regulatory protein with p65-HSF1 significantly increased the induced expression level of the TraR inducible expression system, even though there was no significant difference in the leaky expression values ​​of the two proteins under no induction. Replacing the regulatory protein with TraR (p65) with TraR (p65-HSF1) increased the induced expression level by approximately 6.5-fold. Furthermore, the induced expression level of the TraR inducible expression system using the TraR (p65-HSF1) regulatory protein was approximately 2.4-fold higher than that of the Tet-On (TRE3G) inducible expression system; there was no significant difference in expression level when using the constitutive CMV promoter (P = 0.957).

[0056] The leaky expression levels in the TraR-inducible expression system using the TraR (p65-HSF1) regulatory protein and the TraR (p65) regulatory protein, respectively, without induction, were 9.37E+04 RLU and 7.03E+04 RLU, showing no significant difference (P=0.117). The leaky expression level in the TraR-inducible expression system using the TraR (p65-HSF1) regulatory protein without induction was 14-fold lower than that in the Tet-On (TRE3G) inducible expression system. As previously mentioned, in inducible expression systems, methods commonly used to enhance transcription and expression of target nucleic acid fragments under induction also enhance leaky expression to the same extent in the absence of induction. However, these results surprisingly show that when p65-HSF1 is used instead of p65 as the transcriptional activation domain, while the induced expression level is significantly increased (6.5-fold), the leaky expression level is not significantly increased to the same or similar degree. As shown in Figure 1B and Table 5, the induction / leakage expression ratios for the TraR inducible expression system using the TraR (p65-HSF1) regulatory protein and the TraR (p65) regulatory protein were 160.5 and 31.8, respectively. Substituting the regulatory protein for TraR (p65-HSF1) increased the induction / leakage expression ratio by approximately 5-fold. The induction / leakage expression ratio for the TraR inducible expression system using the TraR (p65-HSF1) regulatory protein was 33-fold higher than the ratio (4.8) for the Tet-On (TRE3G) inducible expression system.

[0057] Table 5. Effects of optimizing the activation domain of regulatory proteins on induced expression and leaky expression

[0058] Example 3: Effect of the composite inducible expression system on the induced expression amount and the non-induced leakage expression amount

[0059] As mentioned above, commonly used methods for controlling non-induced leakage expression activity will correspondingly reduce the maximum expression activity under induction conditions. When two inducible expression systems are combined into a composite inducible expression system, since theoretically the target nucleic acid sequence located downstream of the composite regulatory element can be transcribed only when both induction mechanisms are activated, the composite inducible expression system has lower leakage expression activity compared to a single inducible expression system. However, the mechanism also often results in the composite inducible expression system having a correspondingly lower induced expression amount compared to a single inducible expression system. This embodiment is intended to determine a composite inducible expression system based on the TraR inducible expression system of the present disclosure that has a significantly reduced leakage expression amount when the induced expression amount does not decrease significantly compared to a single TraR inducible expression system.

[0060] LacO, PadRO (RC), and CuO operator sequences were ligated 50 bp from the TATA box sequence following the TraRRE response element, respectively, to obtain a composite response element. Furthermore, a cleavable intron sequence can be placed between the 3' end downstream of the composite response element and the 5' end upstream of the regulated target nucleic acid fragment, thereby obtaining a composite response element with an intron sequence downstream. A Luciferase reporter gene sequence, serving as the regulated target nucleic acid, was ligated downstream of the 3' end of the above response element to construct plasmids pTraRRELacO50-Fluc-Rluc, pTraRREPadRO (RC) 50-Fluc-Rluc, and pTraRRECuO50-Fluc-Rluc, as well as plasmids pTraRRELacO50-BGI-Fluc-Rluc, pTraRREPadRO (RC) 50-BGI-Fluc-Rluc, and pTraRRECuO50-BGI-Fluc-Rluc. The expression levels of TraR (p65-HSF1) and the corresponding repressor proteins were detected. The specific experimental methods are as follows:

[0061] 293T cells (ATCC) are seeded in 96-well plates (black) according to 1.0E+04 individual cells per well, and culture medium is 200 microlitre DMEM complete medium.After cultivating 24 hours, according to the plasmid combination shown in PEI transfection method transfection table 6, every hole adds 20 microlitre transfection reagents during transfection, containing total plasmid amount is 0.3 microgram, wherein response element plasmid (carrying response element and regulated target nucleic acid) transfection amount is 0.05 microgram, in the case of no repressor protein grain (carrying repressor protein coding sequence), response element plasmid and activation protein grain (carrying activation protein coding sequence) mol ratio are 2: 1, in the case of there is repressor protein grain, response element plasmid, activation protein grain and repressor protein grain mol ratio are 2: 1: 1, and remaining plasmid amount is supplemented by 18BF003 empty vector plasmid. The mass ratio of total plasmid amount and PEI MAX is 1: 4, and every kind of combination transfection is 4 holes. Three hours after transfection, complete DMEM medium was replaced, and inducers were added to two wells, depending on the experimental group combination: 20 μM 3-oxo-C8-HSL and 5 mM IPTG were added to the wells containing the TraRRELacO50 complex response element; 20 μM 3-oxo-C8-HSL and 1 mM SF were added to the wells containing the TraRREPadRO(RC)50 complex response element; 20 μM 3-oxo-C8-HSL and 200 μg / ml Cumate were added to the wells containing the TraRRECuO50 complex response element; and 20 μM 3-oxo-C8-HSL was added to the wells containing the TraRRE single regulatory response element. No inducers were added to the other two wells. Twenty-four hours after transfection, relative fluorescence units (RLU) were measured in each well using a fluorescence microplate reader according to the Dual Luciferase Reporter Assay Kit instructions.

[0062] Table 6. Transfection combination list

[0063] The results are shown in Figure 2 and Table 7. Compared with the combination of the TraRRE single regulatory response element and TraR (p65), the combination of the composite response element (TraRRELacO50, TraRREPadRO (RC) 50, and TraRRECuO50) with TraR (p65-HSF1) had a significantly improved induced / leaky expression ratio. The induced / leaky expression ratios of the TraRRELacO50, TraRREPadRO (RC) 50, and TraRRECuO50 composite response elements combined with TraR (p65-HSF1) were approximately 7.02 times, 12.3 times, and 25.7 times, respectively, of the ratios of the TraRRE single regulatory response element combined with TraR (p65); and were approximately 1.4 times, 2.4 times, and 5.1 times, respectively, of the ratios of the TraRRE single regulatory response element combined with TraR (p65-HSF1).

[0064] For the composite response element with LacO and the composite response element with PadRO (RC), the addition of the BGI sequence did not significantly affect the induced / leaked expression ratio (both were significantly higher than the case of a single regulatory response element), but compared with the case without the addition of the BGI sequence, the induced expression value after adding the BGI sequence could be significantly improved.

[0065] As can be seen from the above, compared with a single regulatory response element, the composite response element disclosed herein can have a significantly improved induced / leakage expression ratio.

[0066] It is also worth noting that the CuO-containing composite response elements (TraRRECuO50 (combination 3) and TraRRECuO50-BGI (combination 6)) not only have significantly higher induced / leaked expression ratios than the single regulatory response element (TraRRE (combination 7)), but also have higher induced expression values ​​than the latter. This is an unexpected result, as the addition of a second response element is expected to reduce the induced expression value to a certain extent (at least not higher than the induced expression value of the single regulatory response element) (see Chinese patent application CN202010442506.3).

[0067] Table 7. Effects of the composite inducible expression system on induced expression and non-induced leakage expression

[0068] Example 4: Method for hierarchical induction expression based on composite response elements

[0069] This embodiment is intended to explore a method for hierarchical induction expression based on a composite response element. In this embodiment, using the composite response element disclosed herein, Luciferase is used as a reporter gene, and hierarchical regulation of the expression level of the inducible expression system is achieved by 1) not adding any inducer, 2) adding only the inducer corresponding to the transcription activator protein (3-oxo-C8-HSL), 3) adding only the inducer corresponding to the transcription repressor protein (IPTG, SF, Cumate), and 4) adding two inducers. The specific method is as follows:

[0070] 293T cells were seeded in 96-well plates (black) at a density of 1.0E+04 cells per well in 200 microliters of DMEM complete medium. After 24 hours of culture, the plasmid combinations shown in Table 8 were transfected according to the PEI transfection method. During transfection, 20 microliters of transfection reagent were added to each well, containing 0.3 micrograms of total plasmid, 0.05 micrograms of response element plasmid, 2:1:1 molar ratio of response element plasmid, activation protein plasmid and repression protein plasmid, and the remaining plasmid amount was supplemented by 18BF003 empty plasmid. The mass ratio of total plasmid amount to PEI MAX was 1:4, and 8 wells were transfected with each plasmid. Three hours after transfection, complete DMEM medium was replaced. Two wells were treated with either the repressor inducer (5 mM IPTG, 1 mM SF, or 200 μg / ml Cumate) alone; two wells were treated with the activator inducer (20 μM 3-oxo-C8-HSL alone); and two wells were treated with either 20 μM 3-oxo-C8-HSL and 5 mM IPTG, 20 μM 3-oxo-C8-HSL and 1 mM SF, or 20 μM 3-oxo-C8-HSL and 200 μg / ml Cumate simultaneously. The remaining two wells were then treated with an equal volume of complete DMEM medium. Twenty-four hours after transfection, relative fluorescence units (RLU) were measured in each well using a fluorescence microplate reader using the Dual Luciferase Reporter Assay Kit according to the manufacturer's instructions.

[0071] Table 8. Transfection combination list

[0072] The results are shown in Figure 3. When the TraRRELacO50-BGI composite response element was used, the induced expression value when only the IPTG inducer was added was 1.12E+05RLU, which was 2.2 times higher than that under the uninduced condition; the induced expression value when only the activator inducer 3-oxo-C8-HSL was added was 6.39E+06RLU, which was 116.7 times higher than that under the uninduced condition and 64.9 times higher than that under the addition of IPTG alone; the induced expression value when 3-oxo-C8-HSL and IPTG were added at the same time was 1.22E+07RLU, which was 225.7 times higher than that under the uninduced condition, 123.3 times higher than that under the addition of IPTG alone, and 1.9 times higher than that under the addition of 3-oxo-C8-HSL alone.

[0073] When using the TraRREPadRO(RC)50-BGI composite response element, the induced expression value when only SF was added was 1.08E+05RLU, which was 4.7 times higher than that under the condition without induction; the induced expression value when only 3-oxo-C8-HSL was added was 3.81E+06RLU, which was 162.5 times higher than that under the condition without induction and 40.6 times higher than that when only SF was added; the induced expression value when 3-oxo-C8-HSL and SF were added at the same time was 1.00E+07RLU, which was 428.0 times higher than that under the condition without induction and 109.8 times higher than that when only SF was added; it was 2.7 times higher than that when only 3-oxo-C8-HSL was added.

[0074] When the TraRRECuO50-BGI composite response element was used, the induced expression value when only Cumate was added was 8.81E+04RLU, which was 2.2 times higher than that under the uninduced condition; the induced expression value when only 3-oxo-C8-HSL was added was 4.50E+06RLU, which was 115.2 times higher than that under the uninduced condition and 53.3 times higher than that under the addition of Cumate alone; the induced expression value when 3-oxo-C8-HSL and Cumate were added at the same time was 1.67E+07RLU, which was 426.7 times higher than that under the uninduced condition, 196.6 times higher than that under the addition of Cumate alone, and 3.7 times higher than that under the addition of 3-oxo-C8-HSL alone.

[0075] As can be seen from the above, when a composite response element composed of at least one transcriptional activator response element (e.g., the response element TraRRE of the transcriptional activator TraR (p65) or TraR (p65-HSF1)) and at least one transcriptional repressor response element (the response element LacO of the transcriptional repressor LacI, the response element PadRO (RC) of the transcriptional repressor PadR, or the response element CuO of the transcriptional repressor CymR) is used to induce the expression of the target nucleic acid fragment regulated thereby, at least four levels (levels) of induced expression of the target nucleic acid fragment can be achieved by adding or not adding an inducer corresponding to the transcriptional activator (e.g., 3-oxo-C8-HSL) and / or an inducer corresponding to the transcriptional repressor (e.g., SF, IPTG, or Cumate).

[0076] Example 5: Effect of the distance between CuO operators on the induced expression level and the non-induced leakage expression level

[0077] As observed in Example 3, the composite response elements containing the CuO operator (TraRRECuO50 and TraRRECuO50-BGI) not only exhibited significantly higher induced / leaky expression ratios than the single regulatory response element (TraRRE), but also exhibited higher induced expression values ​​than the single regulatory response element (TraRRE). This example further validates this surprising finding from Example 3.

[0078] The CuO operator sequence was added at the downstream position of the TATA box sequence located downstream of the TraRRE response element, 100bp, 60bp, 50bp, 40bp, 30bp and 14bp respectively, to obtain 6 composite response elements containing CuO. The Luciferase reporter gene sequence as the regulated target nucleic acid was connected to the 3' end downstream of the above composite response element to construct plasmids pTraRRECuO100-Fluc-Rluc, pTraRRECuO60-Fluc-Rluc, pTraRRECuO50-Fluc-Rluc, pTraRRECuO40-Fluc-Rluc, pTraRRECuO30-Fluc-Rluc and pTraRRECuO14-Fluc-Rluc. The expression level was detected using TraR (p65) and CymR repressor proteins. The specific experimental method is as follows:

[0079] 293T cells (ATCC) are seeded in 96-well plates (black) according to 1.0E+04 individual cells per well, and culture medium is 200 microlitre DMEM complete medium.After cultivating 24 hours, according to the plasmid combination shown in PEI transfection method transfection table 9, every hole adds 20 microlitre transfection reagents during transfection, containing total plasmid amount is 0.3 microgram, wherein response element plasmid (carrying response element and regulated target nucleic acid) transfection amount is 0.05 microgram, in the case of no repressor protein grain (carrying repressor protein coding sequence), response element plasmid and activation protein grain (carrying activation protein coding sequence) mol ratio are 2: 1, in the case of there is repressor protein grain, response element plasmid, activation protein grain and repressor protein grain mol ratio are 2: 1: 1, and remaining plasmid amount is supplemented by 18BF003 empty vector plasmid. The mass ratio of total plasmid amount and PEI MAX is 1: 4, and every kind of combination transfection is 4 holes. Three hours after transfection, complete DMEM medium was replaced, and inducers were added to two wells, depending on the experimental group combination: 20 μM 3-oxo-C8-HSL and 200 μg / ml Cumate were added to the wells corresponding to the TraRRECuO50 complex response element, and 20 μM 3-oxo-C8-HSL was added to the wells corresponding to the TraRRE single regulatory response element. No inducer was added to the other two wells. 24 hours after transfection, relative fluorescence units (RLU) of each well were measured using a fluorescence microplate reader according to the Dual Luciferase Reporter Assay Kit instructions.

[0080] Table 9. Transfection combination list

[0081] The results are shown in Table 10. Compared with the TraRRE single regulatory response element, when the CuO operator is located 60 bp, 50 bp and 40 bp downstream of the TATA box in the TraRRE-CuO composite response element, not only the induced / leaked expression ratio is significantly improved, but the induced expression value is also significantly improved (by 27.5%, 22.1% and 19.7%, respectively); when the CuO operator is located 100 bp downstream of the TATA box, the induced / leaked expression ratio does not change significantly, and the induced expression value does not change significantly; when the CuO operator is located 30 bp downstream of the TATA box, although the induced / leaked expression ratio is significantly improved, the induced expression value is significantly reduced; when the CuO operator is located 14 bp downstream of the TATA box, the induced / leaked expression ratio is significantly reduced, and the induced expression value is also significantly reduced. The above results prove that compared with the TraRRE single regulatory response element, in the TraRRE-CuO composite response element, when the CuO operator is located at a specific position downstream of the TATA box (for example, 40-60bp), not only the induced / leaked expression ratio is significantly improved, but the induced expression value also unexpectedly increases instead of decreases.

[0082] Table 10. Effect of the distance between CuO operators on the induced expression level and the non-induced leakage expression level

[0083] In the description of this specification, the reference terms "one embodiment", "some embodiments", "an instance", "some examples", or "example" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0084] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A transcriptional activator protein TraR of the TraR / 3-oxo-C8-HSL inducible expression system, characterized in that: The transcription activator protein TraR has a transcription activation domain, and the transcription activation domain contains an NF-κB p65 sequence and a heat shock factor 1 sequence.

2. The transcription activator protein TraR according to claim 1, characterized in that The NF-κB p65 sequence is a mammalian NF-κB p65 sequence, such as a human NF-κB p65 sequence, and / or the heat shock factor 1 sequence is a mammalian heat shock factor 1 sequence, such as a human heat shock factor 1 sequence.

3. A nucleic acid, characterized in that The nucleic acid encodes the transcription activator protein TraR according to claim 1 or 2.

4. A cell, characterized in that The cell comprises the transcriptional activator protein TraR according to claim 1 or 2 or the nucleic acid according to claim 3.

5. An inducible expression system, characterized in that The inducible expression system comprises the transcriptional activator protein TraR according to claim 1 or 2, the nucleic acid according to claim 3, or the cell according to claim 4.

6. A composite inducible expression system, characterized in that: The composite inducible expression system comprises: a regulatory sequence comprising a first response element containing a tra box sequence, a minimal promoter sequence comprising a TATA box, and a second response element located downstream of the TATA box and capable of binding to a transcriptional repressor protein; and the transcriptional activator protein TraR according to claim 1 or 2, and the transcriptional repressor protein.

7. The composite inducible expression system according to claim 6, characterized in that The second response element capable of binding to the transcription repressor protein is: a CuO operator capable of binding to the transcription repressor protein CymR, a LacO operator capable of binding to the transcription repressor protein LacI, or a PadRO (RC) operator capable of binding to the transcription repressor protein PadR.

8. The composite inducible expression system according to claim 6, characterized in that The regulatory sequence further comprises a spliceable intron sequence located between the downstream of the second response element and the upstream of the regulated nucleic acid sequence.

9. A method for regulating nucleic acid expression in eukaryotic cells, characterized in that: The method comprises using the transcriptional activator protein TraR according to claim 1 or 2, the nucleic acid according to claim 3, and the cell according to claim 4. The inducible expression system according to claim 5, or the composite inducible expression system according to any one of claims 6 to 8.

10. A method for regulating the expression of a nucleic acid sequence at at least four expression levels, characterized in that: The method includes using a composite inducible expression system, the composite inducible expression system comprising a regulatory sequence comprising at least a first response element and a second response element, and a minimal promoter sequence comprising a TATA box located between the two response elements, the first response element being capable of binding to its transcriptional activator protein in the presence of a first inducer, and the second response element being capable of dissociating from its transcriptional repressor protein in the presence of a second inducer, wherein a first expression level is achieved without adding any inducer, a second expression level is achieved with the addition of the second inducer without adding the first inducer, a third expression level is achieved with the addition of the first inducer without adding the second inducer, and a fourth expression level is achieved with the addition of both the first inducer and the second inducer.

11. The method according to claim 10, characterized in that The first response element is a response element containing a tra box sequence, and / or the transcription activator protein capable of binding to the first response element is the transcription activator protein TraR according to claim 1 or 2.

12. A nucleic acid sequence characterized in that The nucleic acid sequence comprises a TraR response element sequence containing a tra box sequence, a minimal promoter sequence comprising a TATA box, and a CuO operator sequence located downstream of the TATA box and capable of binding to the transcription repressor protein CymR. Preferably, the distance between the CuO operator sequence and the TATA box is 40 bp to 60 bp.

13. A vector, cell or inducible expression system, characterized in that The vector, cell or inducible expression system comprises the nucleic acid sequence according to claim 12.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the transcription activator protein TraR according to claim 1 or 2, the nucleic acid according to claim 3, and the cell according to claim 4. The inducible expression system according to claim 5, the composite inducible expression system according to claims 6 to 8, the nucleic acid sequence according to claim 12, or the vector, cell or inducible expression system according to claim 13, and the target gene.

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