Reverse vanillic acid trans transcription factor and application thereof

Abstract

The invention discloses a reverse vanillic acid trans transcription factor. Compared with a vanillic acid trans-transcription factor, the reverse vanillic acid trans-transcription factor has amino acid mutation, and the sites of the amino acid mutation comprise the 31st site, the 97th site, the 127th site, the 137th site, the 165th site and the 183th site. According to the reverse vanillic acid trans-transcription factor, Va is not added to dissociate a vanillic acid operon, so that downstream system expression is closed; va can be added to be combined with a vanillic acid operon, then downstream system expression is started, and higher practicability and safety are achieved.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology and synthetic biology, and specifically relates to a reverse vanillic acid trans-transcription factor and its use. Background Art

[0002] In cell therapy, controlling heterologous transgene expression through small molecule inducers is a common approach. Therefore, the selection and use of inducible switches is crucial, as the small molecules commonly used to control these switches are often themselves antibiotics, steroid hormones, or immunosuppressive drugs, and their presence can affect the physiological activities of the recipient.

[0003] Vanillic acid (Va) is a naturally occurring phenolic compound found in plants and widely used as a food additive (FAO / WHO Expert Committee on Food Additives, JECFA no. 959). It possesses excellent medicinal properties, including anti-cancer, anti-inflammatory, and neuroprotective activities. Furthermore, it exhibits excellent safety, with high doses (no significant adverse reactions were observed in rats after continuous administration of 1000 mg / kg / day for two weeks). Furthermore, it is rapidly metabolized in human blood, preventing long-term accumulation and impacting the physiological activities of the recipient, making it an excellent inducible small molecule.

[0004] Vanillate-responsive transcriptional repressors (VanR) from Caulobacter crescentus have the function of specifically binding to one or several inverted repeat operons VanO (ATTGGATCCAAT) when Va is absent in the environment. When VanR binds to the Va small molecule, it immediately dissociates from VanO.

[0005] The Van-Off system for cell therapy, designed based on Va, VanR, and VanO, is in an on-state when Va is not added. It can only be kept off by continuously adding Va. This logic makes the system difficult to apply in actual treatments. Therefore, it is very important to develop a reverse vanillic acid trans-transcription factor and a reverse-regulated vanillic acid gene circuit control system. Summary of the Invention

[0006] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a reverse vanillic acid trans-transcription factor.

[0007] This application is based on the following findings of the inventors:

[0008] The present invention uses error-prone PCR mutagenesis to randomly mutate the VanR gene over multiple rounds. Using semi-rational assisted optimization of the mutation sites, the researchers screened for a mutant rVanR (i.e., a reverse vanillic acid trans-transcription factor) that, upon binding to vanillic acid, regulates genes in a manner opposite to that of VanR. The mutant rVanR dissociates from VanO (the vanillic acid operator site) when not bound to the small molecule Va. Upon binding to Va, the mutant rVanR binds to the operator site VanO.

[0009] Therefore, the inventors used synthetic biology to design a transgenic expression switch device regulated by Va, namely the vanillic acid gene circuit control system. Engineered cells containing the vanillic acid gene circuit control system are silent in the absence of Va activation. When a certain concentration of Va (0.1-0.5mM) is added, the switch can be activated to express downstream genes.

[0010] Va is a small molecule recognized as safe, non-toxic, rapidly metabolized, and food-safe. The reverse vanillic acid trans-transcription factor and reversely regulated vanillic acid gene circuit control system described in this application both dissociate VanO (the vanillic acid operator site) when not bound to the Va small molecule and bind to VanO at the operator site after binding to Va. Therefore, the reversely regulated vanillic acid gene circuit control system and drug designed based on the reverse vanillic acid trans-transcription factor of this application can maintain the system in a closed state without the addition of Va, and can maintain the system in an activated state with the addition of Va. These drugs have greater practicality and safety, provide new methods and strategies for clinical cell therapy, and have promising clinical application prospects.

[0011] Based on this, in the first aspect of the present application, the present application proposes a reverse vanillic acid trans-transcription factor. According to the embodiments of the present application, compared with the vanillic acid trans-transcription factor, the reverse vanillic acid trans-transcription factor has amino acid mutations, and the sites of the amino acid mutations include positions 97, 127, 137, and 183. The reverse vanillic acid trans-transcription factor of the present application dissociates the vanillic acid operon when Va is not added, thereby shutting down the system; adding Va can bind to the vanillic acid operon VanO, thereby turning on the system. Therefore, the reverse vanillic acid trans-transcription factor of the present application has higher practicality and safety.

[0012] In a second aspect of the present application, the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the reverse vanilloid trans-transcription factor described in the first aspect.

[0013] In a third aspect of the present application, the present application provides a vector. According to an embodiment of the present application, the vector carries the nucleic acid molecule described in the second aspect.

[0014] In a fourth aspect of the present application, a recombinant cell is provided. According to an embodiment of the present application, the recombinant cell comprises the nucleic acid molecule described in the second aspect, or the vector described in the third aspect; or expresses the reverse vanilloid trans-transcription factor described in the first aspect.

[0015] In the fifth aspect of the present application, the present application proposes the use of the reverse vanillic acid trans-transcription factor described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, or the recombinant cell described in the fourth aspect in preparing a vanillic acid gene circuit control system.

[0016] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0018] Figure 1 The rVanR function test results of rVanR01 and VanR-WT in Example 1 of the present application are shown in FIG.

[0019] Figure 2 The results of the rVanR function test of the four mutants 209-1, 210-1 (rVanR02), 211-1, and 212-1 in Example 1 of the present application are as follows;

[0020] Figure 3 The results of the rVanR function test after single-point reverse mutation of rVanR01 and rVanR02 back to the wild-type site in Example 1 of the present application are shown;

[0021] Figure 4 The results of the A137 single-point depth scanning HEK-293 cell experiment in the rVanR02 mutant in Example 1 of this application;

[0022] Figure 5 This is the experimental result of single-point depth scanning of HEK-293 cells in E183 of the rVanR02 mutant in Example 1 of this application;

[0023] Figure 6 The results of the HEK-293 cell experiment were obtained by single-site analysis of the first eight amino acid residues of E183 in the rVanR02 mutant in Example 1 of the present application;

[0024] Figure 7 The results of the detection of rVanR function of the rVanR02 mutant and the rVanR03 mutant in Example 1 of the present application are as follows;

[0025] Figure 8 The results of the detection of rVanR function of rVanR04 and rVanR05 in the HEK-293 cell system in Example 2 of the present application;

[0026] Figure 9 This is the test result of rVanR function after single-point reverse mutation of rVanR05 back to the wild-type site in Example 2 of the present application;

[0027] Figure 10 The results of detecting the rVanR function of each vanillic acid gene circuit control system in the HEK-293 cell system in Example 3 of the present application are as follows;

[0028] Figure 11 The results of detecting the rVanR function of the vanillic acid gene circuit control system in the HEK-293 cell system obtained by adjusting the ratio of the reporter plasmid to the receptor plasmid (SEAP) in Example 3 of the present application are as follows;

[0029] Figure 12 This is the time dynamic observation result when the ratio of reporter plasmid to recipient plasmid is 1:8 in Example 3 of this application;

[0030] Figure 13 The results are as follows: the response multiples of 0-8 mM Va concentration in the vanillic acid gene circuit control system when the ratio of reporter plasmid to receptor plasmid is 1:8 in Example 3 of the present application, and the fluorescence reporter expression results of 0-0.5 mM Va;

[0031] Figure 14 These are the results of reversibility experimental analysis in the vanillic acid gene circuit control system when the ratio of the reporter plasmid to the receptor plasmid is 1:8 in Example 3 of the present application. DETAILED DESCRIPTION

[0032] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0033] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0034] This application details

[0035] Definitions and General Terms

[0036] 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.

[0037] To facilitate understanding of this application, certain technical and scientific terms are defined below. Unless otherwise expressly defined elsewhere herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this application 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] As used herein, the term "having at least 90% sequence similarity" can mean a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%. The sequence identity described herein can be measured using sequence analysis software. For example, the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters, is used. The amino acid sequences described herein are all shown in an N- to C-terminal manner.

[0042] As used herein, the term "variant" or "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid differences (mutations) that differ from a reference sequence. The difference may be a substitution, deletion, or insertion of one or more amino acids.

[0043] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and transfers the inserted nucleic acid molecule into and / or between host cells. The expression vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0044] As used herein, the term "recombinant cell" generally refers to a cell that has been modified or recombined using genetic engineering techniques or cell fusion techniques to modify or reorganize the genetic material of a host cell to obtain a cell with unique traits that are stably inherited. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and can be used for the expression and / or secretion of a target protein.

[0045] Detailed description of the reverse vanillic acid trans-transcription factor and its use

[0046] The present application proposes a reverse vanillic acid trans-transcription factor and its use, nucleic acid molecule, vector, and recombinant cell, which will be described in detail below.

[0047] Reverse vanilloid trans-acting factor

[0048] In a first aspect of the present application, a reverse vanillic acid trans-transcription factor is provided. According to an embodiment of the present application, compared with the vanillic acid trans-transcription factor, the reverse vanillic acid trans-transcription factor has amino acid mutations, and the sites of the amino acid mutations include positions 97, 127, 137, and 183.

[0049] The reverse vanillic acid trans-transcription factor of the present application dissociates the vanillic acid operon in the absence of Va; however, upon addition of Va, it binds to the vanillic acid operon VanO. Thus, the method of the present application can be used to design a reverse-regulated vanillic acid gene circuit control system, enabling the system to remain in an inactive state in the absence of Va and in an active state with the addition of Va, thus improving practicality and safety.

[0050] It should be noted that “the sites of the amino acid mutation include position 97, position 127, position 137, and position 183” include position 97 or its equivalent site, position 127 or its equivalent site, position 137 or its equivalent site, and position 183 or its equivalent site.

[0051] As used herein, the term "equivalent site" refers to a site where the position and amino acid type of a particular site differ due to different origins (e.g., from different provenances or species), types, subclasses, or alleles. The equivalent site of a particular site can be determined by sequence and structural homology comparison.

[0052] According to an embodiment of the present application, the above-mentioned reverse vanilloid trans-transcription factor may further include at least one of the following technical features:

[0054] According to an embodiment of the present application, the amino acid sequence of the vanillic acid trans-transcription factor is shown in SEQ ID NO: 1.

[0055] MTSDMPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEEVRGVLEGFAARRLAERGMTAETHARFVALIAEGEALFAAG RLNGEDLDRYAAYNQAFHDTLVSAAGNGAVESALARNGFEPFAAAGALALDLMDLPAEYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:1).

[0056] Herein, the amino acid numbering of the amino acid sequence of the vanillic acid transactivator (e.g., the amino acid sequence shown in SEQ ID NO: 1) is determined from the N-terminus to the C-terminus, for example, position 97 refers to the 97th position numbered from the first position at the N-terminus;

[0057] In an optional embodiment of the present application, the site of the amino acid mutation is selected from position 97 or its equivalent site, position 127 or its equivalent site, position 137 or its equivalent site and position 183 or its equivalent site.

[0058] In an optional embodiment of the present application, the sites of the amino acid mutation further include position 16 or an equivalent site thereof and position 165 or an equivalent site thereof.

[0059] According to an embodiment of the present application, the sites of the amino acid mutation further include positions 16 and 165.

[0060] In an optional embodiment of the present application, the sites of the amino acid mutation further include at least one of position 7 or its equivalent site, position 31 or its equivalent site, position 103 or its equivalent site, position 113 or its equivalent site, position 162 or its equivalent site, position 164 or its equivalent site, and position 208 or its equivalent site.

[0061] According to an embodiment of the present application, the sites of the amino acid mutation further include at least one of positions 7, 31, 103, 113, 162, 164, and 208.

[0062] In an optional embodiment of the present application, the site of the amino acid mutation is selected from:

[0063] 1) position 97 or its equivalent, position 127 or its equivalent, position 137 or its equivalent, and position 183 or its equivalent, and optionally at least one of position 7 or its equivalent, position 103 or its equivalent, position 113 or its equivalent, position 162 or its equivalent, and position 164 or its equivalent; or

[0064] 2) position 97 or its equivalent, position 127 or its equivalent, position 137 or its equivalent, and position 183 or its equivalent, and optionally at least one of position 31 or its equivalent, position 103 or its equivalent, position 113 or its equivalent, position 162 or its equivalent, position 164 or its equivalent, and position 208 or its equivalent; or

[0065] 3) position 97 or its equivalent, position 127 or its equivalent, position 137 or its equivalent, position 183 or its equivalent, position 16 or its equivalent, and position 165 or its equivalent, and optionally at least one of position 31 or its equivalent, position 103 or its equivalent, position 113 or its equivalent, and position 208 or its equivalent.

[0066] According to an embodiment of the present application, the site of the amino acid mutation is selected from:

[0067] 1) at least one of the 97th, 127th, 137th and 183rd, and optionally the 7th, 103rd, 113th, 162nd and 164th positions; or

[0068] 2) at least one of positions 97, 127, 137, and 183, and optionally positions 31, 103, 113, 162, 164, and 208; or

[0069] 3) at least one of positions 97, 127, 137, 183, 16 and 165, and optionally at least one of positions 31, 103, 113 and 208.

[0070] In an optional embodiment of the present application, the site of the amino acid mutation is selected from position 97 or its equivalent site, position 103 or its equivalent site, position 113 or its equivalent site, position 127 or its equivalent site, position 137 or its equivalent site, position 164 or its equivalent site, and position 183 or its equivalent site.

[0071] According to an embodiment of the present application, the site of the amino acid mutation is selected from position 97, position 103, position 113, position 127, position 137, position 164, and position 183.

[0072] In an optional embodiment of the present application, the site of the amino acid mutation is selected from position 7 or its equivalent site, position 97 or its equivalent site, position 103 or its equivalent site, position 113 or its equivalent site, position 127 or its equivalent site, position 137 or its equivalent site, position 162 or its equivalent site, position 164 or its equivalent site, and position 183 or its equivalent site.

[0073] According to an embodiment of the present application, the site of the amino acid mutation is selected from position 7, position 97, position 103, position 113, position 127, position 137, position 162, position 164, and position 183.

[0074] In an optional embodiment of the present application, the site of the amino acid mutation is selected from position 97 or its equivalent site, position 113 or its equivalent site, position 127 or its equivalent site, position 137 or its equivalent site, and position 183 or its equivalent site.

[0075] According to an embodiment of the present application, the site of the amino acid mutation is selected from position 97, position 113, position 127, position 137, and position 183.

[0076] In an optional embodiment of the present application, the site of the amino acid mutation is selected from position 31 or its equivalent site, position 97 or its equivalent site, position 103 or its equivalent site, position 113 or its equivalent site, position 127 or its equivalent site, position 137 or its equivalent site, position 162 or its equivalent site, position 164 or its equivalent site, position 183 or its equivalent site, and position 208 or its equivalent site.

[0077] According to an embodiment of the present application, the site of the amino acid mutation is selected from position 31, position 97, position 103, position 113, position 127, position 137, position 162, position 164, position 183, and position 208.

[0078] In an optional embodiment of the present application, the site of the amino acid mutation is selected from position 16 or its equivalent site, position 31 or its equivalent site, position 97 or its equivalent site, position 103 or its equivalent site, position 113 or its equivalent site, position 127 or its equivalent site, position 137 or its equivalent site, position 165 or its equivalent site, position 183 or its equivalent site, and position 208 or its equivalent site.

[0079] According to an embodiment of the present application, the site of the amino acid mutation is selected from position 16, position 31, position 97, position 103, position 113, position 127, position 137, position 165, position 183, and position 208.

[0080] In an optional embodiment of the present application, the amino acid mutations include the following sites or their equivalent sites: R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L.

[0081] In this document, the term "R97H" refers to an amino acid sequence of the vanillic acid trans-transcription factor (e.g., an amino acid sequence as shown in SEQ ID NO: 1) in which the arginine at position 97 from the N-terminus is replaced by histidine; the term "A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y" refers to an amino acid sequence of the vanillic acid trans-transcription factor (e.g., an amino acid sequence as shown in SEQ ID NO: 1) in which the alanine at position 137 from the N-terminus is replaced by any one of the amino acids R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y; and the term "E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L" refers to an amino acid sequence of the vanillic acid trans-transcription factor (e.g., an amino acid sequence as shown in SEQ ID NO: 1). The amino acid numbering of the amino acid sequence shown in NO:1 is that the 183rd glutamic acid from the N-terminus is replaced by any one of K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L amino acids.

[0082] According to an embodiment of the present application, the amino acid mutation includes at least one of the following: R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L.

[0083] In an optional embodiment of the present application, the amino acid mutation sites further include M16I or an equivalent site and E165V or an equivalent site.

[0084] According to an embodiment of the present application, the sites of amino acid mutation further include M16I and E165V.

[0085] In an optional embodiment of the present application, the site of the amino acid mutation further includes at least one of R7H or its equivalent site, E31V or its equivalent site, M103L or its equivalent site, A113T or its equivalent site, N162S or its equivalent site, F164Y or its equivalent site, G208S or its equivalent site.

[0086] According to an embodiment of the present application, the site of the amino acid mutation further includes at least one of R7H, E31V, M103L, A113T, N162S, F164Y, and G208S.

[0087] In an optional embodiment of the present application, the amino acid mutation is selected from the following groups of sites or their equivalent sites: 1) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, and optionally at least one of R7H, M103L, A113T, N162S, F164Y; or

[0088] 2) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, and optionally at least one of E31V, M103L, A113T, N162S, F164Y, G208S; or

[0089] 3) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, M16I and E165V, and optionally at least one of E31V, M103L, A113T, G208S.

[0090] According to an embodiment of the present application, the site of the amino acid mutation is selected from:

[0091] 1) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, and optionally at least one of R7H, M103L, A113T, N162S, and F164Y; or

[0092] 2) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, and optionally at least one of E31V, M103L, A113T, N162S, F164Y, G208S; or

[0093] 3) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, M16I and E165V, and optionally at least one of E31V, M103L, A113T, G208S.

[0094] According to an embodiment of the present application, the site of the amino acid mutation is position 137, and the mutated amino acid at position 137 is A137V / E / T / D / M / S / A.

[0095] According to an embodiment of the present application, the site of the amino acid mutation is position 137, and the mutated amino acid at position 137 is A137V.

[0096] According to an embodiment of the present application, the site of the amino acid mutation is position 183, and the mutated amino acid at position 183 is E183K / R / V / I / T.

[0097] In an optional embodiment of the present application, the site of the amino acid mutation is selected from:

[0098] 1) R97H, L127Q, A137 V / E / T / D / M / S / A, and E183 K / R / V / I / TL, and optionally at least one of R7H, M103L, A113T, N162S, F164Y; or

[0099] 2) R97H, L127Q, A137 V / E / T / D / M / S / A, and E183 K / R / V / I / T, and optionally at least one of E31V, M103L, A113T, N162S, F164Y, G208S; or

[0100] 3) R97H, L127Q, A137 V / E / T / D / M / S / A, E183 K / R / V / I / T, M16I and E165V, and optionally at least one of E31V, M103L, A113T, G208S.

[0101] According to an embodiment of the present application, the site of the amino acid mutation is position 183, and the mutated amino acid at position 183 is E183K.

[0102] In an optional embodiment of the present application, the site of the amino acid mutation is selected from R97H or an equivalent site thereof, L127Q or an equivalent site thereof, A137G or an equivalent site thereof, E183K or an equivalent site thereof.

[0103] According to an embodiment of the present application, the site of the amino acid mutation is selected from R97H, L127Q, A137G, and E183K.

[0104] In an optional embodiment of the present application, the site of the amino acid mutation is selected from E31V or its equivalent site, R97H or its equivalent site, L127Q or its equivalent site, A137G or its equivalent site, E165V or its equivalent site, E183K or its equivalent site.

[0105] According to an embodiment of the present application, the site of the amino acid mutation is selected from E31V, R97H, L127Q, A137G, E165V, and E183K.

[0106] In an optional embodiment of the present application, the site of the amino acid mutation is selected from R7H or its equivalent site, R97H or its equivalent site, M103L or its equivalent site, A113T or its equivalent site, L127Q or its equivalent site, A137G or its equivalent site, N162S or its equivalent site, F164Y or its equivalent site, E183K or its equivalent site.

[0107] According to an embodiment of the present application, the site of the amino acid mutation is selected from R7H, R97H, M103L, A113T, L127Q, A137G, N162S, F164Y, and E183K.

[0108] In an optional embodiment of the present application, the site of the amino acid mutation is selected from R97H or its equivalent site, A113T or its equivalent site, L127Q or its equivalent site, A137V or its equivalent site, E183K or its equivalent site.

[0109] According to an embodiment of the present application, the site of the amino acid mutation is selected from R97H, A113T, L127Q, A137V, and E183K.

[0110] In an optional embodiment of the present application, the site of the amino acid mutation is selected from E31V or its equivalent site, R97H or its equivalent site, M103L or its equivalent site, A113T or its equivalent site, L127Q or its equivalent site, A137V or its equivalent site, N162S or its equivalent site, F164Y or its equivalent site, E183K or its equivalent site, G208S or its equivalent site.

[0111] According to an embodiment of the present application, the site of the amino acid mutation is selected from E31V, R97H, M103L, A113T, L127Q, A137V, N162S, F164Y, E183K, and G208S.

[0112] In an optional embodiment of the present application, the site of the amino acid mutation is selected from M16I or its equivalent site, E31V or its equivalent site, R97H or its equivalent site, M103L or its equivalent site, A113T or its equivalent site, L127Q or its equivalent site, A137V or its equivalent site, E165V or its equivalent site, E183K or its equivalent site, G208S or its equivalent site.

[0113] According to an embodiment of the present application, the site of the amino acid mutation is selected from M16I, E31V, R97H, M103L, A113T, L127Q, A137V, E165V, E183K, and G208S.

[0114] In an optional embodiment of the present application, the reverse vanillic acid trans-transcription factor has an amino acid sequence as shown in any one of SEQ ID NO: 3 to SEQ ID NO: 8, or an amino acid sequence having at least 90% sequence similarity thereto.

[0115] It should be noted that, in this application, "the amino acid sequence is as shown in SEQ ID NO: A" includes the amino acid sequence of SEQ ID NO: A or the amino acid sequence of conservatively modified forms of SEQ ID NO: A, all of which are within the scope of protection of this application. For example, "the amino acid sequence of the reverse vanillic acid trans-transcription factor is as shown in any one of SEQ ID NO: 3 to SEQ ID NO: 8" means that the reverse vanillic acid trans-transcription factor has the amino acid sequence as shown in any one of SEQ ID NO: 3 to SEQ ID NO: 8, or the amino acid sequence of conservatively modified forms of any one of SEQ ID NO: 3 to SEQ ID NO: 8, all of which are within the scope of protection of this application.

[0116] As used herein, "conservatively modified forms of an amino acid sequence" refer to amino acid modifications that do not significantly affect or alter the properties of the amino acid sequence comprising the sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the reverse vanilloid trans-transcription factor of the present application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the reverse vanilloid trans-transcription factor of the present application can be replaced by other amino acid residues from the same side chain family, and the modified reverse vanilloid trans-transcription factor can be tested for retention of function using the functional assay described herein. Exemplarily, conservative modifications are present in a number not exceeding 80% of the total number, preferably not exceeding 90% of the total number. In this article, "conservatively modified amino acid sequences" also include amino acid modifications of natural mutations. "Natural mutations" refer to mutations caused by changes in alleles due to individual differences and other reasons during the natural mutation process.

[0117] According to an embodiment of the present application, the amino acid sequence of the reverse vanilloid trans-transcription factor is shown in any one of SEQ ID NO: 3 to SEQ ID NO: 8.

[0118] MTSDMPHIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEEVRGVLEGFAARHLAERGLTAETHARFVTLIAEGEALFAAG RQNGEDLDRYAGYNQAFHDTLVSAAGNGAVESALARSGYEPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:3);

[0119] MTSDMPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGLTAETHARFVTLIAEGEALFAAGRQNGEDLDRYAVYNQAFHDTLVSAAGNGAVESALARSGYEPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:4);

[0120] MTSDMPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGLTAETHARFVTLIAEGEALFAAGRQNGEDLDRYAVYNQAFHDTLVSAAGNGAVESALARSGYEPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAESAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:5);

[0121] MTSDMPRIKPGQRVMIALRKMIASGEIKSGVRIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGLTAETHARFVTLIAEGEALFAAGRQNGEDLDRYAVYNQAFHDTLVSAAGNGAVESALARNGFVPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAESAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:6);

[0122] MTSDMPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEEVRGVLEGFAARHLAERGMTAETHARFVALIAEGEALFAAG RQNGEDLDRYAGYNQAFHDTLVSAAGNGAVESALARNGFEPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:7);

[0123] MTSDMPRIKPGQRVMMALRKMIASGEIKSGVRIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEEVRGVLEGFAARHLAERGMTAETHARFVALIAEGEALFAAG RQNGEDLDRYAGYNQAFHDTLVSAAGNGAVESALARNGFVPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:8).

[0124] Nucleic acid molecules, vectors, and recombinant cells

[0125] In a second aspect of the present application, the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the reverse vanillic acid trans-transcription factor described in the first aspect. The nucleic acid molecule of the present application can encode the reverse vanillic acid trans-transcription factor described in the first aspect.

[0126] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0127] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is provided in most cases, 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 implies disclosure of the other.

[0128] In the third aspect of this application, a vector is provided. According to embodiments of this application, the vector carries the nucleic acid molecule described in the second aspect. When the nucleic acid molecule is linked to an expression vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, that is, not derived from the expression vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked.

[0129] As used herein, "operably linked" means that a foreign gene is linked to an expression vector so that the control elements within the expression vector, such as transcriptional control sequences and translational control sequences, can function as intended to regulate the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids, bacteriophages, and the like.

[0130] According to some specific embodiments of the present application, after the expression vector is introduced into suitable recipient cells, it can effectively achieve the expression of the aforementioned reverse vanillic acid trans-transcription factor under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of the reverse vanillic acid trans-transcription factor.

[0131] In some specific embodiments of the present application, the above-mentioned vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0132] In some specific embodiments of the present application, the above-mentioned vector is a lentiviral vector.

[0133] In an optional embodiment of the present application, the above-mentioned expression vector is a plasmid expression vector.

[0134] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell comprises the nucleic acid molecule described in the second aspect, or the vector described in the third aspect; or alternatively, expresses the reverse-vanillic acid trans-transcription factor described in the first aspect. Under suitable conditions, the recombinant cell can efficiently express the aforementioned vanillic acid trans-transcription factor within the cell.

[0135] It should be noted that "suitable conditions" refer to conditions suitable for the expression of the vanillic acid trans-transcription factor described herein. Those skilled in the art will readily appreciate that suitable conditions for the expression of the vanillic acid trans-transcription factor include, but are not limited to, a suitable transformation or transfection method, suitable transformation or transfection conditions, healthy host cells, suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the vanillic acid trans-transcription factor based on the specific laboratory environment.

[0136] According to an embodiment of the present application, the recombinant cell is obtained by introducing the expression vector described in the eighth aspect into a host cell.

[0137] It should be noted that the recombinant cells described herein are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, among others. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as fall armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described herein are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0138] According to an embodiment of the present application, the recombinant cell is a eukaryotic cell, preferably a mammalian cell.

[0139] use

[0140] In the fifth aspect of the present application, the present application proposes the use of the reverse vanillic acid trans-transcription factor described in the first aspect, the nucleic acid molecule described in the second aspect, the vector described in the third aspect, or the recombinant cell described in the fourth aspect in preparing a vanillic acid gene circuit control system.

[0141] Below in conjunction with embodiment, the scheme of the application will be explained. Those skilled in the art will appreciate that the following examples are merely for illustration of the application and should not be considered as limiting the scope of the application. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents used or instruments not indicated by the manufacturer are conventional products that can be obtained commercially.

[0142] Example 1: Screening of mutation sites of the VanR gene

[0143] This example is based on the vanilloid trans-transcription factor VanR (amino acid sequence shown in SEQ ID NO: 1). Nucleotide mutations were introduced into the VanR gene in vitro by error-prone PCR mutagenesis, and the VanR gene was subjected to multiple rounds of random mutagenesis.

[0144] 1. The randomly selected prokaryotic vector was CTD-TAC promoter-RBS-VanR-Terminator-pVanCC-RiboJ-RBS-EGFP-terminator.

[0145] CTD-TAC promoter sequence:

[0146] TATAGGCAAAAAAGGCCTTGACATCCCACCTCACGTATGCTATAATGTGTGCAGTCTGACGCGG CG (SEQ ID NO: 9), this promoter is a constitutive promoter and can express VanR without the need for induction.

[0147] RBS sequence:

[0148] TTTGTTTAACTTTAAGAAGGAGA (SEQ ID NO: 10), this RBS sequence was designed and optimized by predecessors specifically for VanR expression, and can improve the expression efficiency of VanR.

[0149] The VanR sequence is shown in SEQ ID NO: 1 above.

[0150] pVanCC sequence:

[0151] ATTGGATCCAATTGACAGCTAGCTCAGTCCTAGGTACCATTGGATCCAAT (SEQ ID NO: 11), this promoter contains VanO on both sides. It is an optimized promoter that can work in E. coli and can be regulated by VanR and Va. Its working logic is: when Va is not present in the environment, VanR binds to the VanO regions on both sides, preventing the expression of downstream genes. When a certain amount of Va is added, VanR binds to Va and undergoes conformational change, leaving VanO, allowing downstream genes to start expression.

[0152] RiboJ sequence:

[0153] AGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA (SEQ ID NO: 12) is a 75-nucleotide sequence composed of tobacco ringspot virus (rTRSV) satellite RNA, which can reduce the leaky expression of the system under non-induced conditions.

[0154] EGFP amino acid sequence:

[0155] MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 13) is an enhanced green fluorescent protein. The fluorescence intensity of the green fluorescent protein was detected using a microplate reader (excitation light 470-15 / emission light 515-20) to characterize the expression of the entire system.

[0156] 2. The reaction conditions of the error-prone PCR mutagenesis method are as follows:

[0157] Reagents Dosage 10xPCR Buffer 20uL Taq DNA Polymerase (2.5U) 2uL <![CDATA[MgCl2(100mM)]]> 12uL <![CDATA[MnCl2(10mM)]]> 6uL dATP (0.2 mM) 5uL dTTP (1 mM) 5uL dCTP (1 mM) 5uL dGTP (0.2 mM) 5uL VanR template (10 ng / μL) 4uL Primer-F 4uL Primer-R 4uL <![CDATA[ddH2O]]> 134uL total 200uL

[0158] Upstream primer primer-F: GTTTAACTTTAAGAAGGAGATATACCATGACTAGTG (SEQ ID NO: 14);

[0159] Downstream primer primer-R: CTTGTCGACGGAGCTCGAATTCTTA (SEQ ID NO: 15).

[0160] After mixing the reaction solution, divide it into 10-20uL / tube for PCR, so that the random mutations in each tube are amplified as independent random events, which is beneficial to increase the storage capacity of the total mutation library.

[0161] PCR amplification conditions:

[0162]

[0163]

[0164] After the error-prone PCR amplification product was purified using a DNA purification kit, the error-prone PCR product and expression plasmid pCY146 were digested with restriction endonucleases SpeI and EcoRI, respectively, ligated with T4 DNA Ligase, transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB (containing 100 μg / mL ampicillin) plates, and cultured at 37°C for 12 h to construct the rVanR mutation library.

[0165] 3. The screening method is as follows:

[0166] The rVanR screening strategy: Wild-type VanR will suppress the fluorescence expression of the reporter system PCY146 in the blank group without Va, but will express high-intensity fluorescence in the drug-treated group with 0.5mM Va. Therefore, the fluorescence value of the drug-treated group will be much higher than that of the blank group. The behavior of rVanR must be opposite to that of wild-type VanR, so we need to screen for transformants whose fluorescence value in the drug-treated group is lower than that of the blank group. Because Va has a bactericidal effect, we need to measure the OD value of each group at the same time. 600 To characterize the bacterial density in each group, the fluorescence value of each group was divided by OD 600 As a result, a relative fluorescence value is obtained, which is used to represent the expression status of each group. We will screen and collect transformants whose relative fluorescence values ​​of the drug-treated group are lower than those of the blank group.

[0167] To obtain rVanR mutants, LB plates (containing 100 μg / mL ampicillin) cultured at 37°C for 12 h were illuminated with blue light to identify and mark rVanR transformants that emitted green fluorescence without Va induction. Using a sterile toothpick, 0.5 mL of these marked transformants was inoculated into liquid LB medium containing 100 μg / mL ampicillin and incubated at 37°C at 220 rpm for 2.5 h until the OD600 reached 0.5–0.7.

[0168] Screening of rVanR mutants: Add 0.6 mL of liquid LB medium containing 100 μg / mL ampicillin to a 48-well plate and divide it into two groups. One group is a blank group, to which 1 μL of anhydrous ethanol is added, and the other group is an experimental group, to which 1 μL of a 0.5 M Va solution dissolved in anhydrous ethanol is added, so that the final Va concentration is 0.5 mM. Add 100 μL of transformants with an OD600 of 0.5-0.7 to each of the experimental and control groups. After incubation at 37°C, 220 rpm in a shaker for 16 hours, place the 48-well plate in a microplate reader and measure the OD values ​​of each group. 600 The fluorescence intensity was compared and the relative fluorescence value was calculated. The transformants with absolute fluorescence ratio (blank group > experimental group) were screened out, plasmids were extracted and sequenced, and the fragment was constructed into a eukaryotic vector to verify the performance of its mutant in HEK-293. At the same time, the mutant plasmid was used as a template for the next round of error-prone PCR to construct a mutant library.

[0169] Using the same method as above, multiple rounds of error-prone PCR were performed with the mutant gene as a template to construct a mutant library, ultimately yielding rVanR01 (mutation sites R7H, R97H, A113T, L127Q, N162S, amino acid sequence shown in SEQ ID NO: 2).

[0170] MTSDMPHIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEEVRGVLEGFAARHLAERGMTAETHARFVTLIAEGEALFAAG RQNGEDLDRYAAYNQAFHDTLVSAAGNGAVESALARSGFEPFAAAGALALDLMDLPAEYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:2).

[0171] The rVanR01 obtained above was used to detect the rVanR function of rVanR01 in Escherichia coli and HEK-293 cell systems, respectively. The VanR(WT) fragment of the constructed and functionally verified eukaryotic vector pCY176 CMV-VanR(WT)-VP64-pA was double-digested with NheI and EcoRI. The rVanR01 sequence was then integrated into the plasmid by PCR and homologous recombination and named pCY177CMV-rVanR01-VP64-pA. pCY177 and the constructed reporter plasmid pCY1755*VanO-minP-nanoluc-pA were transfected into HEK-293 cells seeded at 80,000 / well in a 24-well plate 18 hours in advance using PEI transfection reagent. 1uL 0.5M Va was added to the drug-treated group to a final concentration of 0.5mM, and 1uL was added to the control group. DMSO, three replicates per group, 48 hours later, NanoLuc TM Luciferase kit was used for detection, and the test results were as follows Figure 1 shown.

[0172] The results showed that the value of the drug-added group was 1.04 times that of the control group, while the value of the wild-type VanR control group under the same conditions was 30.4 times that of the drug-added group, proving that rVanR01 has begun to exhibit reversal function, but its value in the HEK-293 cell system is only 1.04 times that of the control group, and further iterative optimization is needed.

[0173] 2. Using rVanR01 as a template, error-prone PCR mutagenesis (see step 1 of this example for specific steps) was used for further iterative optimization to obtain four mutants: 209-1, 210-1, 211-1, and 212-1. Using the method of step 1 of this example, the four mutants 209-1, 210-1, 211-1, and 212-1 were cultured in E. coli and HEK-293 cell systems, and their rVanR functions were tested. The test results in the HEK-293 cell system are shown in Figure 2. Figure 2 shown.

[0174] The results showed that both mutants 210-1 and 211-1 exhibited excellent rVanR function in HEK-293 cell experiments, with 210-1 showing a 6.8-fold increase and 211-1 showing a 1.4-fold increase in HEK-293 cell experiments. The 210-1 mutant exhibited even lower leaky expression in the absence of drug, and was used as a template for subsequent optimization. 210-1 was subsequently named rVanR02 (mutation sites: R7H, R97H, M103L, A113T, L127Q, A137G, N162S, F164Y, E183K; amino acid sequence shown in SEQ ID NO: 3).

[0175] 3. The five mutations (R7H, R97H, A113T, L127Q, N162S) in the rVanR02 mutant were reversed to the wild-type site, and the four mutations (M103L, A137G, F164Y, E183K) in the rVanR02 mutant were reversed to the wild-type site. The method of step 1 of this example was used to detect the rVanR function of the mutants that were reversed to the wild-type site in the HEK-293 cell system. The test results are as follows: Figure 3 shown.

[0176] The results showed that R7, A113, N162, M103, and F164 were nonsense mutations;

[0177] R97H and E183K are key mutations in rVanR logic. R97H mutation reduces the ability of rVanR to bind VanO in the absence of Va; E183K mutation determines the existence of rVanR logic.

[0178] A137 G and L127Q are rate-limiting mutations. By changing A137, the binding ability of rVanR can be changed, thereby adjusting the expression level of the reported value.

[0179] Therefore, R97, A137, E183, and L127 are key mutation sites.

[0180] 4. Further, based on the rVanR02 mutant, a deep mutation scan was performed on A137 and E183. First, using point mutation, the amino acid residue A at position 137 was replaced with the remaining 19 amino acid residues to observe whether there are amino acid residues that can improve the function. The results are as follows: Figure 4 and Figure 5 shown.

[0181] turn out:

[0182] Various mutations of A137 (A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y) can all improve the rVanR ability of the mutant. For example, A137V has a higher multiple than A137G. However, A137 itself can also improve the rVanR ability, but the background leakage is too high in the absence of Va stimulation, so it may be suitable for systems that require a larger output value.

[0183] The A137V mutant sequence was selected, and based on this, the same point mutation operation was performed on E183 to observe whether there were mutations in the remaining 19 amino acid residues that were more suitable for our system.

[0184] Various mutations of E183 (E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L) can improve the rVanR ability of the mutants.

[0185] 5. Further, based on the E183K site, by analyzing the protein and molecular docking map, it was found that E183 is close to the folding region and is surrounded by polar negatively charged amino acid residues. Therefore, based on rVanR03, the first 8 amino acid residues of E183 were mutated to K, and the method of step 1 of this example was used to test the rVanR function of the mutant in the HEK-293 cell system. The test results are as follows: Figure 6 shown.

[0186] The results showed that mutating the adjacent amino acid to K did not result in better or worse consequences.

[0187] 6. Based on the key mutation sites, rVanR03 mutants (R97H, A113T, L127Q, A137V, N162S, F164Y, E183K, amino acid sequence as shown in SEQ ID NO: 4) were designed. The rVanR03 mutants were tested for rVanR function in HEK-293 cell system using the method of step 1 of this example. Figure 7 .

[0188] In addition, the present application further reversed the five mutations (R97H, A113T, L127Q, A137V, and E183K) in the rVanR03 mutant to the wild-type sites. The method of step 1 of this example was used to detect the rVanR function of the mutants that had been reversed to the wild-type sites in the HEK-293 cell system. The results showed that A113 was a nonsense mutation, and R97, A137, E183, and L127 were key mutation sites.

[0189] Example 2: Optimization of reverse vanilloid trans-transcription factor

[0190] 1. Since the rVanR function folds of the rVanR01 to rVanR03 mutants in Example 1 are within 10, the rVanR04 mutant (R97H, M103L, A113T, L127Q, A137V, N162S, F164Y, E183K, G208S, amino acid sequence as shown in SEQ ID NO: 5) was further iterated based on rVanR03. The method of step 1 of this example was used to detect its rVanR function in the HEK-293 cell system. The test results are as follows: Figure 7 The results showed that the rVanR04 mutant had a fold increase of 11.32 in the HEK-293 cell experiment.

[0191] 2. Further, based on rVanR04, rVanR05 mutant (M16I, E31V, R97H, M103L, A113T, L127Q, A137V, E165V, E183K, G208S, amino acid sequence as shown in SEQ ID NO: 6) was obtained through iteration and optimization. The method of step 1 of this example was used to detect its rVanR function in the HEK-293 cell system. The test results were as follows: Figure 8 The results showed that the rVanR05 mutant had a fold increase of 35.8 in the HEK-293 cell experiment.

[0192] In addition, the present application further reversed the 4 mutations (M16I, E31V, E165V, G208S) newly introduced in the rVanR05 mutant compared to rVanR03 to the wild-type site. The method of step 1 of this example was used to detect the rVanR function of the mutants reversed to the wild-type site in the HEK-293 cell system. The results are as follows: Figure 9 shown.

[0193] The results showed that M16 and G208 were nonsense mutations, and M31 / E165 were key mutation sites.

[0194] Based on the above experimental results, it was concluded that M31, R97, L127, A137, E165, and E183 are key mutation sites.

[0195] Example 3: Vanillic acid gene circuit control system

[0196] 1. Preparation of vanillic acid gene circuit control system

[0197] This example targets rVanR05 and prepares a vanillic acid gene circuit control system that can be used for cell therapy. The reporter plasmid currently tested is: pCY175 (5*VanO-minP-Secretory(IgK)-nanoluc-pA).

[0198] The DNA sequence of 5*VanO-minP is:

[0199] ATTGGATCCAATGCATTGGATCCAATGGATTGGATCCAATCGATTGGATCCAATtgATTGGAT CCAATTTAATTAAGCAGCTCCTGCAGGTAGAGGGTATATAATGGAAGCTCGACTTCCAG (SEQ ID NO: 16).

[0200] The amino acid sequence of Secretory(IgK)-nanoluc is:

[0201] MTSETDTLLLWVLLLWVPGSTGDASGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQ NLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (SE Q ID NO: 17), wherein Secretory (IgK) is an exocrine short peptide that assists nanoluc in being secreted outside the cell. Secretory (IgK) and nanoluc are connected using a SGGSG flexible short peptide.

[0202] Recipient plasmid: CMV-rVanR-VP64-pA; wherein the amino acid sequence of VP64 is:

[0203] DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML (SEQ ID NO: 18).

[0204] The principle is as follows: minP is the smallest eukaryotic TATA-box promoter, with very low basal leaky expression when not induced. However, it cannot self-activate and express downstream genes in isolation. Only when it binds to transactivation domains such as VP16 (a transactivation domain derived from the Herpes simplex virus), VP64 formed by a tandem complex of four VP16s, and the ternary transcription activator VPR (VP64-p65-Rta), can it recruit key factors of the host transcription complex, such as VP16, VP64, and VPR, to initiate transcription of the minimal promoter and activate downstream gene expression. However, the native VP16 / VP64 / VPR cannot self-localize to minP, so the inventors expressed rVanR by fusion with VP64 and placed the five-repeat vanilloid operon VanO in front of minP. When the small molecule Va is added, rVanR-VP64 binds to VanO, and VP64 then binds to minP and activates the expression of the downstream signaling pathway.

[0205] Furthermore, VP64 in the vanillic acid gene circuit control system was replaced with VPR, which has stronger binding ability. The amino acid sequence of VPR is:

[0206] (SEQ ID NO: 19).

[0207] The method of step 1 of this example was used to detect the rVanR function of the various vanillic acid gene circuit control systems prepared above in the HEK-293 cell system. The results were as follows: Figure 10 shown.

[0208] The results showed that by first replacing VP64 with VPR, which has stronger combining capabilities, the system showed a stronger output multiple (51.9 times).

[0209] 2. The vanillic acid gene circuit control system containing rVanR-VPR (plasmid pCY233) in step 1 of this example was optimized.

[0210] 2.1 Replace the reporter plasmid from pCY175 (5*VanO-minP-Secretory(IgK)-nanoluc-pA) to pSL173 (5*VanO-P hCMVmin -SEAP-pA),

[0211] Among them, P hCMVmin The sequence is as follows:

[0212] TCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGT TTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCGG (SEQ ID NO: 21). P hCMVmin It is the minimal structure of the strong eukaryotic promoter CMV. Its function and activation mode are similar to minP, but it has a higher expression level than minP.

[0213] The amino acid sequence of SEAP is as follows:

[0214] MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTV

[0215] TAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAAR

[0216] FNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGC

[0217] QDIATQLISNMDIDVILGGGRKYMFPMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWN

[0218] RTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDH

[0219] GHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKA

[0220] YTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQE

[0221] QTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPG (SEQ ID NO: 20).

[0222] 2.2 Further optimize the ratio of receptor plasmid to reporter plasmid, with a total transfection concentration of 300 ng. Dynamically adjust the ratio of receptor plasmid to reporter plasmid. For details on the ratios and the test results corresponding to each ratio, see Figure 11 The results showed that the ratios of the various receptor plasmids and reporter plasmids set in this example all had good rVanR functions.

[0223] 2.3 The time dynamic results of the test under the condition of 8:1 ratio of receptor plasmid to reporter plasmid. For specific results, see Figure 12 The results showed that the highest multiple was 69.7 times at 0.5 mM Va concentration after 48 h.

[0224] 3. Based on the optimal vanillic acid gene circuit control system obtained in step 2 of this example (the ratio of the receptor plasmid to the reporter plasmid is 8:1), the response multiple of the entire system was tested from 0 to 8 mM Va concentration, showing that it entered a plateau phase after a concentration of 2 mM; and the fluorescent reporter expression of 0 to 0.5 mM Va was tested. The results are shown in Figure 13 The results showed that the fluorescence results were consistent with those reported by SEAP.

[0225] 4. A reversibility experiment was performed on the superior vanillic acid gene circuit control system (the ratio of the receptor plasmid to the reporter plasmid was 8:1) obtained in step 2 of this example. The specific implementation is as follows:

[0226] The circuit system plasmid was transfected using PEI transfection reagent into HEK-293 cells seeded 18 hours in advance at 80,000 cells / well on a 24-well plate. 8 hours later, the medium was changed and Va was added at a final concentration of 0.5mM. Samples were taken regularly to observe the results from 0 to 48 hours. At 48 hours, the cells were trypsinized, and half of the cells were plated on a 24-well plate without adding Va. Samples were taken regularly to observe the results from 0 to 48 hours. Subsequently, the cells were trypsinized again, and half of the cells were plated on a 24-well plate with Va added at a final concentration of 0.5mM. Samples were taken regularly to observe the results from 0 to 48 hours. This experiment aimed to observe whether the binding of the circuit system and Va is reversible and whether Va permanently affects the switching of the system. The results are shown in [ 15 ]. Figure 12 The results showed that the binding between rVanR and VanO was reversible with and without drug addition.

[0227] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means 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 application. 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.

[0228] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A reverse vanillic acid trans-transcription factor, characterized in that Compared with the vanillic acid trans-transcription factor, the reverse vanillic acid trans-transcription factor has amino acid mutations, and the sites of the amino acid mutations include the 97th, 127th, 137th and 183rd positions.

2. The reverse vanillic acid trans-transcription factor according to claim 1, characterized in that The amino acid mutation sites further include positions 31 and 165; And / or, the amino acid mutation site further includes at least one of position 7, position 31, position 103, position 113, position 162, position 164, and position 208; And / or, the amino acid mutation includes at least one of the following: R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L; And / or, the amino acid mutation sites further include M16I and E165V; And / or, the amino acid mutation site further includes at least one of R7H, E31V, M103L, A113T, N162S, F164Y, and G208S.

3. The reverse vanillic acid trans-transcription factor according to claim 2, characterized in that The amino acid mutation site is selected from: 1) at least one of the 97th, 127th, 137th and 183rd, and optionally the 7th, 103rd, 113th, 162nd and 164th positions; or 2) at least one of the 97th, 127th, 137th and 183rd, and optionally the 31st, 103rd, 113th, 162nd, 164th, 208th; or 3) at least one of the 97th, 127th, 137th, 183rd, 16th and 165th, and optionally the 31st, 103rd, 113th, 208th; And / or, the site of the amino acid mutation is selected from: 1) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, and optionally at least one of R7H, M103L, A113T, N162S, F164Y; or 2) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, and E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, and optionally at least one of E31V, M103L, A113T, N162S, F164Y, G208S; or 3) R97H, L127Q, A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y, E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L, M16I and E165V, and optionally at least one of E31V, M103L, A113T, G208S.

4. The reverse vanillic acid trans-transcription factor according to claim 2 or 3, characterized in that The amino acid mutation site is position 137, and the mutant amino acid at position 137 is A137V / E / T / D / M / S / A, preferably A137V; Optionally, the amino acid mutation site is position 183, and the mutant amino acid at position 183 is E183K / R / V / I / T, preferably E183K; Optionally, the amino acid mutation site is selected from R97H, L127Q, A137G, E183K; And / or, the amino acid mutation site is selected from E31V, R97H, L127Q, A137G, E165V, E183K; And / or, the amino acid mutation site is selected from R7H, R97H, M103L, A113T, L127Q, A137G, N162S, F164Y, E183K; And / or, the amino acid mutation site is selected from R97H, A113T, L127Q, A137V, E183K; and / or, the amino acid mutation site is selected from E31V, R97H, M103L, A113T, L127Q, A137V, N162S, F164Y, E183K, G208S; And / or, the site of the amino acid mutation is selected from M16I, E31V, R97H, M103L, A113T, L127Q, A137V, E165V, E183K, and G208S.

5. The reverse vanillic acid trans-transcription factor according to claim 1, characterized in that The amino acid sequence of the vanillic acid trans-transcription factor is shown in SEQ ID NO:

1.

6. The reverse vanillic acid trans-transcription factor according to claim 1, characterized in that The amino acid sequence of the reverse vanillic acid trans-transcription factor is shown in any one of SEQ ID NO: 3 to SEQ ID NO:

8.

7. A nucleic acid molecule, vector or recombinant cell, characterized in that: The nucleic acid molecule encodes the reverse vanillic acid trans-transcription factor according to any one of claims 1 to 6.

8. A carrier, characterized in that The vector carries the nucleic acid molecule of claim 7.

9. A recombinant cell, characterized in that The recombinant cell comprises the nucleic acid molecule of claim 7, or the vector of claim 8; or expresses the reverse vanillic acid trans-transcription factor of any one of claims 1 to 6.

10. Use of the reverse vanillic acid trans-transcription factor according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the vector according to claim 8 or the recombinant cell according to claim 9 in preparing a vanillic acid gene circuit control system.

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