Method for reverse regulation of vanillic acid gene circuit control system and vanillic acid gene circuit control system

Abstract

The invention discloses a method for reversely regulating and controlling a vanillic acid gene circuit control system, the vanillic acid gene circuit control system comprises a vanillic acid trans-transcription factor, a vanillic acid operon and optional Va, and the method comprises the following steps: carrying out amino acid mutation on the vanillic acid trans-transcription factor, therefore, reverse regulation and control of the vanillic acid gene circuit control system can be realized; wherein the amino acid mutation sites comprise the 97th site, the 127th site, the 137th site and the 183th site. According to the vanillic acid trans-transcription factor, a Va dissociable vanillic acid operon is not added, and a Va combinable vanillic acid operon is added. Therefore, by means of the method, the vanillic acid gene circuit control system can be reversely regulated and controlled, the system can be kept in a closed state without adding Va, the system can be kept in an activated state with adding Va, higher practicability and safety are achieved, a new method and a new strategy are provided for a clinical cell treatment scheme, and good clinical application prospects are achieved.

Description

Technical Field

[0001] This application belongs to the fields of genetic engineering technology and synthetic biology, and particularly relates to a method for reversely regulating a vanillic acid gene circuit control system and a vanillic acid gene circuit control system. Background Art

[0002] Vanillic acid (Va) is a natural phenolic compound in plants and is widely used in food additives (FAO / WHO Expert Committee on Food Additives, JECFA no. 959). On the one hand, it has good medicinal value, such as anti-cancer, anti-inflammatory, and neuroprotective activities. On the other hand, it has excellent safety, with high doses (no obvious adverse reactions were observed after continuous administration to rats at a dose of 1000 mg / Kg / Day for 2 weeks), and its metabolic rate in human blood is fast, and it will not accumulate for a long time to affect the physiological activities of the recipient, making it an excellent inducer small molecule.

[0003] The vanillic acid transcriptional repressor (VanR) derived from Caulobacter crescentus can specifically bind to one or several inverted repeat operators VanO (ATTGGATCCAAT) when Va is absent in the environment, and dissociates from VanO immediately after binding to the Va small molecule.

[0004] The vanillic acid gene circuit control system Van-Off system, a cell therapy switch designed based on Va, VanR, and VanO, is in an open state when Va is not added, and can only be kept in a closed state by continuously adding Va. Such a logic makes it difficult to apply this system in actual treatment. Based on this, it is very important to develop a method for reversely regulating a vanillic acid gene circuit control system or a reversely regulated vanillic acid gene circuit control system. Summary of the Invention

[0005] This application aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, this application provides a method for reversely regulating a vanillic acid gene circuit control system.

[0006] This application is completed based on the following discoveries of the inventors:

[0007] In this application, through error-prone PCR mutagenesis, the VanR gene was subjected to multiple rounds of random mutations, and the mutation sites were semi-rationally assisted and optimized to screen for a mutant rVanR (i.e., reverse vanillic acid transcriptional factor) that can regulate gene logic opposite to VanR after binding to vanillic acid. When the mutant rVanR does not bind to the Va small molecule, it dissociates VanO (vanillic acid operator site), and when the mutant rVanR binds to Va, it binds to the operator site VanO.

[0008] Thus, the inventors designed a transgenic expression switch device regulated by Va through synthetic biology, namely the vanillic acid gene circuit control system. Engineered cells containing the vanillic acid gene circuit control system are in a silent state without Va activation. When a certain concentration (0.1 - 0.5 mM) of Va is added, the switch can be activated to express downstream genes. The method of the reverse-regulated vanillic acid gene circuit control system and the vanillic acid gene circuit control system in this application dissociate VanO (vanillic acid operon) when not binding to the Va small molecule and bind to the vanillic acid operon VanO after binding to Va. Therefore, based on the method of the reverse-regulated vanillic acid gene circuit control system and the reverse-regulated vanillic acid gene circuit control system of this application, the system can remain in the closed state without adding Va and in the activated state when Va is added, having higher practicability and safety, providing new methods and strategies for clinical cell therapy programs and having good clinical application prospects.

[0009] Based on this, in the first aspect of this application, the present application proposes a method for reverse-regulating a vanillic acid gene circuit control system. The vanillic acid gene circuit control system includes a reverse vanillic acid transcriptional factor, a vanillic acid operator, and optionally Va. The method includes: performing amino acid mutations on the vanillic acid transcriptional factor to obtain the reverse vanillic acid transcriptional factor; achieving reverse regulation of the vanillic acid gene circuit control system through the reverse vanillic acid transcriptional factor; wherein the amino acid mutation sites include the 97th, 127th, 137th, and 183rd positions.

[0010] By performing amino acid mutations on the above sites of the vanillic acid transcriptional factor in this application, the invented reverse vanillic acid transcriptional factor can dissociate the vanillic acid operon without adding Va; adding Va can bind to the vanillic acid operon VanO. Thus, the method of this application can design a reverse-regulated vanillic acid gene circuit control system, enabling the system to remain in the closed state without adding Va and in the activated state when Va is added, having higher practicability and safety, providing new methods and strategies for clinical cell therapy programs and having good clinical application prospects.

[0011] In the second aspect of the present application, the present application provides a vanillic acid gene circuit control system. According to an embodiment of the present application, the vanillic acid gene circuit control system includes: a target protein unit, the target protein unit includes a vanillic acid operon, an inducible expression nucleic acid sequence, and a target protein, the 3'-end of the vanillic acid operon is connected to the 5'-end of the inducible expression nucleic acid sequence, and the 3'-end of the inducible expression nucleic acid sequence is connected to the 5'-end of the nucleic acid molecule encoding the target protein; a receptor protein unit, the receptor protein unit includes a reverse vanillic acid trans - acting factor and a transcriptional activator protein, the reverse vanillic acid trans - acting factor is used to bind vanillic acid, and the transcriptional activator protein is used to induce the expression nucleic acid sequence; wherein, when vanillic acid is present, the reverse vanillic acid trans - acting factor binds to the vanillic acid operon; when vanillic acid is absent, the reverse vanillic acid trans - acting factor does not bind to the vanillic acid operon; compared with the vanillic acid trans - acting factor, the reverse vanillic acid trans - acting factor has amino acid mutations, and the sites of the amino acid mutations include the 97th, 127th, 137th, and 183rd positions.

[0012] The reverse vanillic acid trans - acting factor of the present application dissociates from the vanillic acid operon without adding Va; adding Va can bind to the vanillic acid operon VanO. Thus, the vanillic acid gene circuit control system prepared with the above - mentioned reverse vanillic acid trans - acting factor can maintain the system in a closed state without adding Va and maintain the system in an activated state by adding Va, which has higher practicability and safety. It provides new methods and strategies for clinical cell therapy programs and has good clinical application prospects.

[0013] In the third aspect of the present application, the present application provides a genetically engineered cell. According to an embodiment of the present application, the genetically engineered cell includes the vanillic acid gene circuit control system described in the second aspect.

[0014] In the fourth aspect of the present application, the present application provides the use of the vanillic acid gene circuit control system described in the second aspect in the preparation of a drug, and the target protein in the vanillic acid gene circuit control system is selected from drug proteins.

[0015] The additional aspects and advantages of the present application will be partly given in the following description, partly will become obvious from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above - mentioned and / or additional aspects and advantages of the present application will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, wherein:

[0017] Figure 1 It is the detection result of the rVanR function of rVanR01 in Example 1 of the present application and the WT function detection result of VanR;

[0018] Figure 2 Detection results of the rVanR function of four mutants, namely 209-1, 210-1 (rVanR02), 211-1, and 212-1, in Example 1 of this application;

[0019] Figure 3 Detection results of the rVanR function after single-point reverse mutation back to the wild-type site based on rVanR01 and rVanR02 respectively in Example 1 of this application;

[0020] Figure 4 Experimental results of single-point deep scanning of HEK-293 cells at A137 in the rVanR02 mutant in Example 1 of this application;

[0021] Figure 5 Experimental results of single-point deep scanning of HEK-293 cells at E183 in the rVanR02 mutant in Example 1 of this application;

[0022] Figure 6 Experimental results of single-point deep scanning of HEK-293 cells at the first 8 amino acid residues before E183 in the rVanR02 mutant in Example 1 of this application, where the single point is K;

[0023] Figure 7 Detection results of the rVanR function of the rVanR02 mutant and the rVanR03 mutant in Example 1 of this application;

[0024] Figure 8 Detection results of the rVanR function of rVanR04 and rVanR05 in the HEK-293 cell system in Example 2 of this application;

[0025] Figure 9 Detection results of the rVanR function after single-point reverse mutation back to the wild-type site of rVanR05 in Example 2 of this application;

[0026] Figure 10 Detection results of the rVanR function of each vanillic acid gene circuit control system in the HEK-293 cell system in Example 3 of this application;

[0027] Figure 11 Detection results of the rVanR function of the vanillic acid gene circuit control system obtained by adjusting the ratio of the reporter plasmid to the receptor plasmid (SEAP) in the HEK-293 cell system in Example 3 of this application;

[0028] Figure 12 Results of time-course observation when the ratio of the reporter plasmid to the receptor plasmid is 1:8 in Example 3 of this application;

[0029] Figure 13 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 this application, the results of the response multiples to it at Va concentrations of 0 to 8 mM, and the results of the fluorescence reporter expression at 0 to 0.5 mM Va;

[0030] Figure 14 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 this application, the results of the reversibility experiment analysis. Detailed implementation manners

[0031] The embodiments of the present application will be described in detail below. The following described embodiments are exemplary and are only used to explain the present application, and should not be construed as a limitation to the present application.

[0032] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, the meaning of "plurality" is two or more.

[0033] Detailed description of the present application

[0034] Definitions and general terms

[0035] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values and individual point values of each range, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0036] To make it easier to understand the present application, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present application pertains. The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0037] In this document, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0038] As used herein, the terms "optionally", "optional", or "option" generally mean that the subsequent recited event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where it does not.

[0039] 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 are determined by conventional methods to calculate the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences. See, for example, 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, D.C.)). 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. 70:173-187 (1997)); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs using 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.

[0040] In this text, the term "having at least 90% sequence similarity" can refer to having a sequence similarity 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 in this application can be measured using sequence analysis software. For example, the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this application are all shown in the N-terminal to C-terminal manner.

[0041] In this text, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid differences (mutations) and is different from the reference sequence. The difference can be a substitution, deletion or insertion of one or more amino acids.

[0042] This application proposes a method for reverse-regulating a vanillic acid gene circuit control system and a vanillic acid gene circuit control system, which will be described in detail below respectively.

[0043] Method for Reverse-Regulating Vanillic Acid Gene Circuit Control System

[0044] In the first aspect of this application, a method for reverse-regulating a vanillic acid gene circuit control system is proposed. The vanillic acid gene circuit control system includes a reverse vanillic acid trans-acting factor, a vanillic acid operon, and optionally Va. The method includes: performing amino acid mutations on the vanillic acid trans-acting factor to obtain the reverse vanillic acid trans-acting factor; achieving reverse regulation of the vanillic acid gene circuit control system through the reverse vanillic acid trans-acting factor; wherein, the sites of the amino acid mutations include the 97th, 127th, 137th, and 183rd positions.

[0045] By performing amino acid mutations on the above sites of the vanillic acid trans-acting factor in this application, the reverse vanillic acid trans-acting factor can dissociate the vanillic acid operon without adding Va; adding Va can bind to the vanillic acid operon VanO. Thus, the method of this application can reverse-regulate the vanillic acid gene circuit control system, enabling the system to maintain the closed state without adding Va and the activated state when adding Va, having higher practicability and safety, providing new methods and new strategies for clinical cell therapy programs, and having good clinical application prospects.

[0046] It should be noted that "the sites of the amino acid mutations include the 97th, 127th, 137th, and 183rd positions" includes the 97th position or its equivalent position, the 127th position or its equivalent position, the 137th position or its equivalent position, and the 183rd position or its equivalent position.

[0047] In this article, the term "equivalent position" refers to a position where the position and amino acid type of certain specific positions are different due to the existence of different sources (such as from different species or genera), species, subclasses, or alleles. The equivalent positions of the specific positions can be determined by sequence and structural homology alignment.

[0048] Vanillic acid gene circuit control system

[0049] In the second aspect of the present application, the present application proposes a vanillic acid gene circuit control system. According to an embodiment of the present application, the vanillic acid gene circuit control system includes:

[0050] A target protein unit, the target protein unit includes a vanillic acid operon, an inducible expression nucleic acid sequence, and a target protein. The 3' end of the vanillic acid operon is connected to the 5' end of the inducible expression nucleic acid sequence, and the 3' end of the inducible expression nucleic acid sequence is connected to the 5' end of the nucleic acid molecule encoding the target protein;

[0051] A receptor protein unit, the receptor protein unit includes a reverse vanillic acid trans - transcription factor and a transcriptional activation protein. The reverse vanillic acid trans - transcription factor is used to bind vanillic acid, and the transcriptional activation protein is used to induce the expression nucleic acid sequence;

[0052] Wherein, when the vanillic acid is present, the reverse vanillic acid trans - transcription factor binds to the vanillic acid operon;

[0053] When the vanillic acid is absent, the reverse vanillic acid trans - transcription factor does not bind to the vanillic acid operon;

[0054] 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.

[0055] The reverse vanillic acid trans - transcription factor of the present application can dissociate from the vanillic acid operon without adding Va and can bind to the vanillic acid operon when adding Va. Thus, the vanillic acid gene circuit control system prepared by using the above - mentioned reverse vanillic acid trans - transcription factor can maintain the system in a closed state without adding Va and can maintain the system in an activated state when adding Va, having higher practicability and safety. It provides a new method and new strategy for clinical cell therapy programs and has good clinical application prospects.

[0056] It should be noted that "the sites of the amino acid mutations include the 97th, 127th, 137th, and 183rd positions" includes the 97th position or its equivalent positions, the 127th position or its equivalent positions, the 137th position or its equivalent positions, and the 183rd position or its equivalent positions.

[0057] According to an embodiment of the present application, the above vanillic acid gene circuit control system may further include at least one of the following technical features:

[0059] According to an embodiment of the present application, the nucleotide sequence of the vanillic acid operon is as shown in SEQ ID NO:7.

[0060] ATTGGATCCAAT(SEQ ID NO:7).

[0061] According to an embodiment of the present application, in the target protein unit, the vanillic acid operon contains one or more. Exemplarily, the vanillic acid operon contains 5, and its nucleotide sequence is:

[0062] ATTGGATCCAATGCATTGGATCCAATGGATTGGATCCAATCGATTGGATCCAATtgATTGGATCCA AT(SEQ ID NO:8).

[0063] According to an embodiment of the present application, the inducible expression nucleic acid sequence is selected from minP or P hCMVmin sequence. According to an embodiment of the present application, the transcriptional activator protein is selected from at least one of VP16 protein, VP64 protein, and VPR protein.

[0064] According to an embodiment of the present application, the nucleotide sequence of the minimal promoter (minP) sequence is as shown in SEQ ID NO:23.

[0065] TAGAGGGTATATAATGGAAGCTCGACTTCCAG(SEQ ID NO:23).

[0066] According to an embodiment of the present application, the P hCMVmin The nucleotide sequence of the sequence is as shown in SEQ ID NO:24.

[0067] TCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGT TTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG(SEQ ID NO:24).

[0068] According to an embodiment of the present application, the amino acid sequence of the VP64 protein is as shown in SEQ ID NO:18.

[0069] According to an embodiment of the present application, the amino acid sequence of the VPR protein is as shown in SEQ ID NO:19.

[0070] According to an embodiment of the present application, the method for reversely regulating the vanillic acid gene circuit control system described in the first aspect and the vanillic acid gene circuit control system described in the second aspect may further include at least one of the following technical features:

[0071] According to an embodiment of the present application, the amino acid mutation sites of the vanillic acid trans - transcription factor are referenced and located based on the amino acid sequence shown in SEQ ID NO:1.

[0072] MTSDMPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARRLAERGMTAETHARFVALIAEGEALFAAGRLNGEDLDRYAAYNQAFHDTLVSAAGNGAVESALARNGFEPFAAAGALALDLMDLPAEYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:1).

[0073] In this article, the amino acid numbering of the amino acid sequence of the reverse vanillic acid trans - transcription factor (for example, the amino acid sequence shown in SEQ ID NO:1) is determined from the N - terminus to the C - terminus. For example, the 97th position refers to the 97th position starting from the first position at the N - terminus.

[0074] In this article, without special explanation, amino acid mutations are all made with the amino acid sequence shown in SEQ ID NO:1 as the wild - type sequence.

[0075] In an alternative embodiment of the present application, the site of the amino acid mutation is selected from the 97th position or its equivalent position, the 127th position or its equivalent position, the 137th position or its equivalent position, and the 183rd position or its equivalent position.

[0076] In an alternative embodiment of the present application, the site of the amino acid mutation further includes the 16th position or its equivalent position and the 165th position or its equivalent position.

[0077] According to an embodiment of the present application, the site of the amino acid mutation further includes the 16th position and the 165th position.

[0078] In an alternative embodiment of the present application, the site of the amino acid mutation further includes at least one of the 7th position or its equivalent position, the 31st position or its equivalent position, the 103rd position or its equivalent position, the 113th position or its equivalent position, the 162nd position or its equivalent position, the 164th position or its equivalent position, and the 208th position or its equivalent position.

[0079] According to an embodiment of the present application, the site of the amino acid mutation further includes at least one of the 7th position, the 31st position, the 103rd position, the 113th position, the 162nd position, the 164th position, and the 208th position.

[0080] In an alternative embodiment of the present application, the site of the amino acid mutation is selected from:

[0081] 1) the 97th position or its equivalent position, the 127th position or its equivalent position, the 137th position or its equivalent position, and the 183rd position or its equivalent position, and optionally at least one of the 7th position or its equivalent position, the 103rd position or its equivalent position, the 113th position or its equivalent position, the 162nd position or its equivalent position, and the 164th position or its equivalent position; or

[0082] 2) the 97th position or its equivalent position, the 127th position or its equivalent position, the 137th position or its equivalent position, and the 183rd position or its equivalent position, and optionally at least one of the 31st position or its equivalent position, the 103rd position or its equivalent position, the 113th position or its equivalent position, the 162nd position or its equivalent position, the 164th position or its equivalent position, and the 208th position or its equivalent position; or

[0083] 3) the 97th position or its equivalent position, the 127th position or its equivalent position, the 137th position or its equivalent position, the 183rd position or its equivalent position, the 16th position or its equivalent position, and the 165th position or its equivalent position, and optionally at least one of the 31st position or its equivalent position, the 103rd position or its equivalent position, the 113th position or its equivalent position, and the 208th position or its equivalent position.

[0084] According to an embodiment of the present application, the sites of amino acid mutations are selected from:

[0085] 1) the 97th, 127th, 137th, and 183rd positions, and optionally at least one of the 7th, 103rd, 113th, 162nd, and 164th positions; or

[0086] 2) the 97th, 127th, 137th, and 183rd positions, and optionally at least one of the 31st, 103rd, 113th, 162nd, 164th, and 208th positions; or

[0087] 3) the 97th, 127th, 137th, 183rd, 16th, and 165th positions, and optionally at least one of the 31st, 103rd, 113th, and 208th positions.

[0088] In an alternative embodiment of the present application, the sites of amino acid mutations are selected from the 97th position or its equivalent position, the 103rd position or its equivalent position, the 113th position or its equivalent position, the 127th position or its equivalent position, the 137th position or its equivalent position, the 164th position or its equivalent position, and the 183rd position or its equivalent position.

[0089] According to an embodiment of the present application, the sites of amino acid mutations are selected from the 97th, 103rd, 113th, 127th, 137th, 164th, and 183rd positions.

[0090] In an alternative embodiment of the present application, the sites of amino acid mutations are selected from the 7th position or its equivalent position, the 97th position or its equivalent position, the 103rd position or its equivalent position, the 113th position or its equivalent position, the 127th position or its equivalent position, the 137th position or its equivalent position, the 162nd position or its equivalent position, the 164th position or its equivalent position, and the 183rd position or its equivalent position.

[0091] According to an embodiment of the present application, the sites of amino acid mutations are selected from the 7th, 97th, 103rd, 113th, 127th, 137th, 162nd, 164th, and 183rd positions.

[0092] In an alternative embodiment of the present application, the sites of amino acid mutations are selected from the 97th position or its equivalent position, the 113th position or its equivalent position, the 127th position or its equivalent position, the 137th position or its equivalent position, and the 183rd position or its equivalent position.

[0093] According to an embodiment of the present application, the sites of amino acid mutations are selected from the 97th, 113th, 127th, 137th, and 183rd positions.

[0094] In an alternative embodiment of the present application, the sites of amino acid mutation are selected from the 31st position or its equivalent positions, the 97th position or its equivalent positions, the 103rd position or its equivalent positions, the 113th position or its equivalent positions, the 127th position or its equivalent positions, the 137th position or its equivalent positions, the 162nd position or its equivalent positions, the 164th position or its equivalent positions, the 183rd position or its equivalent positions, and the 208th position or its equivalent positions.

[0095] According to an embodiment of the present application, the sites of amino acid mutation are selected from the 31st position, the 97th position, the 103rd position, the 113th position, the 127th position, the 137th position, the 162nd position, the 164th position, the 183rd position, and the 208th position.

[0096] In an alternative embodiment of the present application, the sites of amino acid mutation are selected from the 16th position or its equivalent positions, the 31st position or its equivalent positions, the 97th position or its equivalent positions, the 103rd position or its equivalent positions, the 113th position or its equivalent positions, the 127th position or its equivalent positions, the 137th position or its equivalent positions, the 165th position or its equivalent positions, the 183rd position or its equivalent positions, and the 208th position or its equivalent positions.

[0097] According to an embodiment of the present application, the sites of amino acid mutation are selected from the 16th position, the 31st position, the 97th position, the 103rd position, the 113th position, the 127th position, the 137th position, the 165th position, the 183rd position, and the 208th position.

[0098] In an alternative embodiment of the present application, the amino acid mutations include the following sites or their equivalent positions: 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.

[0099] As used herein, "R97H" means that the 97th amino acid residue from the N-terminus of the amino acid sequence of the reverse vanillic acid transcription factor (e.g., the amino acid sequence shown in SEQ ID NO:1) is replaced by histidine; "A137R / N / D / Q / E / H / L / K / M / S / T / V / A / G / C / F / W / Y" means that the 137th amino acid residue from the N-terminus of the amino acid sequence of the reverse vanillic acid transcription factor (e.g., the amino acid sequence shown in SEQ ID NO:1) is replaced by any one of R / 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" means that the 183rd amino acid residue from the N-terminus of the amino acid sequence of the reverse vanillic acid transcription factor (e.g., the amino acid sequence shown in SEQ ID NO:1) is replaced by any one of K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L.

[0100] 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.

[0101] In an alternative embodiment of the present application, the sites of the amino acid mutation further include M16I or its equivalent site and E165V or its equivalent site.

[0102] According to an embodiment of the present application, the sites of the amino acid mutation further include M16I and E165V.

[0103] In an alternative embodiment of the present application, the sites of the amino acid mutation further include 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.

[0104] According to an embodiment of the present application, the sites of the amino acid mutation further include at least one of R7H, E31V, M103L, A113T, N162S, F164Y, G208S.

[0105] In an alternative embodiment of the present application, the amino acid mutations are selected from the sites of one of the following groups 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

[0106] 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

[0107] 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.

[0108] According to an embodiment of the present application, the sites of the amino acid mutations are selected from:[[]]

[0109] 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

[0110] 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

[0111] 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.

[0112] According to an embodiment of the present application, the site of the amino acid mutation is the 137th position, and the mutated amino acid at the 137th position is A137V / E / T / D / M / S / A.

[0113] According to an embodiment of the present application, the site of the amino acid mutation is the 137th position, and the mutated amino acid at the 137th position is A137V.

[0114] According to an embodiment of the present application, the site of the amino acid mutation is the 183rd position, and the mutated amino acid at the 183rd position is E183K / R / V / I / T.

[0115] In an alternative embodiment of the present application, the sites of the amino acid mutations are selected from:

[0116] 1) R97H, L127Q, A137 V / E / T / D / M / S / A, and E183 K / R / V / I / T L, and optionally at least one of R7H, M103L, A113T, N162S, F164Y; or

[0117] 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

[0118] 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.

[0119] According to an embodiment of the present application, the site of the amino acid mutation is the 183rd position, and the mutated amino acid at the 183rd position is E183K.

[0120] In an alternative embodiment of the present application, the sites of the amino acid mutations are selected from R97H or its equivalent sites, L127Q or its equivalent sites, A137G or its equivalent sites, E183K or its equivalent sites.

[0121] According to an embodiment of the present application, the sites of amino acid mutations are selected from R97H, L127Q, A137G, and E183K.

[0122] In an alternative embodiment of the present application, the sites of amino acid mutations are selected from E31V or its equivalent sites, R97H or its equivalent sites, L127Q or its equivalent sites, A137G or its equivalent sites, E165V or its equivalent sites, and E183K or its equivalent sites.

[0123] According to an embodiment of the present application, the sites of amino acid mutations are selected from E31V, R97H, L127Q, A137G, E165V, and E183K.

[0124] In an alternative embodiment of the present application, the sites of amino acid mutations are selected from R7H or its equivalent sites, R97H or its equivalent sites, M103L or its equivalent sites, A113T or its equivalent sites, L127Q or its equivalent sites, A137G or its equivalent sites, N162S or its equivalent sites, F164Y or its equivalent sites, and E183K or its equivalent sites.

[0125] According to an embodiment of the present application, the sites of amino acid mutations are selected from R7H, R97H, M103L, A113T, L127Q, A137G, N162S, F164Y, and E183K.

[0126] In an alternative embodiment of the present application, the sites of amino acid mutations are selected from R97H or its equivalent sites, A113T or its equivalent sites, L127Q or its equivalent sites, A137V or its equivalent sites, and E183K or its equivalent sites.

[0127] According to an embodiment of the present application, the sites of amino acid mutations are selected from R97H, A113T, L127Q, A137V, and E183K.

[0128] In an alternative embodiment of the present application, the sites of amino acid mutations are selected from E31V or its equivalent sites, R97H or its equivalent sites, M103L or its equivalent sites, A113T or its equivalent sites, L127Q or its equivalent sites, A137V or its equivalent sites, N162S or its equivalent sites, F164Y or its equivalent sites, E183K or its equivalent sites, and G208S or its equivalent sites.

[0129] According to an embodiment of the present application, the sites of amino acid mutations are selected from E31V, R97H, M103L, A113T, L127Q, A137V, N162S, F164Y, E183K, and G208S.

[0130] In an alternative 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.

[0131] 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, G208S.

[0132] In an alternative embodiment of the present application, the reverse vanillic acid transcription factor has an amino acid sequence shown in any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 21 to SEQ ID NO: 22, or an amino acid sequence having at least 90% sequence similarity thereto.

[0133] It should be noted that in the present 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 a conservative modification form of SEQ ID NO: A, both of which are within the protection scope of the present application. Exemplarily, "the amino acid sequence of the reverse vanillic acid transcription factor is as shown in any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 21 to SEQ ID NO: 22" means that the reverse vanillic acid transcription factor is the amino acid sequence shown in any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 21 to SEQ ID NO: 22, or the amino acid sequence of a conservative modification form of any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 21 to SEQ ID NO: 22, both of which are within the protection scope of the present application.

[0134] As used herein, "conservatively modified forms of an amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the properties of the polypeptide containing the amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the reverse vanillic acid transcriptor factor of the present application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (such as lysine, arginine, histidine), amino acids having acidic side chains (such as aspartic acid, glutamic acid), amino acids having uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (such as threonine, valine, isoleucine), and amino acids having aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues in the reverse vanillic acid transcriptor factor of the present application can be replaced with other amino acid residues from the same side chain family, and the modified reverse vanillic acid transcriptor factor can be tested for retained functionality using the functional assays described herein. Exemplarily, conservative modifications are made in an amount not exceeding 80% of the total number, preferably not exceeding 90% of the total number. As used herein, "conservatively modified forms of an amino acid sequence" also includes amino acid modifications resulting from natural mutations, where "natural mutations" refer to mutations caused by changes in alleles and the like due to individual differences and other reasons during the process of natural mutations.

[0135] According to an embodiment of the present application, the amino acid sequence of the reverse vanillic acid transcriptor factor is as shown in any one of SEQ ID NO:3 to SEQ ID NO:6 and SEQ ID NO:21 to SEQ ID NO:22.

[0136] MTSDMPHIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGLTAETHARFVTLIAEGEALFAAGRQNGEDLDRYAGYNQAFHDTLVSAAGNGAVESALARSGYEPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD (SEQ ID NO:3);

[0137] MTSDMPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGLTAETHARFVTLIAEGEALFAAGRQNGEDLDRYAVYNQAFHDTLVSAAGNGAVESALARSGYEPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:4);

[0138] MTSDMPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGLTAETHARFVTLIAEGEALFAAGRQNGEDLDRYAVYNQAFHDTLVSAAGNGAVESALARSGYEPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAESAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:5);

[0139] MTSDMPRIKPGQRVMIALRKMIASGEIKSGVRIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGLTAETHARFVTLIAEGEALFAAGRQNGEDLDRYAVYNQAFHDTLVSAAGNGAVESALARNGFVPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAESAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:6);

[0140] MTSDMPRIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGMTAETHARFVALIAEGEALFAAGRQNGEDLDRYAGYNQAFHDTLVSAAGNGAVESALARNGFEPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD (SEQ ID NO:21);

[0141] MTSDMPRIKPGQRVMMALRKMIASGEIKSGVRIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGMTAETHARFVALIAEGEALFAAGRQNGEDLDRYAGYNQAFHDTLVSAAGNGAVESALARNGFVPFAAAGALALDLMDLPAKYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD (SEQ ID NO:22).

[0142] Genetically engineered cells, use for preparing drugs

[0143] In the third aspect of the present application, a genetically engineered cell is proposed. According to an embodiment of the present application, the genetically engineered cell includes the vanillic acid gene circuit control system described in the second aspect.

[0144] In the fourth aspect of the present application, the use of the vanillic acid gene circuit control system described in the second aspect in the preparation of drugs is proposed, and the target protein in the vanillic acid gene circuit control system is selected from drug proteins.

[0145] The solution of the present application will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only for illustrating the present application and should not be regarded as limiting the scope of the present application. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not indicated with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0146] Example 1: Screening of mutation sites of the VanR gene

[0147] This example is based on the vanillic acid transcriptional repressor (the amino acid sequence is 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. Among them:

[0148] 1. The randomly selected prokaryotic vector is

[0149] CTD-TAC promoter-RBS-VanR-Terminator-pVanCC-RiboJ-RBS-EGFP-terminator.

[0150] The CTD-TAC promoter sequence:

[0151] TATAGGCAAAAAAGGCCTTGACATCCCACCTCACGTATGCTATAATGTGTGCAGTCTGACGCGG CG (SEQ ID NO: 9). This promoter is a constitutive promoter and can express VanR without induction.

[0152] The RBS sequence:

[0153] TTTGTTTAACTTTAAGAAGGAGA (SEQ ID NO: 10). This RBS sequence was specifically designed and optimized for VanR expression by predecessors and can improve the expression efficiency of VanR.

[0154] The VanR sequence is as shown in SEQ ID NO: 1 above.

[0155] The pVanCC sequence:

[0156] ATTGGATCCAATTGACAGCTAGCTCAGTCCTAGGTACCATTGGATCCAAT (SEQ ID NO: 11). Both wings of this promoter contain VanO, which is an optimized promoter that can work in E. coli and can be regulated by VanR and Va. Its working logic is: when Va does not exist in the environment, VanR binds to the VanO region on both wings, preventing the expression of downstream genes. When a certain amount of Va is added, VanR binds to Va and undergoes allosteric changes, leaving VanO, enabling the downstream genes to start expressing.

[0157] The RiboJ sequence:

[0158] AGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA (SEQ ID NO: 12) is a 75 nucleotide sequence consisting of tobacco ringspot virus (rTRSV) satellite RNA, which can reduce the leaky expression of the system under non-induced conditions.

[0159] EGFP amino acid sequence:

[0160] MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 13). This protein 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), thereby characterizing the expression of the entire system.

[0161] 2. The reaction conditions of the error-prone PCR mutagenesis method are as follows:

[0162]

[0163]

[0164] Upstream primer primer-F: GTTTAACTTTAAGAAGGAGATATACCATGACTAGTG (SEQ ID NO: 14);

[0165] Downstream primer primer-R: CTTGTCGACGGAGCTCGAATTCTTA (SEQ ID NO: 15).

[0166] After mixing the reaction solution, divide it into 10-20uL / tube for PCR, so that the random mutation in each tube can be amplified as an independent random event, which is beneficial to increase the storage capacity of the total mutation library.

[0167] PCR amplification conditions:

[0168]

[0169] After the error-prone PCR amplification products were purified using a DNA purification kit, the error-prone PCR products and the expression plasmid pCY146 were digested with the restriction endonucleases SpeI and EcoRI respectively, ligated using T4 DNA Ligase, transformed into Escherichia coli BL21(DE3) competent cells, and spread on an LB (containing 100 μg / mL ampicillin) plate. After culturing at 37 °C for 12 h, an rVanR mutant library was constructed.

[0170] 3. The screening method is as follows:

[0171] rVanR screening idea: Wild-type VanR represses the fluorescence expression of the reporter system PCY146 in the blank group without Va, and highly expresses fluorescence in the drug-added group with 0.5 mM Va added. Therefore, the fluorescence value of the drug-added group will be much higher than that of the blank group; while the performance of rVanR must be opposite to that of wild-type VanR. So we need to screen for transformants with a fluorescence value lower than that of the blank group in the drug-added group. Also, because Va has a bactericidal effect, we need to measure the OD 600 of each group to characterize the bacterial density in each group, and divide the fluorescence value of each group by OD 600 The result is a relative fluorescence value, which is used to represent the expression situation of each group. We will screen and collect the transformants with a relative fluorescence value lower than that of the blank group in the drug-added group.

[0172] Obtaining rVanR mutants: The LB (containing 100 μg / mL ampicillin) plate cultured at 37 °C for 12 h was irradiated with blue light to search for and mark the rVanR transformants that could emit green fluorescence without Va induction. The marked transformants were inoculated with 0.5 mL into a liquid LB medium containing 100 μg / mL ampicillin and cultured at 37 °C in a shaker at 220 rpm for 2.5 h until the OD600 of the bacterial solution was 0.5 - 0.7.

[0173] rVanR mutant screening: 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 the blank group, to which 1 μL of absolute ethanol is added, and the other group is the experimental group, to which 1 μL of a 0.5 M Va solution dissolved in absolute ethanol is added to make the final concentration of Va 0.5 mM. Add 100 μL of the transformants with OD600 = 0.5 - 0.7 to both the experimental group and the control group, and culture them at 37 °C in a shaker at 220 rpm for 16 h. Then place the 48-well plate in a microplate reader and detect the OD 600With fluorescence intensity, calculate the relative fluorescence value, screen out the transformants with an absolute fluorescence ratio (blank group > experimental group), perform plasmid extraction and sequencing, construct this fragment into a eukaryotic vector, and verify the performance of its mutant in HEK-293; at the same time, use this mutant plasmid as the template for the next round of error-prone PCR to construct a mutant library.

[0174] Using the same method as above, perform multiple rounds of error-prone PCR with the mutant gene as the template to construct a mutant library. Finally, rVanR01 (mutation sites are R7H, R97H, A113T, L127Q, N162S, and the amino acid sequence is shown in SEQ ID NO:2) is obtained.

[0175] MTSDMPHIKPGQRVMMALRKMIASGEIKSGERIAEIPTAAALGVSRMPVRTALRSLEQEGLVVRLGARGYAARGVSSDQIRDAIEVRGVLEGFAARHLAERGMTAETHARFVTLIAEGEALFAAGRQNGEDLDRYAAYNQAFHDTLVSAAGNGAVESALARSGFEPFAAAGALALDLMDLPAEYEHLLAAHRQHQAVLDAVSCGDAEGAERIMRDHALAAIRNAEVFEAAASAGAPLGAAWSIRAD(SEQ ID NO:2).

[0176] Detect the rVanR function of rVanR01 obtained above in the E. coli and HEK-293 cell systems respectively. For the eukaryotic vector pCY176 CMV-VanR WT -VP64-pA, use NheI and EcoRI to perform double digestion on the VanR WT fragment, and then integrate the rVanR01 sequence onto the plasmid by PCR and homologous recombination methods, named pCY177CMV-rVanR01-VP64-pA. Mix pCY177 and the pre-constructed reporter plasmid pCY175 5*VanO-nanoluc-pA in a ratio of 200 ng:50 ng, and use the PEI transfection reagent to transfect into HEK-293 cells seeded in 24-well plates at a density of 80,000 cells per well 18 hours in advance. Add 1 μL of 0.5 M Va to the drug treatment group to make the final concentration 0.5 mM, and add 1 μL of DMSO to the control group. Each group has three replicates. After 48 hours, use the NanoLuc TM luciferase kit for detection, and the detection results are as Figure 1 shown.

[0177] It was found that the value of the drug - added group was 1.04 times that of the control group, while under the same conditions, the value of the control group of wild - type VanR was 30.4 times that of the drug - added group, proving that rVanR01 has begun to exhibit reverse - type function. However, its function is only 1.04 times in the HEK - 293 cell system and needs to be continuously iteratively optimized.

[0178] 2. Using rVanR01 as a template, continue iterative optimization by error - prone PCR mutagenesis (the specific steps refer to step 1 of this example) to obtain four mutants: 209 - 1, 210 - 1, 211 - 1, and 212 - 1. Using the method of step 1 of this example, detect the rVanR functions of the four mutants 209 - 1, 210 - 1, 211 - 1, and 212 - 1 in Escherichia coli and HEK - 293 cell systems respectively. The detection results in the HEK - 293 cell system are as Figure 2 shown.

[0179] It was found that the mutants 210 - 1 and 211 - 1 showed excellent rVanR functions in the HEK - 293 cell experiments. The multiple of 210 - 1 in the HEK - 293 cell experiment was 6.8, and the multiple of 211 - 1 in the HEK - 293 cell experiment was 1.4. Among them, the leaky expression of the 210 - 1 mutant without adding drugs was lower. It was used as a template for subsequent optimization and was named rVanR02 later (the mutation sites are R7H, R97H, M103L, A113T, L127Q, A137G, N162S, F164Y, E183K, and the amino acid sequence is as shown in SEQ ID NO:3).

[0180] 3. Reverse - mutate 5 mutations (R7H, R97H, A113T, L127Q, N162S) in the rVanR02 mutant back to the wild - type sites respectively, and reverse - mutate 4 mutations (M103L, A137G, F164Y, E183K) in the rVanR02 mutant back to the wild - type sites respectively. Using the method of step 1 of this example, detect the rVanR functions of the mutants with reverse - mutated wild - type sites in the HEK - 293 cell system. The detection results are as Figure 3 shown.

[0181] It was found that: R7, A113, N162, M103, and F164 are nonsense mutations;

[0182] R97H and E183K are key mutations for rVanR logic. Among them, the R97H mutation reduces the ability of rVanR to bind VanO without Va; the E183K mutation determines the existence of rVanR logic;

[0183] A137G and L127Q are rate-limiting mutations. By changing A137, the binding ability of rVanR can be altered, thereby adjusting the expression level of the reported value.

[0184] Therefore, R97, A137, E183, and L127 are key mutation sites.

[0185] 4. Further, based on the rVanR02 mutant, deep mutational scanning was performed on A137 and E183. First, using site-directed mutagenesis, the amino acid residue A at position 137 was replaced with the other 19 amino acid residues to observe whether there were amino acid residues that made the function better. The results are as Figure 4 and Figure 5 shown.

[0186] The results found that:

[0187] Multiple mutation methods 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 is also acceptable, but the background leakage without Va stimulation is too high, which may be suitable for systems that require a larger output value.

[0188] Select the A137V mutant sequence. On this basis, the same site-directed mutagenesis operation was also performed on E183 to observe whether there were more suitable mutations for our system among the other 19 amino acid residues.

[0189] Multiple mutation methods of E183 (E183K / A / Q / T / N / C / I / M / F / P / S / Y / V / R / L) can all improve the rVanR ability of the mutant.

[0190] 5. Further, based on the E183K site, by analyzing the protein and molecular docking map, it was found that E183 is adjacent to the folded region and surrounded by polar negatively charged amino acid residues. Therefore, on the basis of rVanR03, the first 8 amino acid residues before E183 were each mutated to K, and the method of step 1 of this example was used to detect the rVanR function of the mutant after mutation in the HEK-293 cell system. The detection results are as Figure 6 shown.

[0191] The results showed that mutating the adjacent amino acids to K did not result in better or worse outcomes.

[0192] 6. Based on the key mutation sites, rVanR03 mutants (R97H, A113T, L127Q, A137V, N162S, F164Y, E183K, with the amino acid sequence shown in SEQ ID NO:4) were designed. Using the method of step 1 of this example, the rVanR function of the rVanR03 mutants was detected in the HEK-293 cell system, and the detection results Figure 7 .

[0193] Moreover, in this application, 5 mutations (R97H, A113T, L127Q, A137V, E183K) in the rVanR03 mutants were respectively reverse-mutated back to the wild-type sites. Using the method of step 1 of this example, the mutants with the reverse-mutated wild-type sites were detected for their rVanR function in the HEK-293 cell system. It was found that A113 was a nonsense mutation, and R97, A137, E183, and L127 were key mutation sites.

[0194] Example 2: Optimization of the reverse vanillic acid transcription factor

[0195] 1. Since the rVanR function multiples of the rVanR01-rVanR03 mutants in Example 1 were within 10. Further, based on rVanR03, the rVanR04 mutant (R97H, M103L, A113T, L127Q, A137V, N162S, F164Y, E183K, G208S, with the amino acid sequence shown in SEQ ID NO:5) was iteratively generated. Using the method of step 1 of this example, its rVanR function in the HEK-293 cell system was detected, and the detection results are as Figure 7 shown. It was found that the multiple of the rVanR04 mutant in the HEK-293 cell experiment was 11.32.

[0196] 2. Further, based on rVanR04, through iteration and optimization, the rVanR05 mutant (M16I, E31V, R97H, M103L, A113T, L127Q, A137V, E165V, E183K, G208S, with the amino acid sequence shown in SEQ ID NO:6) was obtained. Using the method of step 1 of this example, its rVanR function in the HEK-293 cell system was detected, and the detection results are as Figure 8 shown. It was found that the multiple of the rVanR05 mutant in the HEK-293 cell experiment was 35.8.

[0197] Furthermore, in the present application, reverse mutations were separately made for the 4 mutations (M16I, E31V, E165V, G208S) newly introduced in the rVanR05 mutant compared to rVanR03 back to the wild-type sites. Using the method of step 1 of this example, the mutants with reverse mutations back to the wild-type sites were tested for their rVanR function in the HEK-293 cell system, and the results are as Figure 9 shown.

[0198] It was found that: M16 and G208 are nonsense mutations, and M31 / E165 are key mutation sites.

[0199] Based on the above experimental results, the conclusion is drawn that: M31, R97, L127, A137, E165, E183 are key mutation sites.

[0200] Example 3: Vanillic acid gene circuit control system

[0201] 1. Preparation of vanillic acid gene circuit control system

[0202] In this example, for rVanR05, a vanillic acid gene circuit control system that can be used for cell therapy was prepared. The currently tested reporter plasmid: pCY175 (5*VanO-minP-Secretory(IgK)-nanoluc-pA). Among them,

[0203] The DNA sequence of 5*VanO-minP is:

[0204] ATTGGATCCAATGCATTGGATCCAATGGATTGGATCCAATCGATTGGATCCAATtgATTGGAT CCAATTTAATTAAGCAGCTCCTGCAGGTAGAGGGTATATAATGGAAGCTCGACTTCCAG(SEQ ID NO:16).

[0205] The amino acid sequence of Secretory(IgK)-nanoluc is:

[0206] MTSETDTLLLWVLLLWVPGSTGDASGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA(SEQ ID NO:17), where Secretory(IgK) is an exocrine short peptide that aids in the secretion of nanoluc outside the cell. Secretory(IgK) and nanoluc are linked by a SGGSG flexible short peptide.

[0207] Receptor plasmid: CMV-rVanR-VP64-pA; among them, the amino acid sequence of VP64:

[0208] DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML(SEQ ID NO:18).

[0209] The principle is as follows: minP is the smallest eukaryotic TATA-box promoter, which has very low basal leaky expression when not induced to start, but cannot self-activate the expression of downstream genes alone. Only when combined with trans-activation domains such as VP16 (the trans-activation domain derived from Herpes simplex virus), VP64 formed by tandemly linking 4 VP16 complexes, and the ternary transcription activator VPR (VP64-p65-Rta), can it recruit key factors of the host transcription complex through VP16, VP64, VPR, etc., initiate the transcription of the minimal promoter, and then activate the expression of downstream genes. However, natural VP16 / VP64 / VPR cannot self-localize to minP. Therefore, the inventor fused rVanR and VP64 for expression, and placed 5 repeated VanR operator VanO in front of minP. When the small molecule Va is added, rVanR-VP64 will bind to VanO, and VP64 will then bind to minP and activate the downstream signaling pathway for expression.

[0210] Furthermore, replace VP64 in the above vanillic acid gene circuit control system with VPR, which has a stronger binding ability. The amino acid sequence of VPR:

[0211] DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSRSSGSPKKKRKVGSQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF(SEQ ID NO:19).

[0212] Using the method of step 1 of this example, the rVanR function of the above-prepared multiple vanillic acid gene circuit control systems was detected in the HEK-293 cell system, and the results are as Figure 10 shown.

[0213] It was found that when VP64 was first replaced with VPR with stronger binding ability, the system showed a stronger output multiple (51.9-fold).

[0214] 2. The vanillic acid gene circuit control system containing rVanR-VPR (plasmid pCY233) in step 1 of this example was optimized.

[0215] 2.1 The reporter plasmid was replaced from PCY175 (5*VanO-minP-Secretory(IgK)-nanoluc-pA) to pSL173 (5*VanO-Ph CMVmin -SEAP-pA), where the nucleotide sequence of P hCMVmin is as shown in SEQ ID NO:24. P hCMVminIt is the minimum structure of the eukaryotic cell strong promoter CMV, and its function and activation mode are similar to those of minP, but it has a higher expression level compared to minP.

[0216] The amino acid sequence of SEAP is as follows:

[0217] MLLLLLLLGLRLQLSLGIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTV

[0218] TAARILKGQKKDKLGPEIPLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAAR

[0219] FNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGC

[0220] QDIATQLISNMDIDVILGGGRKYMFPMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYVWN

[0221] RTELMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDH

[0222] GHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKA

[0223] YTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLVHGVQE

[0224] QTFIAHVMAFAACLEPYTACDLAPPAGTTDAAHPG(SEQ ID NO:20).

[0225] 2.2 Further optimize the ratio of the receptor plasmid to the reporter plasmid. The total transfection concentration is 300 ng, and the ratio of the receptor plasmid to the reporter plasmid is dynamically adjusted. For the specific ratio and the corresponding detection results, please refer to Figure 11 . It was found that various ratios of the receptor plasmid to the reporter plasmid set in this example all had good rVanR functions.

[0226] 2.3 Time dynamic results tested under the condition that the ratio of the recipient plasmid to the reporter plasmid is 8:1. For specific results, see Figure 12 . The results show that the highest multiple at 48 h is 69.7 times at a Va concentration of 0.5 mM.

[0227] 3. Based on the optimized vanillic acid gene circuit control system (the ratio of the recipient plasmid to the reporter plasmid is 8:1) obtained in step 2 of this example, the response multiples of the entire system were tested from 0 to 8 mM Va concentration, showing that it enters a plateau phase after a concentration of 2 mM; and the fluorescence reporter expression of 0 to 0.5 mM Va was tested. For specific results, see Figure 13 . The results show that the fluorescence results are consistent with the SEAP report results.

[0228] 4. Perform a reversibility experiment analysis on the optimized vanillic acid gene circuit control system (the ratio of the recipient plasmid to the reporter plasmid is 8:1) obtained in step 2 of this example. The specific implementation is as follows:

[0229] Transfect the circuit system plasmid into HEK-293 cells seeded in a 24-well plate at a density of 80,000 cells / well 18 hours in advance using PEI transfection reagent. After 8 hours, change the medium and add Va with a final concentration of 0.5 mM. Samples were taken at regular intervals to observe the results from 0 to 48 hours. At the 48th hour, digest the cells with trypsin, take half of the cells and seed them in a 24-well plate without adding Va, and sample at regular intervals to observe the results from 0 to 48 hours; subsequently, digest the cells with trypsin again, take half of the cells and seed them in a 24-well plate, and add Va with a final concentration of 0.5 mM at the same time, and sample at regular intervals to observe the results from 0 to 48 hours. This experiment aims to observe whether the binding of this circuit system to Va is reversible and whether Va will permanently affect the switch of the system. For specific results, see Figure 12 . The results show that the binding between rVanR and VanO is reversible with or without adding the drug.

[0230] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this 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 a suitable manner in any one or more embodiments or examples. In addition, without conflict, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0231] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present application.

Claims

1. A method for reversely regulating a vanillic acid gene circuit control system, the vanillic acid gene circuit control system comprising a reverse vanillic acid trans-transcription factor, a vanillic acid operon, and optionally Va, characterized in that: The method comprises: Carrying out amino acid mutation on the vanillic acid trans-transcription factor to obtain the reverse vanillic acid trans-transcription factor; Reverse regulation of the vanillic acid gene circuit control system is achieved through the reverse vanillic acid trans-transcription factor; Among them, the sites of amino acid mutation include position 97, position 127, position 137 and position 183.

2. A vanillic acid gene circuit control system, characterized in that: include: A target protein unit, the target protein unit comprising a vanillic acid operon, an inducible expression nucleic acid sequence and a target protein, wherein the 3' end of the vanillic acid operon is connected to the 5' end of the inducible expression nucleic acid sequence, and the 3' end of the inducible expression nucleic acid sequence is connected to the 5' end of a nucleic acid molecule encoding the target protein; A receptor protein unit, the receptor protein unit comprising a reverse vanillic acid trans-transcription factor and a transcription activator protein, the reverse vanillic acid trans-transcription factor is used to bind vanillic acid, and the transcription activator protein is used to induce expression of a nucleic acid sequence; Wherein, when the vanillic acid is present, the reverse vanillic acid trans-transcription factor binds to the vanillic acid operon; When the vanillic acid is absent, the reverse vanillic acid trans-transcription factor does not bind to the vanillic acid operon; 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.

3. The method according to claim 1 or the vanillic acid gene circuit control system according to claim 2, 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.

4. The method or vanillic acid gene circuit control system according to claim 3, 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.

5. The method and vanillic acid gene circuit control system according to claim 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 mutated 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.

6. The method according to claim 1 or the vanillic acid gene circuit control system according to claim 2, characterized in that: The amino acid mutation site of the vanillic acid trans-transcription factor is located with reference to the amino acid sequence shown in SEQ ID NO:

1.

7. The method according to claim 1 or the vanillic acid gene circuit control system according to claim 2, 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:6 and SEQ ID NO:21 to SEQ ID NO:

22.

8. The vanillic acid gene circuit control system according to claim 2, characterized in that: The nucleotide sequence of the vanillic acid operon is shown in SEQ ID NO:7; and / or, in the target protein unit, the vanillic acid operon contains one or more; And / or, the inducible expression nucleic acid sequence is selected from a minP sequence or a P hCMVmin sequence; The transcriptional activator protein is selected from at least one of VP16 protein, VP64 protein, and VPR protein; Optionally, the nucleotide sequence of the minP sequence is as shown in SEQ ID NO: 23; and / or, the P hCMVmin The nucleotide sequence of the sequence is shown in SEQ ID NO:24; And / or, the amino acid sequence of the VP64 protein is shown in SEQ ID NO: 18; And / or, the amino acid sequence of the VPR protein is shown in SEQ ID NO:

19.

9. A genetically engineered cell, characterized in that: The invention comprises the vanillic acid gene circuit control system according to any one of claims 7 to 8.

10. Use of the vanillic acid gene circuit control system according to any one of claims 7 to 8 in the preparation of drugs, wherein the target protein in the vanillic acid gene circuit control system is selected from drug proteins.

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