CN116083392A - Mammalian quantitative expression system - Google Patents
Mammalian quantitative expression system Download PDFInfo
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- CN116083392A CN116083392A CN202310136734.1A CN202310136734A CN116083392A CN 116083392 A CN116083392 A CN 116083392A CN 202310136734 A CN202310136734 A CN 202310136734A CN 116083392 A CN116083392 A CN 116083392A
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Abstract
The present invention is in the field of synthetic biology and bioengineering, and relates to systems and methods for the accurate quantitative expression of one or more genes of interest in mammalian cells. Specifically, precise control and quantitative expression of one or more genes of interest in mammalian cells is achieved by the combined use of a chimeric capping enzyme-RNA polymerase and a promoter recognized by the monomeric RNA polymerase. The expression system of the present invention can be advantageously used for advanced vaccine engineering, cell fate programming, and other therapeutic uses.
Description
Technical Field
The present invention is in the field of synthetic biology and bioengineering, and relates to systems and methods for the accurate quantitative expression of one or more genes of interest in mammalian cells.
Background
In the case of a polygenic expression system, maintaining the ratio and amount of each protein in an optimal state is critical for the stability of the multimeric protein complex in mammalian cells and for the synergistic function of each enzyme in biological pathways. Changes in the number of proteins due to chromosomal deletions, duplications or ectopic may cause abnormal aggregation of large amounts of proteins, which in turn may lead to destructive consequences such as cell dysfunction, e.g. tumors or neurodegenerative disorders. In cells, in order to maintain the protein at the desired ratio and amount, there are a variety of mechanisms to fine-tune and buffer protein levels to mitigate the effects of fluctuations in environmental and genetic levels, including negative feedback, mRNA nucleation restriction, targeted non-exponential degradation of unassembled subunits in the complex, and the like. In particular, it has been found that human cells and other eukaryotic cells regulate the number of doses of an obligatory subunit (obligate subset) in a protein complex by controlling the rate of synthesis (rather than negative feedback) to regulate the proportion of each subunit.
The importance of metering in mammalian polygenic systems has not been widely accepted until recently. For example, the ratio of the four reprogramming factors Oct4, sox2, KLF4, and c-Myc greatly affects the efficiency and quality of induced pluripotent stem cells in the stem cell fate decision process. At present, some libraries of promoters have been characterized in the art that are capable of quantitative expression in mammalian hosts, but the expression intensity of these expression systems is highly correlated with sequences near the expression cassette, and thus precise fine tuning of the metering is difficult to achieve. In the invention, chimeric capping enzyme-RNA polymerase (capping-RNAP) is constructed by fusing monomeric RNA polymerase (RNAP) from phage with mRNA capping enzyme (capping), and transcription of homologous promoters is driven by RNAP domains in the fusion protein, so that the expression quantity and the expression proportion of each protein in a protein complex in the background of mammalian cells are predicted and accurately controlled.
Disclosure of Invention
In a first aspect, the present invention relates to a system for expressing one or more genes of interest in a mammalian cell, the system comprising:
a chimeric capping enzyme-RNA polymerase comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7RNA polymerase (T7 RNAP), T3 RNA polymerase (T3 RNAP), K11 RNA polymerase (K11 RNAP), K1.5RNA polymerase (K1.5RNAP), and SP6RNA polymerase (SP 6 RNAP); the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
A promoter recognized by a monomeric RNA polymerase, wherein the promoter recognized by the T7 RNA polymerase is selected from the group consisting of SEQ ID NO:1-SEQ ID NO:54, a group of two or more of the group consisting of (a) and (b); the promoter recognized by the T3 RNA polymerase is selected from the group consisting of SEQ ID NO:161-SEQ ID NO: 172; the promoter recognized by the K11 RNA polymerase is selected from the group consisting of SEQ ID NO:55-SEQ ID NO:112, a group of two or more of the group consisting of 112; the K1.5RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NOs: 183-SEQ ID NO:234, a group of elements; the promoter recognized by the SP6 RNA polymerase is selected from the group consisting of SEQ ID NO:113-SEQ ID NO: 160.
In a second aspect, the present invention relates to a system for expressing one or more genes of interest in a mammalian cell, the system comprising:
one or more chimeric capping enzyme-RNA polymerase gene expression cassettes, each independently comprising a chimeric capping enzyme-RNA polymerase coding sequence, each independently comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5RNA polymerase, and SP6 RNA polymerase; the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
One or more gene expression cassettes of interest, each independently comprising a promoter recognized by a monomeric RNA polymerase and a gene sequence of interest, wherein the promoter recognized by the T7 RNA polymerase is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO:54, a group of two or more of the group consisting of (a) and (b); the promoter recognized by the T3 RNA polymerase is selected from the group consisting of SEQ ID NO:161-SEQ ID NO: 172; the promoter recognized by the K11 RNA polymerase is selected from the group consisting of SEQ ID NO:55-SEQ ID NO:112, a group of two or more of the group consisting of 112; the K1.5RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NOs: 183-SEQ ID NO:234, a group of elements; the promoter recognized by the SP6 RNA polymerase is selected from the group consisting of SEQ ID NO:113-SEQ ID NO: 160.
In a third aspect, the present invention relates to a method for expressing one or more genes of interest in a mammalian cell, the method comprising:
constructing one or more chimeric capping enzyme-RNA polymerase gene expression cassettes, each independently comprising a chimeric capping enzyme-RNA polymerase coding sequence, each independently comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5RNA polymerase, and SP6 RNA polymerase; the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
Constructing one or more gene expression cassettes of interest, each independently comprising a promoter recognized by a monomeric RNA polymerase and a gene sequence of interest, wherein the promoter recognized by the T7RNA polymerase is selected from the group consisting of SEQ ID NOs: 1-SEQ ID NO:54, a group of two or more of the group consisting of (a) and (b); the promoter recognized by the T3 RNA polymerase is selected from the group consisting of SEQ ID NO:161-SEQ ID NO: 172; the promoter recognized by the K11 RNA polymerase is selected from the group consisting of SEQ ID NO:55-SEQ ID NO:112, a group of two or more of the group consisting of 112; the K1.5RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NOs: 183-SEQ ID NO:234, a group of elements; the promoter recognized by the SP6 RNA polymerase is selected from the group consisting of SEQ ID NO:113-SEQ ID NO: 160.
In a fourth aspect, the present invention relates to the use of the system according to the first aspect or the method according to the second aspect for the preparation of a vaccine.
Advantageous effects
In mammalian cells, most of the transcription is environmentally dependent, i.e. the intensity of the transcription translation differs when the same expression cassette is inserted at different positions in the genome. This environmental dependent property greatly hampers quantitative studies of transcriptional regulation in mammalian hosts. In particular, for biological processes requiring simultaneous control of multiple gene expression levels, it is difficult in the prior art to precisely control each gene expression level or the expression ratio thereof.
The expression system of the invention combines the non-mammal-derived monomer RNA polymerase-capping enzyme fusion protein and the promoter identified by the monomer RNA polymerase, thereby avoiding the interference of cis-form and trans-form action elements on transcription in a mammal host and realizing the quantitative and precise control of the expression of mammal genes. The non-environment dependent expression system can advantageously be used for precise control of the expression level and/or the expression ratio of one or more proteins in a mammalian cell, and said expression level/expression ratio can be precisely predicted by the model of the invention. As demonstrated in the examples, the intensity of expression of a single gene in a mammalian host depends on the binding affinity of the RNAP subunit in the fusion protein to its cognate promoter; the binding affinity is readily determined in prokaryotes, and the binding affinity in mammalian hosts is proportional to that in prokaryotic hosts. According to the resource competition model of the invention, for a multi-gene expression system driven by a single capping-RNAP, the relative expression level of each gene is proportional to the binding affinity of each homologous promoter. This quantitative result was observed in different types of mammalian cells (e.g., human embryonic kidney cell HEK293T cell line, chinese hamster ovary CHO cell line). It can be seen that the quantitative expression system of the present invention can be applied to various types of mammalian cells, tissues or organisms in a modular manner to achieve quantitative fine regulation of one or more genes of interest. In this regard, the present invention extends to gene regulatory elements that can be used in mammals.
The RNA polymerase domain in the chimeric RNA polymerase-capping enzyme of the invention can be monomeric RNA polymerases of different phage sources, each RNA polymerase having a cognate promoter pool specifically recognized. There is little signal crosstalk between each group of RNA polymerase/homologous promoters, so that multiple chimeric RNA polymerase-capping enzymes can be expressed in the same host, and the expression of target genes is controlled by using homologous promoters recognized by each single RNA polymerase, so that the expression of each target gene is independently controlled. The gene expression cassettes in different working environments can be rationally designed according to specific requirements, and gene lines with different expression amounts and diversity can be built by means of the expression system. For a specific promoter library, the invention comprises 54T 7 promoters, 12T 3 promoters, 58K 11 promoters, 52K 1.5 promoters and 48 SP6 promoters, and a person skilled in the art can accurately select the promoters according to specific strength requirements, so that rational design of a gene line is realized.
The invention also provides methods for quantitatively controlling the relative expression intensity of two or more genes using a single species of chimeric RNA polymerase-capping enzyme. Without wishing to be bound by theory, since in this embodiment each promoter transcription is driven by the same transcription machinery (RNAP), the expression intensity of each specific promoter controlled by a single species of chimeric RNA polymerase-capping enzyme is positively correlated with the relative binding affinity of that promoter to RNAP, and thus two or more genes of interest can be linked downstream of each specific promoter controlled by a single species of RNA polymerase-capping enzyme fusion protein, respectively, and the expression ratio of each gene of interest can be precisely regulated. In addition, the transcription intensity of each promoter can be accurately and quantitatively predicted by a resource competition model without free parameters. As demonstrated in the examples, we used a single RNA polymerase-capping enzyme fusion protein in combination with the corresponding three orthogonal promoters to optimize the ratio of expression of the three proteins in the influenza a virus-like particles (VLP) complex in a predictable manner and greatly increase the yield of the complete VLP complex. In addition, when the double-gene or triple-gene expression system is adopted, multiple antigens can be expressed in a precise quantitative mode or in a constant proportion at the same time, and multivalent vaccines can be realized.
In a preferred embodiment, the promoters recognized by the RNAPs of the present invention may be engineered to be inducible by adding an operator sequence upstream and downstream of these promoters. For example, one or more sets of operator sequences may be added upstream and downstream of the RNAP recognized promoter and the corresponding transactivator expressed in the host (including but not limited to RpaR, braR, bjaR, tetR, C1434, phlf, HKCl, lmrA, bm3R, TP901CI, cymR, ttgR, and LexA), to construct an inducible promoter capable of responding to an inducer in a mammalian host. These inducible promoters can be used to expand the dynamic range of inducible expression, as demonstrated in the examples, to construct transcriptional and expression elements with different properties. In a preferred embodiment, the first promoter may also comprise a homologous promoter sequence (e.g., a dual promoter) such that positive feedback is applied to the expression of the capping-RNAP using the homologous promoter. This positive feedback to the RNAP arrangement may further increase the transcript level of the RNAP, thereby increasing the total expression of one or more genes of interest.
Because the expression system of the invention has the excellent property of environmental insensitivity, avoids the interference of an endogenous transcription machine, and can quantitatively and coexpression a plurality of proteins, the system and the method of the invention can be advantageously used for advanced vaccine engineering, complex protein drug preparation, cell fate programming and other medical purposes.
Drawings
FIG. 1 is a schematic diagram of the construction and testing of a single reporter gene expression system according to an embodiment of the invention. (A) Comparison of a prokaryotic transcription translation machine with a eukaryotic transcription translation machine. In different species, binding of RNAP to its cognate promoter is the rate limiting step in the transcription process, the rate of reaction of which is dependent on the binding affinity of RNAP to its cognate promoter (with K A Representation). The capping enzyme domain in the fusion protein facilitates translation of the transcript in mammalian cells. Schematic of a single reporter gene expression system. The chimeric capping enzyme-RNA polymerase (capping-RNAP) in the single reporter gene expression system is driven by a constitutive promoter to express, and a library of homologous promoters recognized by the RNA polymerase domain drives expression of the reporter gene yfp. (C) - (D) fluorescence intensity of reporter gene in flow cytometry assayed T7 single reporter gene expression system. The abscissa indicates the fluorescence intensity in E.coli, and the ordinate indicates the fluorescence intensity in CHO cells. The fusion proteins used in the experiments were capping-T7 RNAP, and the promoter sources were pT7WT, pT7M1, pT7M11, pT7M14, pT7M15, pT7M17, pT7M21, pT7M22, pT7M25, pT7M28, pT7M29, pT7M30, pT7M35, pT7M38, pT7M41, pT7M45, pT7M48, pT7M49, pT7M52, pT7M55 and pT7M6. The data points are the average fluorescence values of three independent repeated experiments, and the error bars are Standard deviation. (E) Principle of orthogonality testing of transcription machinery in combination with promoters. The capping-RNAP and its cognate promoter are located on separate plasmids. (F) The heat map shows the fluorescence intensity of the reporter gene in the orthogonality test. The RNAPs tested were T7 RNAP, K1.5RNAP, K11 RNAP, phi15 RNAP, T3 RNAP and SP6 RNAP, respectively; homologous promoters are pT7WT, pK1.5WT, pK11WT, pPhi15WT, pT3WT and pSP6WT, respectively. (G) Flow cytometry assayed SP6 and K11 single reporter gene expression systems report fluorescence intensity of genes in HEK293T, CHO and e. The SP6 promoters tested were pSP6m41, pSP6m24, pSP6m29, pSP6m06, pSP6m34, pSP6m30, pSP6m09, pSP6m18, pSP6m19, pSP6m17, pSP6m42, pSP6m28, pSP6m22, pSP6m47 and pSP6WT. The K11 promoters tested were pK11m11, pK11m01, pK11m16, pK11m04, pK11m07, pK11m13, pK11m10, pK11m19, pK11m28, pK11m25, pK11m34, pK11m31, pK11m50, pK11m46, pK11m49, pK11m52, pK11WT. Data points are average fluorescence values of three independent repeated experiments, and error bars are standard deviations.
FIG. 2 is a schematic diagram of the construction and testing of a dual reporter gene expression system according to an embodiment of the invention. (A) schematic diagram of a dual reporter gene expression system. The capping-RNAP in the dual-reporter gene expression system is expressed by the drive of a constitutive promoter, and the homologous promoter P identified by the RNAP yfp And P mCherry The expression of the reporter gene yfp and mcherry was driven separately. (B) The heat map shows the reported fluorescence intensities of 49T 7 dual reporter gene expression systems in HEK293T cells as determined experimentally and as a result of model fitting. pT7WT, pT7M17, pT7M21, pT7M25, pT7M35, pT7M41 and pT7M49 were used as P respectively yfp And P mCherry Connected to the reporter coding sequence yfp and upstream of mcherry, 7×7 dual reporter gene expression systems were constructed. Fluorescence intensities of YFP and mCherry at each combination were determined as P by flow cytometry yfp P mCherry Characterization of intensity. The fitting graph is obtained by fitting the free parameters of the formulas (2) - (6) according to the experimentally measured double fluorescence intensity. (C) The principle of a resource competition model is shown, where two homologous promoters compete for free RNAP. (D) Free RNA under steady state for 49 double reporter gene expression systemsResults of P concentration simulation.
FIG. 3 is a schematic diagram of the construction and testing of a tri-reporter expression system according to an embodiment of the invention. (A) A genetic map for studying the effect of genomic background on reporter gene expression. The yfp, mcherry and bfp coding sequences were ligated downstream of each pT7WT promoter in 6 orders. (B) Fluorescence intensities of three reporter proteins in CHO cells and HEK293T cells as determined by flow cytometry. The heat map shows fluorescence intensity as an average value of three experiments, and CV as a coefficient of variation. Schematic of a three reporter gene expression system. The capping-RNAP in the three-reporter gene expression system is expressed by the drive of a constitutive promoter, and the RNAP recognizes the promoter P bfp 、P yfp And P mCherry The expression of the reporter genes bfp, yfp and mcherry are driven, respectively. Determination of fluorescence intensities of BFP, YFP and mCherry as P by flow cytometry bfp 、P yfp P mCherry Characterization of intensity. (D) The relative intensity of each promoter in the 29 three reporter gene expression systems (1 in pT7WT intensity). (E) Measurement and prediction of the strength of each promoter of 29 three-reporter gene expression systems in HEK293T cells. The intensity of each promoter is characterized by the fluorescence intensity of the corresponding reporter gene, the fluorescence intensity is shown as the average value of three experiments, and the error bars correspond to standard deviations. R is R 2 Is the correlation between the measured value and the predicted value.
FIG. 4 is a schematic representation of the preparation of recombinant virus-like particles (VLPs) of influenza A H1N1 virus using the three-gene expression system of the invention. (a) schematic representation of recombinant VLPs. The recombinant VLP is formed by self-assembly of three antigens, namely Hemagglutinin (HA), neuraminidase (NA) and matrix protein 1 (M1). In the present invention, the mcherry coding sequence was fused to amino acids 1-61 at the N-terminus of NA (NA-mcherry), and EGFP was fused to M1 (gfp-M1), thereby enabling observation of VLPs using a fluorescence microscope. And (B) a transmission electron microscopy image of VLP particles, scale bar 50nm. (C) The VLPs observed by fluorescence microscopy are superimposed, FITC field of view and TRITC field of view in order from left to right. The scale bar is 10 μm. (D) schematic design of recombinant VLP expression system. The capping-RNAP in the expression system is driven to express by a constitutive promoter, and the T7RNAP recognizes the promoter Son P HA 、P M1 And P NA The expression of the HA-encoding gene, gfp-M1 encoding gene and NA-mcherry gene are driven separately. (E) Relative yields of VLPs prepared from 21 expression systems were compared to the promoters CMV, SV40 and EF1 a commonly used in mammalian cells. Will P HA 、P M1 And P NA The yields were set to 100% for pT7 WT. (F) Yield of VLPs under 157, 464 promoter combinations predicted from the resource competition model. The magnified part is a combination of promoter intensities that enables higher than 100% vlp yields. (G) Correlation of model predicted VLP yield with experimentally measured VLP yield. Data points are average value + -SD of at least three repeated experiments, correlation of measured value and predicted value R 2 0.49.
FIG. 5 is an inducible expression system according to the present invention. (A) schematic representation of an inducible expression system according to the present invention. The chimeric capping enzyme-RNA polymerase and the repressor in the inducible expression system are driven to express by a constitutive promoter, and the promoter for driving the reporter gene comprises an RNA polymerase domain recognition site and a repressor recognition site, and the capping-RNAP expression cassette, the reporter gene expression cassette and the repressor expression cassette are respectively arranged on different vectors. (B) Dose response curves for 9 induction systems in CHO cells measured by flow cytometry. (C) Schematic of a linear induction system constructed in accordance with the present invention. (D) Dose response curve of linear induction system measured by flow cytometry. Data points are mean ± SD of at least three replicates.
FIG. 6 is a nucleic acid vaccine and its induced immune response according to the present invention. (A) nucleic acid vaccine Gene route schematic. In PFB-HA construction, the constitutive promoter hEF1a and the RNAP homologous promoter drive the capping-RNAP expression, and the RNAP homologous promoter drives the Hemagglutinin (HA) expression. In RNAP-HA construction, the constitutive promoter hEF1a drives the capping-RNAP expression and the RNAP homologous promoter drives HA expression. In O-HA constructs, the RNAP homologous promoter drives expression of HA, and the construct does not express capping-RNAP. (B) Plasmid DNA of each construct based on SP6RNAP was transfected to HA levels after DC2.4 cells as detected by Western blot. NC is a blank. (C) Constructs comprising positive feedback based on SP6RNAP, K1E RNAP, and K1.5RNAP expression systems were transfected into DC2.4 cells and HA levels detected using Western blot. NC is a blank. (D) Schematic of the operation of nucleic acid vaccines with DC2.4 cells as vector. DC2.4 cells were first plated for culture, transfected with plasmid DNA comprising the construct of the invention after 16h, the transfected cells were collected after 48h and injected into 6-8 week old female mice (n=5) for immunization, serum samples were collected after 21 days of immunization, and levels of specific antibodies and cytokines were determined. (E) HA-specific IgG concentration in mouse serum samples assayed by ELISA. (F) IL-2, IL-4, TNF- α and IFN- γ levels in mouse serum samples as determined by ELISA. NC is a blank. PC was the positive control. (E) In (F), hEF1a-HA is a construct of which the constitutive hEF1a promoter drives HA expression, and SP6-RNAP-HA and SP6-PFB-HA are RNAP-HA and PFB-HA constructs based on the SP6 expression system, respectively. K1E-RNAP-HA and K1E-PFB-HA are RNAP-HA and PFB-HA constructs, respectively, based on the K1E expression system. K1.5-RNAP-HA and K1.5-PFB-HA are RNAP-HA and PFB-HA constructs, respectively, based on the K1.5 expression system.
Detailed Description
The terms "genetic background", "expression background", "environment" as used herein refer to DNA sequences upstream and/or downstream of a gene of interest and its promoter. It is known in the art that the environment in which a promoter is located may influence the level of gene expression it drives, and thus the predictability of the intensity of expression. In the present invention, by using heterologous capping enzyme-RNA polymerase fusion proteins and their cognate promoters, host endogenous gene network interference is avoided. Thus, the system of the present invention may be modularized.
The term "chimeric enzyme" as used herein is different from enzymes found in nature and may include catalytic domains of different origin (from different enzymes) or enzymes whose catalytic domains are of the same origin but arranged in a different manner from nature. The term "monomeric enzyme" refers to an enzyme having only one subunit, the monomeric enzyme comprising only one polypeptide chain.
Chimeric enzymes
The term "chimeric capping enzyme-RNA polymerase (RNA P)" as used herein comprises a monomeric RNA polymerase domain and a viral capping enzyme domain. The monomeric RNA polymerase may be a phage or mitochondrial derived nucleotide transferase that recognizes a promoter having a specific sequence and synthesizes single stranded RNA complementary to a double stranded template DNA sequence in the 5 'to 3' direction, thereby driving transcription of genes downstream of the promoter. The monomeric RNA polymerase is small in size, does not place excessive burden on the host's endogenous machinery, and facilitates assembly of the expression cassette to a plasmid or integration into the host genome. The capping enzyme domain comprises at least one RNA triphosphatase catalytic domain, at least one guanyltransferase catalytic domain, and at least one N7-guanine methyltransferase catalytic domain, which catalyzes the capping process of mRNA. The working principle is that firstly, RNA triphosphatase removes gamma-phosphate from 5 '-triphosphoric acid to generate 5' -diphosphate RNA; the second step is the transfer of GMP groups from GTP to 5' -diphosphate by RNA guanylate transferase via lysine-GMP covalent intermediates; thirdly, adding a methyl group on N7 amine of the guanine cap by purine-N7 methyltransferase to form a basic cap structure; the fourth step is to methylate ribonucleotides at the 2' O position by an independent m7G specific 2' O-methyltransferase to generate the cap structure m7GpppN at the 5' end of the mRNA. The m7 gppppn cap is critical for mRNA stability and transport from the nucleus to the cytoplasm. In this way, RNA molecules with a 5' end m7 gppppn cap are synthesized in mammalian host cells, which molecules can be recognized and translated by eukaryotic translation mechanisms without cytotoxicity and without induction of apoptosis. In the present invention, the chimeric capping enzyme-RNA polymerase is also referred to as a capping enzyme-RNA polymerase fusion protein or RNA polymerase-capping enzyme fusion protein. Chimeric capping enzyme-RNA polymerase Proprinciples and partial examples can be found in EP2377938A1.
Preferably, the RNA polymerase domain in the chimeric RNA polymerase-capping enzyme of the present invention may use T7 RNAP, T3 RNAP, K11 RNAP, K1.5RNAP or SP6RNAP. The RNA polymerases are monomeric RNA polymerases derived from bacteriophage, each RNA polymerase has a specific recognition promoter series, signal crosstalk hardly exists, multiple chimeric RNA polymerase-capping enzymes can be expressed in the same host, and the corresponding specific promoters are connected to multiple target genes to independently control the expression of the target genes. The phage-derived RNA polymerase reacts independently of RNA polymerase II in eukaryotic cells, and is simpler in structure than eukaryotic RNA polymerase, typically a single subunit enzyme. They are highly specific, do not require any additional protein factors to complete the transcription loop (transcription cycle), and can produce very long transcripts about five times faster than the E.coli RNA polymerase extension mechanism. In embodiments of the invention, the monomeric RNA polymerase may be wild-type T7 RNA polymerase (T7 RNAP, NCBI genome ID NC_001604; gene ID 1261050; uniProtKB/Swiss-Prot protein ID P00573), wild-type T3 RNA polymerase (T3 RNAP, NCBI genome ID NC_003298; gene ID 927437; uniProtKB/Swiss-Prot protein ID Q778M 8), wild-type K11RNA polymerase (NCBI genome ID NC_011043; protein ID YP_ 002003793.1), wild-type K1.5RNA polymerase (K1.5 RNAP, NCBI genome IDNC_008152; gene ID 5075932; uniProtKB/Swiss-Prot protein ID Q8SCG 8), wild-type SP6RNA polymerase (SP 6RNAP, NCBI genome ID NC_004831; gene ID 14878; uniProtKB/Sws-Prot protein Q7Y 1), or mutated versions thereof may also be used. For example, the T7 RNA polymerase may be T7 RNAP comprising mutations such as R551S, F644A, Q S, G645A, R627S, I S and D812E (Makarova et al, 1995) or K631M (Osumi-Davis et al, 1992; osumi-Davis et al, 1994).
The viral capping enzyme is selected from the group consisting of a wild-type bluetongue virus (blue tongue virus) capping enzyme, a wild-type african swine fever virus (African swine fever virus) capping enzyme, a wild-type acanthamoduo-phaga mimetic virus (acanthamoeba polyphaga mimivirus) capping enzyme, a wild-type bamboo mosaic virus (bamboo mosaic virus) capping enzyme, and a wild-type vaccinia virus capping enzyme; capping enzyme mutants that retain the activity of removing the 5' -triphosphate gamma phosphate of the pre-mRNA, transferring GMP from GTP to the newly generated RNA end, and adding the N7 methyl group of guanine to the GpppN cap can also be used. The NCBI genome ID of the bluetongue virus (BTV) serotype 10 is Y00421; the capping enzyme gene ID is 2943157; the protein ID was YP_052969.2. NCBI genome ID of bamboo mosaic virus (BMV isolate BaMV-O) is NC_001642; the gene ID of the capping enzyme was 1497253; uniProtKB/Swiss-Prot protein ID is Q65005. NCBI genome ID of African Swine Fever Virus (ASFV) strain BA71V is NC_001659; the UniProtKB/Swiss-Prot protein ID of the NP868R capping enzyme is P32094; genbank ID is L07263.1 or U18466.2. NCBI genome ID of acanthamoeba autophagy mimicry virus (APMV) is NC_014649; the capping enzyme gene ID is 3162607 (mimi_r382); protein ID is YP_142736.1; uniProtKB/Swiss-Prot protein ID is Q5UQX1. NCBI genome ID of Vaccinia virus strain (WesternReserve, WR) was NC_006998, and Vaccinia virus (Vaccinia) capping enzyme was D1R or D12L. The NCBI gene ID of D1R was 3707562 and the protein ID was YP_ 232988.1. The NCBI gene ID of D12L was 3707515 and the protein ID was YP_232999.1. The viral capping enzyme is preferably African swine fever virus NP868R capping enzyme.
It will be appreciated by those skilled in the art that proteins substantially homologous to the capping enzymes or RNA polymerases described above can also be employed in connection with the protein domains of the present invention. The term "substantially homologous" refers to protein sequences that are at least 95%, at least 98% or at least 99% identical at the level of the protein sequence. Homologous sequences can be functionally identical sequences in different species. Homologs of the genes or proteins of the invention can be readily determined by one skilled in the art. Furthermore, codons may be optimized for humanization based on the protein sequence, increasing the strength of expression of the protein in a heterologous host, as is well known in the art.
The RNA polymerase domain is preferably located at the C-terminus of the chimeric protein. Preferably, the RNA polymerase domain is linked to the C-terminus of the capping enzyme domain by means of a linking peptide. It is known in the art that a linker peptide is typically a plurality of serine and glycine in tandem, providing flexibility without affecting the activity of each domain. The sequence of the connecting peptide may be (G) 4 S) 4 . The term "domain" as used herein is a fundamental unit of protein structure, folding, function, evolution and design. The fusion protein comprises at least two domains, each encoded by a separate gene and linked so as to be transcribed and translated as a unit to produce a polypeptide.
Promoters recognized by monomeric RNA polymerase
The term "promoter" refers to a region of DNA that drives transcription of a nucleic acid sequence, located upstream of the synonymous strand of the gene transcription initiation site. The transcription machinery (RNA polymerase) is able to bind to nucleic acids at the promoter. In the present invention, the promoter recognized by RNA polymerase comprises the RNAP recognition region and a transcription initiation site. Promoters recognized by RNA polymerase are also known as homologous promoters (cognate promoters) or specific promoters due to their specific recognition by the corresponding RNAPs. "homologous promoter", "homologous RNAP" indicates that the promoter is of the same origin as its transcription machinery. For example, T7 RNAP is a homologous RNAP of the T7 promoter; pK11 is a homologous promoter of K11 RNAP. As demonstrated by the examples of the present invention, the interaction of different RNAP recognition with its cognate promoter is without signal crosstalk, so the pool of promoters specifically recognized by the same RNAP is also referred to as the orthogonal pool of promoters (orthorgonal promoter library).
The promoter recognized by the RNA polymerase may be a constitutive promoter or an inducible promoter. Constitutive promoters which can be used as promoters recognized by the monomeric RNA polymerase of the present invention are shown in Table 1. In addition, an operator sequence may be added upstream and/or downstream of the RNAP recognition region to construct an inducible promoter for RNA polymerase recognition; and expresses the corresponding repressor protein in the host cell, and dynamically controls the intensity of the promoter recognized by the single RNA polymerase. Useful repressor proteins are, for example, rpaR, braR, bjaR, tetR, CI434, phlf, HKCl, lmrA, bm3R, TP901CI, cymR, ttgR and the operator sequence rpaO, braO, bjaO, tetO of LexA, rpaR, braR, bjaR, tetR, see chinese patent application CN202111150967.4. Other repressor proteins have the corresponding operator sequences of SEQ ID NO:174-182.
TABLE 1 homologous promoters for each RNAP and their relative Activity
Quantitative expression system
The term "quantitative" as used herein refers to the ability to parameterize the functional characteristics of elements in a system (e.g., the behavior of a promoter), and specific values can be obtained by fitting experimental data to a model. In the present invention, the relative expression intensities of promoters can be precisely predicted using a model without free parameters to achieve rational design of expression systems, and thus the systems and methods according to the present invention are quantitative systems and quantitative methods. "number" or "dose" refers to the number or concentration of proteins. "relative expression intensity" also referred to as the number of counts (stoichiometries) refers to the ratio of the number of more than two proteins of interest produced by transcription and translation. As demonstrated in the examples, the strength of an individual gene expressed using the expression system of the present invention depends on the binding affinity between the RNAP subunit in the fusion protein and its cognate promoter. Binding affinity in mammalian host cells is proportional to that in prokaryotic hosts. The expression intensities of these homologous promoters in prokaryotic cells have been determined so that the relative expression intensities of the fusion protein-driven homologous promoters in mammalian cell hosts are deduced from the expression intensities. For different species of mammalian cells, the relative expression intensity is almost unchanged. In the present invention, as shown in Table 1, the relative expression intensities of the homologous promoters were normalized by the expression intensities of the other homologous promoters under the condition that only 1 wild-type homologous promoter was contained in the host cell. When two or more homologous promoters recognized by the same RNAP are used in combination, the relative intensities of the respective promoters are the ratios of the respective relative intensities in Table 1. In the present invention, "relative expression strength" is also referred to as "relative activity". "almost unchanged", "almost identical" means that the actual measured value of a certain parameter under different systems differs from the value given in the present invention by no more than a factor of 10, a factor of 3, a factor of 2, a factor of 1, a factor of 80, a factor of 50, a factor of 20, a factor of 15, and even no more than a factor of 10, a factor of 5, a factor of 3, a factor of 1.
In the present invention, the expression system comprising a T7 RNA polymerase domain and a T7 promoter as the promoter recognized by the RNA polymerase domain in the chimeric capping enzyme-RNA polymerase is also referred to as a T7 expression system; an expression system in which the chimeric capping enzyme-RNA polymerase comprises a T3 RNA polymerase domain and the promoter recognized by the RNA polymerase domain is a T3 promoter is also referred to as a T3 expression system; an expression system in which the promoter recognized by the SP6 RNA polymerase domain and the promoter recognized by the RNA polymerase domain in the chimeric capping enzyme-RNA polymerase is the SP6 promoter is also referred to as an SP6 expression system; an expression system in which the chimeric capping enzyme-RNA polymerase comprises a K11 RNA polymerase domain and the promoter recognized by the RNA polymerase domain is a K11 promoter is also referred to as a K11 expression system; the expression system comprising K1.5RNA polymerase domain and the promoter recognized by the RNA polymerase domain in the chimeric capping enzyme-RNA polymerase is the K1.5 promoter, also referred to as the K1.5 expression system.
The term "expression cassette (expression cassette)" refers to a DNA fragment consisting of one or more genes and sequences controlling the expression of the genes, such that the proteins encoded by the genes are capable of being expressed in a desired host cell (i.e., mammalian cell). The expression control sequences contained in the expression cassette are known in the art and optionally include promoter sequences, 3' non-coding regions, transcription termination sites, and/or polyadenylation sequences, and the like. In the present invention, an expression cassette comprising a promoter and a coding sequence may be integrated into the genome or present in a plasmid vector.
In the present invention, the first promoter driving expression of the capping-RNAP may be any promoter active in mammalian cells, may be a constitutive or inducible promoter, or may be a tissue specific promoter. For example, the first promoter may be a constitutively expressed CMV1, CMV3G, CMVmini, EF a, EF1a core promoter, TRE3G promoter; or inducible promoters commonly used in mammals, such as those comprising the CMVmini core sequence and comprising tetO or cymO manipulation sites. It will be appreciated by those skilled in the art that in embodiments where the first promoter is an inducible promoter, the chimeric capping enzyme-RNA polymerase expression cassette further comprises a transcription factor coding sequence that modulates the manipulation site. The sequence of the above promoter is given, for example, in chinese patent application CN 202111150967.4. Such sequences are well known to those skilled in the art. In a preferred embodiment, the first promoter may comprise a promoter recognized by a monomeric RNA polymerase, such that the production of the capping-RNAP has positive feedback. In this way, the total expression level can be increased while maintaining the expression ratio of each target gene.
Mammalian cells as hosts include, but are not limited to, CHO cells (chinese hamster ovary cells), MDCK (a highly differentiated endothelial cell line derived from MadIin-Darby canine kidney), COS cells (african green monkey kidney fibroblasts transformed with SV40 virus genes), HEK293 human embryonic kidney cells, and the like. The expression system of the present invention may be a transient expression system or a stable expression system. The term "transient expression system" refers to a system that is not selectively cultured after the expression vector is introduced into a host cell. The term "stable expression system" refers to the introduction of a vector into a host cell and the screening culture such that the vector DNA is stably present in the cell. Based on this, the methods and systems of the present invention are also advantageous in constructing animal models of artificial tissues, artificial organs, human diseases, as well as in gene function analysis, biopharmaceutical, vaccine preparation, gene therapy, and the like.
Embodiments of the aspects described herein may be illustrated by the following numbered paragraphs:
1. a system for expressing one or more genes of interest in a mammalian cell, the system comprising:
a chimeric capping enzyme-RNA polymerase comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5RNA polymerase, and SP6 RNA polymerase; the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
The promoter recognized by the monomeric RNA polymerase, wherein the promoter recognized by the T7 RNA polymerase is selected from the group consisting of SEQ ID NO:1-SEQ ID NO:54, a group of two or more of the group consisting of (a) and (b); the promoter recognized by the T3 RNA polymerase is selected from the group consisting of SEQ ID NO:161-SEQ ID NO: 172; the promoter recognized by the K11 RNA polymerase is selected from the group consisting of SEQ ID NO:55-SEQ ID NO:112, a group of two or more of the group consisting of 112; the K1.5RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NOs: 183-SEQ ID NO:234, a group of elements; the promoter recognized by the SP6RNA polymerase is selected from the group consisting of SEQ ID NO:113-SEQ ID NO: 160.
2. A system for expressing one or more genes of interest in a mammalian cell, the system comprising:
one or more chimeric capping enzyme-RNA polymerase gene expression cassettes, each independently comprising a chimeric capping enzyme-RNA polymerase coding sequence, each independently comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5RNA polymerase, and SP6RNA polymerase; the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
One or more gene expression cassettes of interest, each independently comprising a promoter recognized by a monomeric RNA polymerase and a gene sequence of interest, wherein the promoter recognized by the T7 RNA polymerase is selected from the group consisting of seq id NO:1-SEQ ID NO:54, a group of two or more of the group consisting of (a) and (b); the promoter recognized by the T3 RNA polymerase is selected from the group consisting of SEQ ID NO:161-SEQ ID NO: 172; the promoter recognized by the K11 RNA polymerase is selected from the group consisting of SEQ ID NO:55-SEQ ID NO:112, a group of two or more of the group consisting of 112; the K1.5RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NOs: 183-SEQ ID NO:234, a group of elements; the promoter recognized by the SP6 RNA polymerase is selected from the group consisting of SEQ ID NO:113-SEQ ID NO: 160.
3. The system of paragraph 2, the system comprising:
a chimeric capping enzyme-RNA polymerase gene expression cassette comprising a first promoter sequence and a chimeric capping enzyme-RNA polymerase coding sequence, the chimeric capping enzyme-RNA polymerase comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; and
one or more gene expression cassettes of interest, each independently comprising a second promoter sequence and a gene sequence of interest, wherein each second promoter is independently a promoter recognized by the monomeric RNA polymerase.
4. The system of paragraph 3 wherein the number of genes of interest is 1-10, preferably 1-3.
5. The system of paragraph 3 wherein the first promoter is selected from the group consisting of CMV1, CMV3G, EF1a, CMVini or EF1a core promoters.
6. The system of paragraph 3 wherein the first promoter is a dual promoter and comprises a promoter recognized by the monomeric RNA polymerase.
7. The system of paragraph 3, whereinThe chimeric capping enzyme-RNA polymerase has a (G) between the monomeric RNA polymerase domain and the viral capping enzyme domain 4 S) 4 And (3) connecting peptides.
8. The system of paragraph 3 wherein the viral capping enzyme is African swine fever NP868 capping enzyme.
9. The system of paragraph 2, the system comprising:
a first chimeric capping enzyme-RNA polymerase gene expression cassette comprising a first promoter sequence and a first chimeric capping enzyme-RNA polymerase coding sequence, the first chimeric capping enzyme-RNA polymerase comprising a first monomeric RNA polymerase domain and a first viral capping enzyme domain;
a first gene expression cassette of interest comprising a second promoter sequence and a first gene sequence of interest, wherein the second promoter is a promoter recognized by the first monomeric RNA polymerase;
A second chimeric capping enzyme-RNA polymerase gene expression cassette comprising a third promoter sequence and a second chimeric capping enzyme-RNA polymerase coding sequence, the second chimeric capping enzyme-RNA polymerase comprising a second monomeric RNA polymerase domain and a second viral capping enzyme domain; and
a second gene expression cassette comprising a fourth promoter sequence and a second gene sequence of interest, wherein the fourth promoter is a promoter recognized by the second monomeric RNA polymerase;
wherein the first monomeric RNA polymerase and the second monomeric RNA polymerase are each independently selected from the group consisting of T7RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5RNA polymerase, and SP6 RNA polymerase; the first viral capping enzyme and the second viral capping enzyme are each independently selected from the group consisting of bluetongue viral capping enzyme, african swine fever viral capping enzyme, acanthamoidovorous pseudoviral capping enzyme, bamboo mosaic viral capping enzyme, and poxviral capping enzyme.
10. The system of paragraph 9, wherein the first and third promoters are each independently selected from CMV1, CMV3G, EF a, CMVmini or EF1a core promoters.
11. The system of paragraph 9, wherein the first promoter and/or third promoter is a dual promoter and comprises a promoter recognized by the monomeric RNA polymerase.
12. The system of paragraph 9 wherein the first monomeric RNA polymerase domain has (G) between the first viral capping enzyme domain 4 S) 4 A linker peptide; the second monomeric RNA polymerase domain has (G) between the second monomeric RNA polymerase domain and the second viral capping enzyme domain 4 S) 4 And (3) connecting peptides.
13. The system of paragraph 9 wherein the first viral capping enzyme and the second viral capping enzyme are african swine fever NP868 capping enzymes.
14. The system of paragraph 2, the system further comprising:
a repressor expression cassette selected from one or more of RpaR, braR, bjaR, tetR, CI434, phlf, HKCl, lmrA, bm3R, TP901CI, cymR, ttgR, and LexA;
wherein the promoter recognized by the monomeric RNA polymerase comprises the manipulation site of the repressor protein.
15. The system of any of paragraphs 1-14, wherein the system is a transient expression system or a stable expression system.
16. A method for expressing one or more genes of interest in a mammalian cell, the method comprising:
Constructing one or more chimeric capping enzyme-RNA polymerase gene expression cassettes, each independently comprising a chimeric capping enzyme-RNA polymerase coding sequence, each independently comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5RNA polymerase, and SP6 RNA polymerase; the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
constructing one or more gene expression cassettes of interest, each independently comprising a promoter recognized by a monomeric RNA polymerase and a gene sequence of interest, wherein the promoter recognized by the T7RNA polymerase is selected from the group consisting of seq id NO:1-SEQ ID NO:54, a group of two or more of the group consisting of (a) and (b); the promoter recognized by the T3 RNA polymerase is selected from the group consisting of SEQ ID NO:161-SEQ ID NO: 172; the promoter recognized by the K11 RNA polymerase is selected from the group consisting of SEQ ID NO:55-SEQ ID NO:112, a group of two or more of the group consisting of 112; the K1.5RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NOs: 183-SEQ ID NO:234, a group of elements; the promoter recognized by the SP6 RNA polymerase is selected from the group consisting of SEQ ID NO:113-SEQ ID NO: 160.
17. The method of paragraph 16, the method comprising:
constructing a chimeric capping enzyme-RNA polymerase gene expression cassette comprising a first promoter sequence and a chimeric capping enzyme-RNA polymerase coding sequence, wherein the chimeric capping enzyme-RNA polymerase comprises a monomeric RNA polymerase domain and a viral capping enzyme domain; and
one or more gene expression cassettes of interest are constructed, each independently comprising a second promoter sequence and a gene sequence of interest, wherein each second promoter is independently a promoter recognized by the monomeric RNA polymerase.
18. The method of paragraph 17 wherein the number of genes of interest is 1-10, preferably 1-3.
19. The method of paragraph 17 wherein the first promoter is selected from the group consisting of CMV1, CMV3G, EF1a, CMVini or EF1a core promoters.
20. The method of paragraph 17 wherein the first promoter is a dual promoter and comprises a promoter recognized by the monomeric RNA polymerase.
21. The method of paragraph 17 wherein the chimeric capping enzyme-RNA polymerase has a single RNA polymerase domain and a viral capping enzyme domain (G 4 S) 4 And (3) connecting peptides.
22. The method of paragraph 17 wherein the viral capping enzyme is African swine fever NP868 capping enzyme.
23. The method of paragraph 16, the method comprising:
constructing a first chimeric capping enzyme-RNA polymerase gene expression cassette comprising a first promoter sequence and a first chimeric capping enzyme-RNA polymerase coding sequence, the first chimeric capping enzyme-RNA polymerase comprising a first monomeric RNA polymerase domain and a first viral capping enzyme domain;
constructing a first target gene expression cassette, wherein the first target gene expression cassette comprises a second promoter sequence and a first target gene sequence, and the second promoter is a promoter recognized by the first single RNA polymerase;
constructing a second chimeric capping enzyme-RNA polymerase gene expression cassette comprising a third promoter sequence and a second chimeric capping enzyme-RNA polymerase coding sequence, the second chimeric capping enzyme-RNA polymerase comprising a second monomeric RNA polymerase domain and a second viral capping enzyme domain; and
constructing a second target gene expression cassette, wherein the second target gene expression cassette comprises a fourth promoter sequence and a second target gene sequence, and the fourth promoter is a promoter recognized by the second single RNA polymerase;
Wherein the first monomeric RNA polymerase and the second monomeric RNA polymerase are each independently selected from the group consisting of T7RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5RNA polymerase, and SP6 RNA polymerase; the first viral capping enzyme and the second viral capping enzyme are each independently selected from the group consisting of bluetongue viral capping enzyme, african swine fever viral capping enzyme, acanthamoidovorous pseudoviral capping enzyme, bamboo mosaic viral capping enzyme, and poxviral capping enzyme.
24. The method of paragraph 23, wherein the first and third promoters are each independently selected from the group consisting of CMV1, CMV3G, EF a, CMVmini, or EF1a core promoters.
25. The method of paragraph 23, wherein the first promoter and/or third promoter is a dual promoter and comprises a promoter recognized by the monomeric RNA polymerase.
26. The method of paragraph 23 wherein the first monomeric RNA polymerase domain has (G) between the first viral capping enzyme domain 4 S) 4 A linker peptide; the second monomeric RNA polymerase domain has (G) between the second monomeric RNA polymerase domain and the second viral capping enzyme domain 4 S) 4 And (3) connecting peptides.
27. The method of paragraph 23 wherein the first viral capping enzyme and the second viral capping enzyme are african swine fever NP868 capping enzymes.
28. The method of paragraph 16, the method further comprising:
constructing a repressor expression cassette selected from one or more of RpaR, braR, bjaR, tetR, CI434, phlf, HKCl, lmrA, bm3R, TP901CI, cymR, ttgR, and LexA;
wherein the promoter recognized by the monomeric RNA polymerase comprises the manipulation site of the repressor protein.
29. The method of any one of paragraphs 16-28, wherein each expression cassette is transiently or stably transfected into a mammalian cell.
30. Use of the expression system of any one of paragraphs 1-15 or the method of any one of paragraphs 16-29 for the preparation of a vaccine.
31. The use of paragraph 30 wherein the DC cells are employed as a delivery vehicle for the vaccine.
32. The use of paragraph 31 wherein the vaccine is a multivalent vaccine.
Example 1: construction and characterization of quantitative expression systems
1 Single reporter gene expression System
Protein expression in mammalian cells is more complex than in simple prokaryotic cells. As shown in fig. 1A, in the nucleus, a precursor RNA (premature RNA) is transcribed as a DNA template, and the precursor RNA needs to undergo a series of post-transcriptional modifications in the nucleus to produce mature mRNA, which is then transferred to the cytoplasm to complete the subsequent translation process to produce the corresponding protein. The structure of mature mRNA has five main parts, including from 5 'to 3': a 5'cap structure (5' cap), a 5 'untranslated region (5' UTR), a coding region, a 3 'untranslated region (3' UTR), and a poly-A (PolyA) tail. In our design, a monomeric RNA polymerase is fused to a capping enzyme, the RNA polymerase domain of the fusion protein is used as a transcription machinery to drive transcription of the cognate promoter, and modification of the resulting precursor RNA by the capping enzyme is accomplished. As shown in FIG. 1B, the chimeric capping enzyme-RNA polymerase and RNA polymerase domain recognition promoter (P y f p ) In a different carrier. The design facilitates testing the strength of each promoter in the promoter library and the orthogonality of the single gene expression systems using different RNAPs.
First, we studied the relationship between transcriptional activity of homologous promoters and binding affinity between homologous promoter-RNAP in mammalian systems. We have characterized the relative binding affinities of multiple orthogonal promoters in prokaryotic hosts (Table 1), which provides the basis for precise control of protein expression levels and expression ratios in mammalian hosts. As shown in FIG. 1B, to construct a quantitative assay system, we constructed a T7 single gene expression system. The pOMC2 plasmid was constructed by ligating the constitutive promoter EF 1. Alpha. To NP868R-T7 RNAP upstream. The plasmid also constitutively expresses red fluorescence mcherry, indicating transfection status. 21T 7 promoters (pT 7WT, pT7M1, pT7M11, pT7M14, pT7M15, pT7M17, pT7M21, pT7M22, pT7M25, pT7M28, pT7M29, pT7M30, pT7M35, pT7M38, pT7M41, pT7M45, pT7M48, pT7M49, pT7M52, pT7M55 and pT7M 6) were ligated upstream of the reporter gene yfp to construct the pOMC1 plasmid. Two of them are combinedThe individual plasmids were co-transfected into chinese hamster ovary Cells (CHO) and human embryonic kidney 293T (HEK 293T). The yellow fluorescence intensity of YFP, i.e., the expression intensity of the corresponding expression system, was determined by flow cytometry and reacted with the activity of each T7 promoter under construction. As shown in FIGS. 1C and 1D, the relative strength of the promoter in E.coli (horizontal axis) and the relative strength of the promoter in CHO cells (vertical axis) are linearly related within 100 times of the expression intensity, and the correlation coefficient R is related at different plasmid concentrations 2 0.9350 and 0.9205, respectively. It can be seen that the relative binding affinity of each T7 promoter to T7RNAP in mammalian hosts was consistent with that in E.coli, each promoter retained the fold change in expression observed in prokaryotic cells, and that high concentrations of capping-T7RNAP (0.7. Mu.g plasmid) were not toxic to cells. This linear correlation also exists in HEK293T cells. The results show that the relative intensities of promoters in the homologous promoter library selected by the invention are consistent in a mammalian host and in prokaryotic cells, and the expression system can be utilized to finely regulate and control the expression of target genes in mammalian cells.
We also tested 15K 11 single gene expression systems and 13 SP6 single gene expression systems, respectively, using the human embryonic kidney cell lines HEK293T and CHO cell lines as hosts. The results showed that the relative activities of the pK11 and pSP6 promoters were identical in HEK293T, CHO cell line and e.coli, indicating that the relative intensities of the promoters in the promoter pool constructed according to the present invention were identical in different genomic contexts (fig. 1G).
The activity of a single promoter can be predicted by a simple MM equation:
where α is the maximum expression of the promoter in the presence of sufficient homologous RNAP. Beta is the background expression of the promoter in the absence of RNAP. K (K) A Relative binding affinity for homologous RNAP; k (K) D ＝1/K A 。[RNAP]Is the concentration of RNAP (i.e., the concentration of chimeric enzyme) [ P ]]To reportConcentration of protein. n is the Hill coefficient. For all promoters, α and β can be obtained by leakage and saturation expression measurements of the reporter gene, which are slightly different in CHO and HEK293T cells. Homologous promoters and RNAP K in mammalian systems A Linear correlation with binding affinity measured in the prokaryotic system. Therefore, the expression amount of the protein controlled by each homologous promoter can be predicted according to the formula (1).
Furthermore, we constructed a capping-RNAP (its RNAP domains are T7 RNAP, K1.5RNAP, K11 RNAP, phi15 RNAP, T3RNAP, SP6RNAP, respectively) expression cassette with different transcription machinery, and a reporter ypf expression cassette driven by the WT promoter recognized by each RNAP, different combinations comprising the two constructed plasmids were co-transfected into CHO host cells for orthogonality testing (FIG. 1E). As shown in FIG. 1F, each RNAP specifically activates a corresponding homologous promoter. Although K1.5RNAP activated the pSP6WT promoter to some extent, the transcription strength of pSP6WT was significantly lower than that of the homologous promoter pK1.5WT. The results indicate that phage-derived monomeric RNA polymerase T7 RNAP, K1.5RNAP, K11 RNAP, phi15 RNAP, T3RNAP, SP6RNAP can orthogonally recognize their cognate promoters. Orthogonal expression systems can be constructed under mammalian systems using these promoters and capping-RNAP.
2 double reporter gene expression system
When a plurality of genes are simultaneously expressed in mammalian cells, the expression intensity and proportion of each gene are affected by the genetic background and the cellular environment. The background effect (context effect) is due to the competition of different genes for transcription key resources to some extent. The system of the present invention uses a nonhost-derived capping-RNAP as the transcription machinery without concern for competition of the phage-derived RNAP by the host promoter in the mammalian cell. Therefore, the expression intensities and the expression ratios of two or more genes expressed by the expression system of the present invention can be quantitatively predicted and quantitatively controlled.
FIG. 2A shows a T7 dual gene expression system of the present invention. pT7WT, pT7M17, pT7M21, pT7M25, pT7M35, pT7M41 and pT7M49 were used as P respectively yfp And P mCherry Connected to the reporter gene yfp and upstream of mcherry, 7×7 dual reporter gene expression systems were constructed. Based on the measurement results under the single reporter gene system, the normalized expression intensities of pT7WT, pT7M17, pT7M21, pT7M25, pT7M35, pT7M41 and pT7M49 were 1.0000, 0.4539, 0.2423, 0.1814, 0.0495, 0.0379 and 0.0158, respectively, and the difference in the expression intensities was about 100-fold. The expression system also comprises the transcription machinery NP868R-T7RNAP controlled by the constitutive promoter EF 1. Alpha. On the same plasmid. The pOMC16 plasmid containing the above elements was transfected into CHO or HEK293T cells cultured in 24 well plates at a plasmid transfection amount of 0.5. Mu.g per well. Fluorescence intensities of YFP and mCherry under each construction were determined as P by flow cytometry yfp P mCherry Characterization of intensity. FIG. 2B is a heat map showing fluorescence intensity in HEK293T host cells for a 7X 7 promoter combined dual reporter gene expression system. As shown in fig. 2B, at P yfp In the case of a strong promoter, no matter P cherry The expression level of mCherry is greatly reduced (first column of experimental heat map); similarly, at P mCherry In the case of a strong promoter, the amount of YFP expression was also greatly reduced (first line of the experimental heat map). These results indicate that two reporter genes are competing for certain limited resources. We also observed similar phenomena in the yfp-bfp double expression system. Interestingly, however, this competing effect was greatly alleviated in CHO cell background and other cell lines over-expressing RNAP. We speculate that limited resources for promoter competition are therefore available RNAP. The RNAP that can be used is total RNAP minus RNAP that has been occupied by the promoter. In the present invention, the available RNAP is also referred to as free RNAP. To describe this resource competition effect, we extended the simple model with free RNAP instead of total RNAP:
[RNAP] free ＝[RNAP] tot /(1+[DNA] yfp free /K yfp +[DNA] mcherry free /K mcherry ) (4)
in the formulae (2) to (6), the definitions of variables are the same as those of the formula (1), and the subscripts indicate the genes corresponding to the parameters. [ RNAP ] free Concentration of free RNAP, [ RNAP ]] tot Is the total RNAP concentration. For the yfp-mcherry dual reporter system, [ RNAP ]] free ＝[RNAP] tot -[RNAP] yfp -[RNAP] mcherry (FIG. 2C). Wherein [ RNAP] yfp [ RNAP ]] mcherry Respectively is with P yfp Or P mCherry Concentration of bound RNAP. New added variable [ DNA ]] tot Is the concentration of transfected plasmid. Since no free variables exist in the formulae (2) - (6), the gene expression amount of any dual gene expression system according to the present invention can be predicted according to the formulae (2) - (6).
Numerical simulations of 49 dual expression systems showed [ RNAP] free The concentration varies greatly in different systems. When P yfp And P mCherry In the case of strong promoters (e.g., pT7 WT) [ RNAP ]] free Minimum; when the strength of each promoter decreases, [ RNAP ]] free Lifting; [ RNAP] free The largest case is P yfp And P mCherry Are all weak promoters pT7M49 (FIG. 2D).
However, since RNAP < K and the leaky expression of β is negligible with respect to the expression intensity when the promoter is in the open (on) state, [ RNAP] free /K yfp [ RNAP ]] free /K mCherry All < 1, therefore [ YFP ]]/[mcherry]≈α e ·(K mCherry /K yfp ). Wherein alpha is e Is equal to alpha yfp 、α mCherry [ DNA ]] tot A related constant. Although under different systems [ RNAP] free The concentrations were different, but the expression ratio of YFP to mCherry was hardly changed.
3 three reporter gene expression system
According to literature [1-3]The DNA sequence (i.e., the local genetic background) in the vicinity of the gene expression cassette and the sequence of the gene arrangement also affect the intensity and proportion of expression of the individual genes. To investigate the effect of the expression environment on the promoter strength of the expression system of the present invention, we connected bfp, yfp and mCherry to the pT7WT promoter downstream in different sequences, respectively, constructed all 6 possible sequential combinations (fig. 3A), measured the expression levels of each fluorescent protein under the construction using flow cytometry, and calculated the coefficient of variation CV of each promoter strength under different constructions. As shown in FIG. 3B, the promoter strength variation coefficient was small in each alignment (CV in HEK293T cells yfp ＝0.07，CV mCherry ＝0.08，CV bfp =0.12; CV in CHO cells yfp ＝0.13，CV mCherry ＝0.03，CV bfp =0.11). The results demonstrate that under the construction of the present invention, the promoter activity and the expression ratio of the multiple genes are independent of the local genetic background and the reporter gene sequence.
As shown in FIG. 3C, to further investigate the properties of the three reporter gene expression system, we selected the promoters pT7WT (1), pT7M17 (2), pT7M21 (3), pT7M25 (4), pT7M35 (5), pT7M41 (6) and pT7M49 (7) as P, respectively bfp 、P yfp And P mChe rr y Linked upstream of the reporter genes bfp, yfp and mCherry, driving the reporter gene expression. The expression system also comprises the transcription machinery NP868R-T7RNAP controlled by the constitutive promoter EF 1. Alpha. On the same plasmid. The promoter combination comprises: B1C1M1, B2C1M1, B3C1M1, B4C1M1, B6C1M1, B7C1M1, B1C2M2, B3C1M3, B3C2M2, B4C2M2, B5C2M2, B6C2M2, B7C2M 7, B4C2M7, B3C2M6, B5C2M6, B2C7M2, B4C7M2, B5C3M2, B2C4M4, B2C4M5, B2C7M7, B2C6M4, B3C4M4, B4C7M7, B3C5M4, B7C5M4, B6C 46M6 (B is P) bfp Promoter, C is P yfp M is P mCherry The numbers are those of the 7 promoters mentioned above, e.g.B5C2M6 denotes P bfp Is pT7M35, P yfp Is pT7M17, P mCherry A combination of pT7M 41).
BFP, YFP and mCherry fluorescence intensities of HEK293T cells containing the respective expression systems were determined as P by flow cytometry (FIG. 3D) bfp 、P yfp P mCherry Characterization of intensity (normalized to pT7WT intensity). In addition, using the intensities of the promoters measured by the dual reporter gene expression system (FIG. 2B, table 2) as parameters, we predicted the expression intensities of the promoters in the triple reporter gene expression system using the resource competition model above. As shown in fig. 3E, the resource competition model enables accurate prediction of the relative expression intensity of each promoter in the three-reporter gene expression system. In both HEK293T and CHO cell lines, the pearson correlation coefficient for the expression levels of the three reporter genes reached 0.81 to 0.88 (fig. 3E). An interesting phenomenon is that in both cell lines, the predicted activity of the promoter was higher than the actual measurement when the strongest promoter pT7WT was used for both promoters, probably due to cytotoxicity of the cells caused by overexpression of the reporter protein.
In summary, in the gene expression system constructed by the invention, the promoter strength is precisely predictable, irrespective of the genetic background. The expression system of the present invention can be advantageously used for simultaneous quantitative expression of a plurality of proteins, such as expression of a multi-subunit protein, establishment of a protein complex network formed by a plurality of proteins, and vaccine production.
Optimization of 4 VLP yield
As an application of the present invention we use a three gene expression system for virus-like particles (VLPs) expressing influenza a virus H1N 1. A virus-like particle (VLP) is a non-pathogenic, non-replicable protein nanoparticle. They have a broad application potential in, for example, vaccine and drug delivery. Influenza A Hemagglutinin (HA), neuraminidase (NA) and matrix protein 1 (M1) are expressed in B9 cells in appropriate ratios and these three subunits can spontaneously assemble to be stableFixed VLPs (fig. 4B). VLP particles can be isolated and purified by sucrose density gradient centrifugation. Studies have shown that the stoichiometry of the protein subunits is critical for the efficiency of assembly and immunogenicity of VLPs [ 4-5)]. Thus, by means of the model of the invention, the expression ratios of the three subunits can be precisely controlled by selecting promoters of appropriate expression strengths from the promoter library of the invention depending on the desired protein ratio. With reference to previous studies, we fused the mCherry coding sequence to amino acids 1-61 of the N-terminus of NA (NA-mCherry) and EGFP to M1 (GFP-M1), enabling qualitative and quantitative measurements of intact VLPs formed by self-assembly of HA protein, NA-mCherry protein and EGFP-M1 protein using fluorescence microscopy. As shown in FIG. 4C, most of the GFP-M1 observed in the FITC field was co-localized with NA-mCherry in the TxRed field, while some VLP particles in the TxRed field were not observed in the FITC field, indicating that particles with EGFP-M1 contained mostly NA-mCherry. Thus, we selected green fluorescence as an indicator and quantitatively measured VLP yield using flow cytometry. To compare the yields of VLPs at different expression ratios, we selected 21 promoter combinations for testing, with the virus-like particles produced by the wild-type T7 promoter combinations (111, numbered from left to right corresponding to the promoter numbers of HA, GFP-M1 and NA-mCherry) as reference (100%). Cells were not treated (cells), phosphate Buffered Saline (PBS) and cells expressing EGFP-M1 protein only (M1) served as controls. As shown in fig. 4E, the strongest promoter combination (111) did not achieve the highest VLP yield, while the weak promoters of certain subunits produced more VLPs. The promoters tested were: pT7WT (1), pT7M14 (2), pT7M17 (3), pT7M21 (4) and pT7M25 (5). Three digits are numbered P HA 、P M1 And P NA Promoters employed, e.g.123P HA 、P M1 And P NA pT7WT, pT7M14 and pT7M17, respectively.
The three-gene resource competition model of the present invention can be used to predict VLP production. Numerical modeling of 54×54×54= 157,464 promoter combinations using models showed that high expression of HA protein was detrimental to the production of intact VLPs, and that appropriate combinations of three genes could produce VLP yields 2-fold of the strongest promoter combination (the magnified region in fig. 4F, hereinafter "reference region"). As shown in fig. 4G, we have verified that some combinations of promoters predicted to be high yield from this region species, all of which produce VLPs higher than the strongest promoter combination (111), and whose yields are consistent with model predictions without free parameters. In this example, the expression ratios of the three antigens of VLPs were adjusted in a predictable and precisely adjustable manner based on the model-optimized promoter combinations of the present invention, achieving a substantial increase in VLP yield.
5 inducible expression System
In the above single, double or triple gene systems, the expression systems of the present invention are all constitutive expression systems. The expression system of the invention may also be used to construct inducible expression systems. As shown in FIG. 5A, a pOMC1 plasmid was constructed by ligating the constitutive promoter EF1 alpha upstream of the NP868R-T7 RNAP. The constitutive promoter EF1 a was ligated upstream of the repressor proteins CI434, phlf, HKCI, lmrA, bm3R, TP901CI, cymR, ttgR or LexA coding sequence, respectively, replacing the fluorescent reporter sequence in the pnmc 1 backbone plasmid. The 9 control sites corresponding to the repressor proteins are connected to the downstream of the T7 core promoter-TRE 3G promoter, and the downstream of the promoter is connected with a reporter gene yfp to replace the fluorescent reporter gene sequence in the pOMC1 skeleton plasmid. In this induction system, the transcriptional machinery NP868R-T7 RNAP competes with the repressor protein for the corresponding binding site on the promoter. Three plasmids were co-transfected into CHO cells and the reporter fluorescence intensity was measured by flow cytometry at a concentration of 10 ng-1. Mu.g of the repressor plasmid (both the fluorescence reporter plasmid concentration and the capping-RNAP plasmid concentration were 0.3. Mu.g), respectively, to give an induction curve for the system. As shown in fig. 5B, at low doses of repressor protein, transcription machinery mediated transcriptional activation predominates, while at high doses, repressor protein transcriptional inhibition predominates.
The regulatory regions of the induction system may also be linearized. It is known in the art that adding negative feedback to an induction system enables linear transformation of the reporter level. As shown in FIG. 5C, expression of NP868R-T7 RNAP was driven by the pC-dox promoterExpression of the reporter gene Citrine was driven by either pT7wt or pT7M 25. pC-dox is a double-input promoter which is an inducible promoter P rpaO-CMV1 Is constructed by inserting a tetO manipulation site into the upstream and downstream of the gene, and can respond to pC-HSL and doxycycline dual signals. The expression of the repressor RpaR recognizing rpaO is driven by the constitutive promoter EF 1. Alpha., the repressor recognizing the repressor tetO is driven by the promoter P tet-CMV D2i Expression is initiated and the promoter region contains the tetO site. The export plasmid also constitutively expresses mCherry to monitor transfection. The principle and sequence of the pC-dox system are described in Chinese patent application CN 201910411123.7. To characterize the concentration of NP868R-T7 RNAP, an input plasmid was also constructed containing the same RpaR expression cassette, tetR expression cassette, and the reporter gene citrine driven by pC-dox as the output plasmid. The input plasmid and the output plasmid were transfected into CHO cells separately. As shown in FIG. 5D, the fluorescence intensity of the reporter gene on the input and output plasmids was linearly changed within a 100-fold range under the induction of doxycycline (dox) at 0-20ng/mL, p-coumaroyl-homoserine lactone (pC-HSL) at 200 nM-10. Mu.M, indicating that the system was able to achieve a linear response. The results indicate that RNAP binds to the promoter with little synergy and that protein expression is not hypersensitive (Hill coefficient n.apprxeq.1).
Summary 6
The present invention has developed a novel method for precisely controlling the stoichiometry of polygenic expression in mammalian cells using an expression system with orthogonal transcription machinery. The expression level of the single gene can be directly calculated from the binding affinity; the ratio of the amount of expression of multiple genes can be predicted by a resource competition model that includes binding affinity. According to a simplified model, the expression level of each gene in a multi-gene expression system is proportional to the binding strength of its cognate promoter-RNAP over a wide range. As an example, the number of doses of three gene expression of influenza a VLPs was systematically screened and optimized to obtain higher VLP yields. These results demonstrate the potential of the expression system of the invention in basic research and medical applications.
Endogenous expression systems rely on host transcription machinery, and it is difficult to precisely control the expression ratio of multiple genes using the natural or engineered promoters of the endogenous transcription machinery, both of which are affected by the sequence of the genes, the genomic environment, epigenetic modifications, and the host cell type [6-8]. According to literature reports, the gene arrangement sequence of multiple genes has a great influence on the activity of an engineering promoter. Expression of the upstream gene often results in reduced expression of the downstream gene [6]. In addition, studies of natural constitutive promoters have shown that patterns of gene expression exhibit cell-specific, unpredictable silencing in certain cell types. For example, in primary motor neurons, the CMV promoter is initially able to drive high expression, however its activity subsequently decreases [10]. However, in our system, the relative activity of the orthogonal promoter library is independent of its order on the genome and the genetic environment, and the system of the present invention can be advantageously transferred to other types of mammalian cells.
The number of measures of polygenic expression is critical for biological function and evolutionary fitness. However, there is still a lack of effective tools in the art for precise control of stoichiometry. Recent studies have recognized this core technical problem and proposed a CRISPR-Cas9 based metrology control approach [8-9]. This high throughput approach is suitable for systematically assessing the phenotype of individual genes. In the methods of the invention, the expression system is more easily predicted by the model because it does not occupy endogenous transcription machinery and mRNA capping machinery. One problem that the system of the present invention may face is that when the protein expression level is too high, excessive translation resources may be occupied, causing a burden or toxicity to the host cell.
In summary, the present invention establishes an environment-independent orthogonal expression platform in mammalian cells that can precisely control the expression level of a single gene or the expression ratio of a plurality of genes in a gradual manner. Precise control of the expression ratio enables engineering of stem cell differentiation and transdifferentiation and synergistic expression of polyproteins or subunits.
7 materials and methods
Table 1 Induction System involved in the examples
Cell lines, culture media and reagents. At 5% CO 2 The chinese hamster ovary cells (CHO, national Infrastructure Cell Line Resource, NICR), the capping-RNAP stable cell line (B9) and the human embryonic kidney cells HEK293T cells (ATCC) were cultured in high glucose DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco) and 100U/ml penicillin-streptomycin (Hyclone) at 37 ℃. Unless otherwise indicated, the enzymes used for molecular cloning were purchased from NEB, the compounds were purchased from Sigma-Aldrich, and the DNA synthesis service was provided by GENEWIZ. Transfection reagent (Lipofectamine 3000) was from Invitrogen.
Plasmids and sequences. Plasmid sequences are described in http:// bdainformatics.org/datarepositisource. The plasmid used for transfection of mammalian cells was plasmid PB531A-1 based on the PiggyBac transposon system. For expression in mammalian cells, the TRE3G promoter is ligated downstream of the T7 core promoter and is named according to the T7 core promoter. For the T3 promoter, the K1.5 promoter, the K11 promoter, the Phi15 promoter, and the SP6 promoter, the TRE3G promoter was ligated to each promoter core sequence in a similar manner. The sequence of pPhi15WT is ACCAGATTTAAAAACCCACACAATAGACAGA (SEQ ID NO: 173). The manipulation sequences corresponding to CI434, phlf, HKCI, lmrA, bm3R, TP901CI, cymR, ttgR and LexA are SEQ ID NO:174-182. The sequences of the respective T7 promoter, T3 promoter, SP6 promoter, K1.5 promoter and K11 promoter are given in Table 1.
The monomer RNA polymerase domain T7 RNAP (UniProtKB/Swiss-ProtID Q7Y5R 1), K1.5RNAP (UniProtKB/Swiss-ProtID Q8SCG 8), african swine fever capping enzyme NP868R, repressor protein CI434 (SEQ ID NO:2 of Chinese patent application CN 201610289098.6), phlf (UniProtIDQ 9RF 02), HKCI (NCBIID_ 024226607.1), lmrA (NCBIID_ 003246449.1), bm3R (UniProtKB/Prot ID P43506), TP CI (UniProtKB/Swiss-ProtID Q7Y5R 1), and DNA fragment (UniProtKB/Swiss-Prot ID Q8SCG 8), and DNA fragment code for UniProtID 4R (UniProtID 48) from UniProtKB/Swiss-Prot ID Q8SCG8, ttCI 434 (SEQ ID NO:2 of Chinese patent application CN 201610289098.6), phlf (UniProtIDQ 9RF 02), HKB (NCBIID_ 024226607.1), lmKB 3R (UniProtKB/Prot ID P43506), and DNA fragment (UniProtID No. 29 to be synthesized from UniProtKB/Swiss-ProtID Q7R (UniprotID No. 29).
For the capping-RNAP expression plasmid, NP868R capping enzyme was first ligated to (G 4 S) 4 The 5 'end of the flexible peptide linker and the RNAP fragment was ligated to the 3' end of the flexible peptide linker to construct a capping-RNAP coding sequence. IRES-mcherry fragments of PB531A-1 plasmid were replaced with capping-RNAP using Gibson assembly. The EF1 alpha promoter carried on PB531A-1 drives the expression of capping-RNAP. The reporter gene used in the present invention is reporter gene yfp, mcherry, tagBFP (bfp) and egfp (gfp). The three antigen sequences of VLPs are hemagglutinin (HA/Brisbane, H1N1, genBank IDACA 28844.1), neuraminidase (NA/WSN, H1N1, genBank IDACF 54601.1) and matrix protein 1 (M1, H1N1, genBank ID NP 040978.1), respectively. GFP-M1 sequences with markers are described [11 ] ]The method comprises the steps of carrying out a first treatment on the surface of the NA-mCherry [12 ]]. For single reporter genes, the coding sequence is ligated downstream of the T7 promoter and a synthetic terminator is added downstream of the coding sequence [13 ]]. For the dual reporter system, the triple reporter system and the VLP expression system, the corresponding expression cassettes were assembled to the mapping-T7 RNAP expression plasmid (pOMC 16) by means of the golden gate method using the Sap1 enzyme and ligase. All of the protein coding sequences described above are followed by a poly (A) sequence. The repressor expression plasmid skeleton is pOMC1, and the promoter is EF1 alpha (also called EF1a promoter or hEF1a promoter, SEQ ID NO:40 of Chinese patent application CN 202111150967.4).
And (5) transfection. Cells were cultured to 70-80% confluency and transfected with Lipofectamine 3000 according to manufacturer's instructions. The same batch of purified plasmid was adjusted to the same concentration value and the same reagents required for each transfection were aliquoted to reduce errors. For the T7 single reporter expression system, 0.3. Mu.g of each T7 promoter-yfp plasmid was co-transfected with 0.7. Mu.g or 0.07. Mu.g of the capping-T7 RNAP expression plasmid into CHO or HEK293T cells in 24 well plates (Corning). For RNAP orthogonality analysis, 0.5. Mu.g of each capping-RNAP expression plasmid and 0.5. Mu.g of reporter plasmid were co-transfected into CHO or HEK293T cells in 24 well plates. For dual or triple reporter expression systems, transfection was performed in 24 well plates with 0.5 μg plasmid. For VLP purification by sucrose density gradient centrifugation, 50 μg of VLP expression plasmid was transfected into CHO cells in 15cm dishes. For quantitative measurement of VLPs using an Apogee nanoscale flow cytometer, 2 μg VLP expression plasmid was transfected into CHO cells in a 6-well plate.
Flow cytometry. Samples were prepared in 96-well plates according to the following procedure: cells were isolated 2 days after transfection with pancreatin-EDTA (Gibco). The medium is then added to inactivate the pancreatin. Cells were transferred to 96-well plates, transiently centrifuged at 200g for 5 min, and fixed with 4% paraformaldehyde (PFA, boster biotechnology). Collection of 10 from each sample 5 Individual cells were detected using a Beckman Coulter CytoFlex S flow cytometer or FITC channel, ECD channel, or DAPI channel of BD Fortessa SORP and analyzed using CytoExpert (Beckman Coulter) or FlowJo (V10) software.
construction of a capping-T7 RNAP stable cell line. Stable cell lines were generated using the piggyBac transposon system according to the manufacturer's instructions. In a 6-well plate, 0.25. Mu.g of the calling-T7 RNAP expression plasmid was co-transfected with 0.75. Mu.g of the transposon plasmid into CHO cells. Two days after transfection, cells were transferred to three 48-well plates for continued culture. When confluence reached 30-50%, positive clones were screened by adding 400. Mu.g/ml hygromycin. The T7 report expression level of positive clones was verified by means of a flow cytometer (Beckman, cytoFlex S). The B9 clone was selected for subsequent experiments with VLPs.
Purification of VLPs. VLP samples were purified using a sucrose buffer method like Buffin [14 ]. Cell culture broth was collected 72 hours after transfection, and centrifuged at 300g for 5 min, followed by 3000g for 10 min. After filtration through a 0.45 μm filter (Millipore), the supernatant was added to the 30% sucrose solution upper layer (PBS dilution) and ultracentrifuged at 30000rpm at 4℃for 3h using a P32ST (Himac) bucket centrifuge rotor. The VLP particles were resuspended with PBS.
Transmission Electron Microscopy (TEM). Mu.l of sucrose density gradient centrifugation purified VLP samples were deposited on a carbon-coated copper mesh (Beijing, beijing Kogyo Corp.) for 10 min at room temperature. Excess sample was aspirated from the grid with filter paper, and the sample on the grid was then stained with 2% phosphotungstic acid for 1 min at room temperature. Excess stain was sucked off with filter paper. After drying overnight, the copper mesh was scanned with a transmission electron microscope (FEI Tecnai G2F 20).
Microscopic imaging and co-localization analysis of fluorescent VLPs. Based on the Gonzalez-domiiguez [15] method, 1.5. Mu.l of the collected fluorescent VLP sample was placed on a clean slide and carefully spread with a cover slip (15 mm diameter). TRITC channels and FITC channels were imaged with an ANDOR (ZYLA) camera using a 100-fold oleoscopic lens observation of a nikon Ti2-E microscope. The spot identification was analyzed by means of Imaris software (oxford instruments) to detect co-localization of green and red signals.
Flow cytometry determination of fluorescent VLPs. Cell culture broth was collected 72 hours after transfection, centrifuged at 300g for 5 minutes, and at 3000g for 10 minutes in that order, and cell debris was removed. The signals at 488nm excitation light were determined by a procedure of analyzing extracellular vesicles with an Apogee nanoscale flow cytometer (Apogee Flow Systems) filtered with a 0.45 μm filter (Millipore). The data was analyzed using histogram software (Apogee Flow Systems).
And (5) numerical simulation. Both numerical modeling and prediction were done using MATLAB R2018a software. Alpha, beta and Hill coefficients n were obtained from control experiments using fminearch function fitting, the binding affinity (K value) of RNAP to the corresponding promoter was from literature [16], and the other parameters were obtained from function fitting in MATLAB.
TABLE 2 parameters in formula (1)
The YFP-mCherry and YFP-BFP (BFP was used instead of mCherry in the equation for YFP-mCherry) dual reporting subsystems were fitted separately using formulas (2) - (6). In predicting the expression of three reporters in any cell line, the parameters of YFP and mCherry are derived from the fit of YFP-mCherry data, while the BFP corresponding parameters are derived from the fit of YFP-BFP data. The relevant parameters of YFP are consistent between the two sets of data.
TABLE 3 parameters (T7 expression System) in formulas (2) - (6)
For VLP production, the yield of VLPs was predicted using the following equation.
n(VLP)＝min(n 1(HA) ，n 2(M1) ，n 3(NA) )
TABLE 4 VLP yield prediction model parameters
| RNAP tot | DNA tot | α1/f1 | α2/f2 |
| 530.2632335 | 553.9613943 | 0.019729646 | 0.853326577 |
| α3/f3 | R1 | R2 | R3 |
| 1 (normalization) | 0.031747869 | 2.775271807 | 2.838294149 |
The parameters in the formula are shown in Table 4. Where α represents the maximum production rate of each type of protein. In one VLP particle, f units of the corresponding type of protein are required for assembly. The number of VLPs is the minimum of n1, n2 and n 3. Calculations were performed using Python 3.8, and the "Optimize minimize" function in the SciPy software package was used to search for a set of parameters that minimize L2 loss between experimental data and calculation results. For VLP yield prediction, [16] was used ]K,54 of all available T7 promoters 3 Prediction of seed combinations all uses the fitting parameters described above, andthe 14 promoter combinations were experimentally verified.
The VLP particles were visualized by electron microscopy. 50. Mu.g of plasmids expressing three influenza A virus antigens HA, NA and M1 were transfected with Lipofectamine 3000 (Thermo Scientific), the plasmid DNA and liposome complexes were added to 15cm dish-cultured CHO cells, and after 3 days the cell culture supernatants were harvested. After removing cell debris by centrifugation at 300g for 5 minutes, impurities were further removed by centrifugation at 3000g for 10 minutes. Filtration was performed with a 0.45 μm filter (millipore). 3mL of a 30% sucrose solution prepared with phosphate buffer PBS was added to the bottom of the ultracentrifuge tube, and the filtered cell supernatant was superimposed on sucrose, and the sample was concentrated by centrifugation at 30000rpm (Himac Hitachi horizontal rotor P32 ST) for 3 hours and dissolved with PBS. VLP samples were dropped onto a copper mesh (mesoscopic instrument) for 10 minutes and then washed twice with PBS and stained with 2% phosphotungstic acid for 1 minute. PBS was washed twice and after overnight drying, observed under an electron microscope.
Example 2: nucleic acid vaccine
As an application, we use the expression system for the construction of novel nucleic acid vaccines. Traditional mRNA vaccines are typically produced by in vitro transcription of T7RNAP, followed by purification by 5' end capping and other modifications (e.g., base substitutions) to produce mRNA molecules having a structure similar to that of nature. The immune effect of the mRNA molecules depends on the stability and structure of the mRNA. In the system of the invention, by expressing the capping-RNAP in mammalian cells, both transcription and translation processes are performed in vivo, and the vaccine design solves the problems of unstable structure and need for low temperature storage of the existing nucleic acid vaccine (e.g., mRNA vaccine). It is contemplated that the vaccine preparation system of the present invention should comprise at least three coding sequences, RNA polymerase, capping enzyme and antigen, and that their inclusion into liposomes would result in oversized liposomes, resulting in reduced efficiency of the coating. For the purpose of improving the delivery efficiency, we used DC cells as delivery vehicles, first transfected the coding sequences comprising the three proteins into a dendritic cell line (DC 2.4), and then injected the DC cells into animals to induce immune responses. As illustrated in fig. 6D and 6E, the nucleic acid vaccine of the present invention is capable of eliciting both cellular and humoral immunity, similar to an mRNA vaccine.
The capping-RNAP and antigen co-expression system was first tested in HEK293T cells. The constitutive promoter hEF1a was ligated upstream of NP868R-SP6 RNAP, pSP6WT was ligated upstream of hemagglutinin (HA, A/Brisbane/59/2007 (H1N 1), NCBI ID: ACA 28844), and the SP6-RNAP-HA plasmid was constructed. In this system, expression of HA is driven by SP6 RNAP. To increase the expression level of RNAP, as an improvement, the constitutive promoters hEF1a and pSP6WT were ligated upstream of the NP868R-SP6 RNAP fusion protein, pSP6WT was ligated upstream of HA, and the SP6-PFB-HA plasmid was constructed. In this construction, transcription of the calling-RNAP has positive feedback regulation. As a negative control, an SP6-O-HA plasmid was constructed that did not express the capping-RNAP, with SP6-O-HA having only pSP6 WT-driven HA, and no capping-RNAP was expressed. The blank (NC) is untreated DC cells. As shown in fig. 6B, western blot results demonstrate that neither the blank nor the negative control cells expressed HA protein. The positive feedback regulation of the capping-RNAP increases the expression level of HA by 6 times. According to our further investigation, the optimal expression ratio of antigenic protein to capping-RNAP was about 1:3. In the case of a defined level of antigen protein expression, higher levels of RNAP expression do not drive more antigen expression, whereas lower RNAP does not saturate the antigen expression level.
To verify the vaccine use of the expression system of the present invention, we also constructed a nucleic acid vaccine based on the K1E expression system as well as on the K1.5 expression system. In the K1E-RNAP-HA plasmid, NP868R-K1ERNAP expression was driven by the constitutive promoter hEF1a (K1E Genbank ID: 3837329), and HA protein expression was driven by the wild-type pK1.5WT promoter. In the K1E-PFB-HA plasmid, the expression of NP868R-K1E RNAP is driven by hEF1a and pK1.5WT double promoters, and the expression of HA protein is driven by pK1EWT promoter (SEQ ID NO:5 of Chinese patent application CN 202211269476.6). In the K1.5-RNAP-HA plasmid, the expression of NP868R-K1.5RNAP was driven by the constitutive promoter hEF1a and the expression of the HA protein was driven by the wild-type pK1.5WT promoter. In the K1.5-PFB-HA plasmid, NP868R-K1.5RNAP was expressed by hEF1a and pK1.5WT double promoters, and HA protein expression was driven by the pK1.5WT promoter. As shown in fig. 6C, each plasmid was transfected into DC2.4 cells, and antigen-presenting DC2.4 cells were injected into mice to induce immune responses in the mice. Mice injected subcutaneously with 250 μl of physiological saline from the back were used as negative controls. Mice injected subcutaneously on the back with 25. Mu.g (in 250. Mu.l saline) of recombinant Hemagglutinin (HA) (Sino Biological, 11052-V08H) were used as positive controls. Determining the serum of the mouse, wherein the amount of HA-specific IgG reflects the intensity of the induced humoral immunity; cytokines IL-2, IL-4, TNF- α and IFN- γ levels reflect the intensity of the induced cellular immunity. As shown in fig. 6D and 6E, nucleic acid vaccines based on the three expression systems SP6, K1E and K1.5 were able to elicit both humoral and cellular immunity, and positive feedback of RNAP resulted in both increased HA-specific IgG titers and increased levels of individual cytokines in mouse serum under the three systems, reflecting both humoral and cellular immunity boosting. It can be seen that the nucleic acid vaccine of the present invention can be advantageously used for immunization against viruses. Furthermore, given the use of DC delivery in cancer therapy, the nucleic acid vaccine may also be used in the treatment of tumors or other diseases. In animal experiments, the negative control was mice injected subcutaneously with 250. Mu.l saline from the back, and the PC was mice injected subcutaneously with 25. Mu.g (dissolved in 250. Mu.l saline) of recombinant Hemagglutinin (HA) (Sino Biological, 11052-V08H) from the back.
Method
A cell line. At 5% CO 2 Human embryonic kidney cells HEK293T (ATCC) were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) at 37 ℃. Mouse dendritic cell line DC2.4 (Merck) was cultured in RPMI-1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). The backbone of the plasmid was PB531A-1.
And (3) a mouse. Female BALB/c mice of 6-8 weeks old were purchased from Peking Vitre Liwa laboratory animal technologies Co. All mice used in this study were pathogen free (SPF) grade and well-conditioned. Mice were fed in SPF laboratory animal facilities with 5 companion animals per cage. All mice had sufficient water and standard feed to provide 12 hours of light and dark cycles (temperature: 20-25 ℃, humidity: 40% -70%).
Western blot. At 8×10 5 Density of wells HEK293T or DC2.4 cells were seeded in 12 well plates (Corning) at 5% co 2 Culturing at 37℃for 16 hours. The plasmid (1. Mu.g per well) was mixed with 200. Mu.L of Opti-MEM medium (Gibco), followed by 1. Mu.L of Lipo8000 (Beyotime) was added to the mixture and thoroughly mixed. The transfection mixture was added to the wells and gently mixed with the cells, and then placed into an incubator for further culture. After 48 hours, wash with pre-chilled PBS and digest with 0.25% trypsin. Whole cell lysates were prepared with RIPA lysate (Yeasen) and then separated on SDS-PAGE gels and transferred to PVDF membrane. The primary antibody used in Western blotting is directed against H1N1 influenza A HA protein and anti-GAPDH antibody, and the secondary antibody is goat anti-mouse IgG or goat anti-rabbit IgG conjugated with horseradish peroxidase, and is detected by means of enhanced chemiluminescence. The X-ray film was analyzed to determine the linear range of chemiluminescent signals and quantified using a densitometer. All antibodies were purchased from Beijing Yiqiao Shenzhou technologies and technologies Inc.
Flow cytometry. At 8×10 5 Density of wells HEK293T or DC2.4 cells were seeded in 24 well plates (Corning) at 5% co 2 Culturing at 37℃for 16 hours. The plasmid (0.5. Mu.g per well) was mixed with 100. Mu.L of Opti-MEM medium (Gibco), followed by 0.5. Mu.L of Lipo8000 (Beyotime) was added to the mixture and thoroughly mixed. The transfection mixture was added to the wells and gently mixed with the cells, and then placed into an incubator for further culture. After 48 hours, the cells were washed twice with pre-chilled PBS and digested with 0.25% trypsin. Cells were collected and fluorescence signals of all samples were detected with FITC channel and ECD channel of Beckman CytoFLEX S flow cytometer.
Ethical statement. Animal research was approved by the ethical committee of animal experiments in the Shenzhen advanced technology research institute and followed the recommended rules in the ethical committee of Shenzhen advanced technology research institute, guidelines for nursing and use of laboratory animals.
Mice experiments. Mice were immunized with DC2.4 cells. DC2.4 cells were seeded onto 10cm plates (timing) (5X 10) 6 Plate) was incubated at 37℃under 5% CO2 for 16 hours, followed by 10. Mu.g of plasmidTransfection was performed. Cells were collected after 48 hours and resuspended in 0.9% nacl solution. For positive control, recombinant HA protein was diluted with 0.9% nacl solution and mixed with an equal volume of complete freund's adjuvant (FCA) and emulsified by vortexing. The mixture was injected subcutaneously into the back of BALB/c mice and vaccinated. 3 weeks after immunization, serum samples were obtained by orbital vein collection of mice, centrifugation at 3000g for 15 minutes.
Enzyme-linked immunosorbent assay (ELISA). Recombinant HA protein (10. Mu.g/mL in PBS) was attached to ELISA plates. After overnight incubation at 4 ℃, the plates were blocked with 5% skim milk (PBS in solvent) for 2 hours. The mouse serum samples were diluted and added to each well of the ELISA plate and incubated at 37℃for 2h. Subsequently, PBST was washed 5 times and incubated with the secondary antibody again for 2 hours, and 3,3', 5' -Tetramethylbenzidine (TMB) substrate was added to carry out the color reaction, and the reaction was terminated with 2M hydrochloric acid. Absorbance at 450nm was read with a microplate reader.
Multiplex cytokine analysis (CBA). Diluted serum samples were mixed with antibody-modified (IL-4, IL-2, TNF- α, IFN- γ, available from Biolegend) microbeads in a V-bottom microplate, the microplate was fixed on a plate shaker and mixed for 2h (500 rpm) at room temperature. 1,000Xg room temperature centrifugation for 5 minutes, using a multichannel pipette to remove the supernatant, and to each well, add detection antibody, at room temperature mixing for 1h (500 rpm). The SA-PE reagent was then added to each well and the plate was shaken at about 500rpm for 0.5h at room temperature. The plates were then washed twice with wash buffer and the supernatant removed, and the PE and APC channel fluorescence signals of all samples were read using a flow cytometer, after centrifugation at 1,000Xg for 5 minutes.
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Claims (9)
1. A system for expressing one or more genes of interest in a mammalian cell, the system comprising:
a chimeric capping enzyme-RNA polymerase comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5 RNA polymerase, and SP6 RNA polymerase; the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
a promoter recognized by a monomeric RNA polymerase, wherein the promoter recognized by the T7 RNA polymerase is selected from the group consisting of SEQ ID NO. 1-SEQ ID NO. 54; the T3 RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NO. 161-SEQ ID NO. 172; the K11 RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NO. 55-SEQ ID NO. 112; the promoter recognized by the K1.5 RNA polymerase is selected from the group consisting of SEQ ID NO. 183-SEQ ID NO. 234; the promoter recognized by the SP6 RNA polymerase is selected from the group consisting of SEQ ID NO. 113-SEQ ID NO. 160.
2. A system for expressing one or more genes of interest in a mammalian cell, the system comprising:
one or more chimeric capping enzyme-RNA polymerase gene expression cassettes, each independently comprising a chimeric capping enzyme-RNA polymerase coding sequence, each independently comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5 RNA polymerase, and SP6 RNA polymerase; the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
one or more gene expression cassettes of interest, each independently comprising a promoter recognized by a monomeric RNA polymerase and a gene sequence of interest, wherein the promoter recognized by the T7 RNA polymerase is selected from the group consisting of SEQ ID NOs 1-54; the T3 RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NO. 161-SEQ ID NO. 172; the promoter recognized by the K11 RNA polymerase is selected from the group consisting of SEQ ID NO. 55-SEQ ID NO. 112; the promoter recognized by the K1.5 RNA polymerase is selected from the group consisting of SEQ ID NO 183-SEQ ID NO 234; the promoter recognized by the SP6 RNA polymerase is selected from the group consisting of SEQ ID NO. 113-SEQ ID NO. 160.
3. The system of claim 2, the system comprising:
a chimeric capping enzyme-RNA polymerase gene expression cassette comprising a first promoter sequence and a chimeric capping enzyme-RNA polymerase coding sequence, the chimeric capping enzyme-RNA polymerase comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; and
one or more gene expression cassettes of interest, each independently comprising a second promoter sequence and a gene sequence of interest, wherein each second promoter is independently a promoter recognized by the monomeric RNA polymerase;
preferably, the number of the target genes is 1 to 10, more preferably 1 to 3;
preferably, the first promoter is selected from the group consisting of CMV1, CMV3G, EF1a, CMVmini or EF1a core promoters;
preferably, the first promoter is a dual promoter and comprises a promoter recognized by the monomeric RNA polymerase;
preferably, the chimeric capping enzyme-RNA polymerase has a (G 4 S) 4 A linker peptide;
preferably, the viral capping enzyme is african swine fever NP868 capping enzyme;
Preferably, the system further comprises a repressor expression cassette selected from one or more of RpaR, braR, bjaR, tetR, CI434, phlf, HKCl, lmrA, bm3R, TP901CI, cymR, ttgR, and LexA; wherein the promoter recognized by the monomeric RNA polymerase comprises an operator site for the repressor protein;
preferably, the system is a transient expression system or a stable expression system.
4. The system of claim 2, the system comprising:
a first chimeric capping enzyme-RNA polymerase gene expression cassette comprising a first promoter sequence and a first chimeric capping enzyme-RNA polymerase coding sequence, the first chimeric capping enzyme-RNA polymerase comprising a first monomeric RNA polymerase domain and a first viral capping enzyme domain;
a first gene expression cassette of interest comprising a second promoter sequence and a first gene sequence of interest, wherein the second promoter is a promoter recognized by the first monomeric RNA polymerase;
a second chimeric capping enzyme-RNA polymerase gene expression cassette comprising a third promoter sequence and a second chimeric capping enzyme-RNA polymerase coding sequence, the second chimeric capping enzyme-RNA polymerase comprising a second monomeric RNA polymerase domain and a second viral capping enzyme domain; and
A second gene expression cassette comprising a fourth promoter sequence and a second gene sequence of interest, wherein the fourth promoter is a promoter recognized by the second monomeric RNA polymerase;
wherein the first monomeric RNA polymerase and the second monomeric RNA polymerase are each independently selected from the group consisting of T7RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5 RNA polymerase, and SP6 RNA polymerase; the first viral capping enzyme and the second viral capping enzyme are each independently selected from the group consisting of bluetongue viral capping enzyme, african swine fever viral capping enzyme, acanthamoidovora pseudoviral capping enzyme, bamboo mosaic viral capping enzyme, and poxviral capping enzyme;
preferably, the first promoter and/or the third promoter are each independently selected from CMV1, CMV3G, EF a, CMVmini or EF1a core promoters;
preferably, the first promoter and/or the third promoter is a double promoter and comprises a promoter recognized by a monomeric RNA polymerase;
preferably, the first monomeric RNA polymerase domain has a (G) between the first viral capping enzyme domain 4 S) 4 A linker peptide; the second monomeric RNA polymerase domain has (G) between the second monomeric RNA polymerase domain and the second viral capping enzyme domain 4 S) 4 A linker peptide;
preferably, the first viral capping enzyme and the second viral capping enzyme are african swine fever NP868 capping enzymes;
preferably, the system further comprises a repressor expression cassette selected from one or more of RpaR, braR, bjaR, tetR, CI434, phlf, HKCl, lmrA, bm3R, TP901CI, cymR, ttgR, and LexA; wherein the promoter recognized by the first and/or second monomeric RNA polymerase comprises an manipulation site of the repressor protein;
preferably, the system is a transient expression system or a stable expression system.
5. A method for expressing one or more genes of interest in a mammalian cell, the method comprising:
constructing one or more chimeric capping enzyme-RNA polymerase gene expression cassettes, each independently comprising a chimeric capping enzyme-RNA polymerase coding sequence, each independently comprising a monomeric RNA polymerase domain and a viral capping enzyme domain; wherein the monomeric RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5 RNA polymerase, and SP6 RNA polymerase; the virus capping enzyme is selected from the group consisting of bluetongue virus capping enzyme, african swine fever virus capping enzyme, acanthamoidovora pseudovirus capping enzyme, bamboo mosaic virus capping enzyme and poxvirus capping enzyme; and
Constructing one or more gene expression cassettes of interest, each independently comprising a promoter recognized by a monomeric RNA polymerase and a gene sequence of interest, wherein the promoter recognized by the T7 RNA polymerase is selected from the group consisting of SEQ ID NOs 1-54; the T3 RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NO. 161-SEQ ID NO. 172; the K11 RNA polymerase recognizes a promoter selected from the group consisting of SEQ ID NO. 55-SEQ ID NO. 112; the promoter recognized by the K1.5 RNA polymerase is selected from the group consisting of SEQ ID NO. 183-SEQ ID NO. 234; the promoter recognized by the SP6 RNA polymerase is selected from the group consisting of SEQ ID NO. 113-SEQ ID NO. 160.
6. The method of claim 5, the method comprising:
constructing a chimeric capping enzyme-RNA polymerase gene expression cassette comprising a first promoter sequence and a chimeric capping enzyme-RNA polymerase coding sequence, wherein the chimeric capping enzyme-RNA polymerase comprises a monomeric RNA polymerase domain and a viral capping enzyme domain; and
constructing one or more gene expression cassettes of interest, each of which independently comprises a second promoter sequence and a gene sequence of interest, wherein each second promoter is independently a promoter recognized by the monomeric RNA polymerase;
Preferably, the number of the target genes is 1 to 10, more preferably 1 to 3;
preferably, the first promoter is selected from the group consisting of CMV1, CMV3G, EF1a, CMVmini or EF1a core promoters;
preferably, the first promoter is a dual promoter and comprises a promoter recognized by the monomeric RNA polymerase;
preferably, the chimeric capping enzyme-RNA polymerase has a (G 4 S) 4 A linker peptide;
preferably, the viral capping enzyme is african swine fever NP868 capping enzyme;
preferably, the system further comprises a repressor expression cassette selected from one or more of RpaR, braR, bjaR, tetR, CI434, phlf, HKCl, lmrA, bm3R, TP901CI, cymR, ttgR, and LexA; wherein the promoter recognized by the monomeric RNA polymerase comprises an operator site for the repressor protein;
preferably, the system is a transient expression system or a stable expression system.
7. The method of claim 5, the method comprising:
constructing a first chimeric capping enzyme-RNA polymerase gene expression cassette comprising a first promoter sequence and a first chimeric capping enzyme-RNA polymerase coding sequence, the first chimeric capping enzyme-RNA polymerase comprising a first monomeric RNA polymerase domain and a first viral capping enzyme domain;
Constructing a first target gene expression cassette, wherein the first target gene expression cassette comprises a second promoter sequence and a first target gene sequence, and the second promoter is a promoter recognized by the first single RNA polymerase;
constructing a second chimeric capping enzyme-RNA polymerase gene expression cassette comprising a third promoter sequence and a second chimeric capping enzyme-RNA polymerase coding sequence, the second chimeric capping enzyme-RNA polymerase comprising a second monomeric RNA polymerase domain and a second viral capping enzyme domain; and
constructing a second target gene expression cassette, wherein the second target gene expression cassette comprises a fourth promoter sequence and a second target gene sequence, and the fourth promoter is a promoter recognized by the second single RNA polymerase;
wherein the first monomeric RNA polymerase and the second monomeric RNA polymerase are each independently selected from the group consisting of T7RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, K1.5 RNA polymerase, and SP6 RNA polymerase; the first viral capping enzyme and the second viral capping enzyme are each independently selected from the group consisting of bluetongue viral capping enzyme, african swine fever viral capping enzyme, acanthamoidovora pseudoviral capping enzyme, bamboo mosaic viral capping enzyme, and poxviral capping enzyme;
Preferably, the first promoter and/or third promoter is selected from CMV1, CMV3G, EF1a, CMVmini or EF1a core promoters;
preferably, the first promoter and/or the third promoter is a double promoter and comprises a promoter recognized by a monomeric RNA polymerase;
preferably, the first monomeric RNA polymerase domain has a (G) between the first viral capping enzyme domain 4 S) 4 A linker peptide; the second monomeric RNA polymerase domain has (G) between the second monomeric RNA polymerase domain and the second viral capping enzyme domain 4 S) 4 A linker peptide;
preferably, the first viral capping enzyme and the second viral capping enzyme are african swine fever NP868 capping enzymes;
preferably, the system further comprises a repressor expression cassette selected from one or more of RpaR, braR, bjaR, tetR, CI434, phlf, HKCl, lmrA, bm3R, TP901CI, cymR, ttgR, and LexA; wherein the promoter recognized by the first and/or second monomeric RNA polymerase comprises an manipulation site of the repressor protein;
preferably, the system is a transient expression system or a stable expression system.
8. Use of the expression system of any one of claims 1-4 or the method of any one of claims 5-7 in the preparation of a vaccine.
9. Use according to claim 8, wherein DC cells are employed as delivery vehicle for the vaccine, preferably the vaccine is a multivalent vaccine.
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