Promoter and application thereof

Abstract

The invention discloses a promoter and application thereof, and discovers a promoter P0 of EPAS1, and the promoter can effectively promote the expression of a gene.

Description

Technical Field

[0001] The present invention relates to the field of protein expression, and in particular to a novel promoter and application thereof. Background Art

[0002] Oxygen is an indispensable basis for the survival of most organisms. When exercising in a hypoxic environment or undergoing long-term high-intensity training, the internal environment of the organism will change and the oxygen balance will be destroyed, and the cells will be in a state of hypoxia. Hypoxia will change the physiological functions of the body and cells, and in severe cases, it can cause a series of injuries and diseases. Therefore, biological organisms have formed a complete set of oxygen sensing mechanisms and gene expression regulation mechanisms under different oxygen environments to adapt to the hypoxic environment. Among the various regulatory mechanisms, hypoxia-inducible factor (HIF) is the most important transcription factor family, which is closely related to the growth, development and pathogenesis of some diseases of the organism. HIF is a transcriptionally active heterodimer composed of an active regulatory subunit α and a structural expression subunit β. Under aerobic conditions, HIF will be controlled by the α subunit of a specific amino acid residue that is hydroxylated after transcription and lose its activity. At present, there are three types of α subunits, namely HIF-1α, HIF-2α and HIF-3α. Although these three α subunits have different functions, they have similar structures, and all three are regulated by oxygen.

[0003] Endothelial PAS1 protein (EPAS1), also known as hypoxia-inducible factor-2 (HIF-2), is a key transcription factor for responding to hypoxia. Most of the changes in enzymes or factors caused by hypoxia act through EPAS1. The target genes of EPAS1 are mainly genes related to energy metabolism, iron metabolism, vascular growth, vasoconstriction, and bone marrow hematopoiesis, including vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), lactate dehydrogenase A (LDHA), erythropoietin (EPO), and inducible nitric oxide synthase (NOS2). These target genes all contain hypoxia response elements (HRE). EPAS1 recognizes the core sequence 5'-TACGTGCT-3' and binds to HRE, thereby inducing the transcription of target genes and mediating the cell's response to hypoxia.

[0004] The regulation of gene expression mainly occurs at the transcription level, and the promoter plays a key role in the regulation of transcription level. The strength of promoter activity directly affects the level of gene expression. Promoter refers to a DNA sequence in a gene that can be combined with RNA polymerase and other trans factors that affect transcription to achieve accurate and effective initiation of transcription. Promoter can guide the correct combination of the same template, activate RNA polymerase, start gene transcription, thereby controlling the start time and expression degree of gene expression (transcription). However, when different target genes are connected behind the current promoter, different starting effects will be presented, so it is still necessary to develop a promoter that is compatible with the target gene. Summary of the invention

[0005] In view of this, the present invention is proposed. The purpose of the present invention is to provide a novel promoter and its application. The present invention discovered the promoter P0 of EPAS1, which can effectively promote the expression of genes.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a promoter, which comprises a nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence of a variant, homologue, fragment or derivative thereof.

[0008] In the present invention, "promoter" includes a Pribnow box, a TATA box or a TATA box-like region located about 10 to 30 base pairs upstream of the transcription start point (+1), and is responsible for enabling RNA polymerase to initiate transcription from the correct position.

[0009] Furthermore, the promoter may also include a region necessary for binding of proteins other than RNA polymerase for controlling expression.

[0010] In the present invention, the method for obtaining the promoter is not particularly limited, and the promoter can be obtained by a common chemical synthesis method or a genetic engineering method.

[0011] Furthermore, the promoter of the present invention can be produced by introducing a mutation into the nucleotide sequence represented by SEQ ID NO: 1 of the present invention.

[0012] In the present invention, mutation refers to a change in the nucleotide sequence, including deletion, substitution, addition and / or insertion of the nucleotide sequence.

[0013] Furthermore, methods for introducing mutations include, but are not limited to, ultraviolet irradiation or site-specific mutation introduction.

[0014] Furthermore, methods for introducing site-specific mutations include, but are not limited to, overlap extension PCR, ODA method, and Kunkel method.

[0015] Furthermore, mutation can also be performed using a commercially available kit for site-specific mutagenesis introduction such as Site-Directed Mutagenesis System Mutan-SuperExpressKm Kit, Transformer™ Site-Directed Mutagenesis Kit, or KOD-Plus-Mutagenesis Kit.

[0016] Furthermore, the promoter includes a nucleotide sequence in which one or more nucleotides are deleted, substituted, added and / or inserted into the nucleotide sequence of SEQ ID NO: 1.

[0017] Furthermore, "1 or several" used in reference to deletion, substitution, addition and / or insertion of nucleotides in a nucleotide sequence includes 1 to 20.

[0018] Furthermore, "1 or several" used in reference to deletion, substitution, addition and / or insertion of nucleotides in a nucleotide sequence includes 1 to 10.

[0019] Furthermore, "1 or several" used in reference to deletion, substitution, addition and / or insertion of nucleotides in a nucleotide sequence includes 1 to 5.

[0020] Furthermore, "1 or several" used in reference to deletion, substitution, addition and / or insertion of nucleotides in a nucleotide sequence includes 1 to 3.

[0021] Furthermore, "1 or several" used in reference to deletion, substitution, addition and / or insertion of nucleotides in a nucleotide sequence includes 1 or 2.

[0022] Further, the promoter comprises a nucleotide sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO:1.

[0023] Further, the promoter comprises a nucleotide sequence that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO:1.

[0024] Furthermore, the promoter also includes a nucleotide sequence that is complementary to the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence of a variant, homolog, fragment or derivative thereof.

[0025] Furthermore, the promoter also includes a nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence of a variant, homolog, fragment or derivative thereof.

[0026] Furthermore, the promoter also includes a nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence complement of a variant, homolog, fragment or derivative thereof.

[0027] The second aspect of the present invention provides a DNA, wherein the DNA comprises the promoter described in the first aspect of the present invention.

[0028] Furthermore, the DNA also includes a target gene connected downstream of the promoter.

[0029] Furthermore, the target gene is not particularly limited. The target gene is a gene encoding a target substance and / or an enzyme related to its synthesis. The target gene may be a heterologous gene encoding a heterologous expression product, or a gene of the same origin introduced from the outside, or a gene encoding an expression product originally possessed by the host cell, or a gene encoding any other protein, peptide, nucleic acid, etc.

[0030] Furthermore, examples of target substances encoded by the target gene include enzymes, hormones, cytokines, other physiologically active peptides, transporters, non-coding RNAs, selectable markers, and the like.

[0031] Furthermore, examples of enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, synthetases, glycolytic enzymes, pentose phosphate cycle enzymes, TCA cycle enzymes, and enzymes involved in the synthesis of aromatic compounds.

[0032] Furthermore, the target gene includes a nucleic acid encoding a protein, a nucleic acid encoding an antisense RNA, a nucleic acid encoding an enzyme and / or a nucleic acid encoding a selectable marker.

[0033] Furthermore, the target gene is selected from nucleic acids encoding proteins.

[0034] Furthermore, the target gene is a nucleic acid encoding EPAS1.

[0035] Furthermore, the target gene originates from, including but not limited to, microorganisms (such as bacteria, yeast, actinomycetes, filamentous fungi, ascomycetes or basidiomycetes), plants, insects, and animals.

[0036] Furthermore, the target gene also includes artificially synthesized genes.

[0037] The third aspect of the present invention provides a gene expression unit, which comprises the DNA described in the second aspect of the present invention and a terminator.

[0038] Furthermore, the gene expression unit also includes a cis-acting element that increases the transcriptional activity of the promoter.

[0039] Furthermore, the cis-acting element includes an enhancer, a regulatory sequence or an inducible element.

[0040] The fourth aspect of the present invention provides a vector, which comprises the promoter described in the first aspect of the present invention or the DNA described in the second aspect of the present invention or the gene expression unit described in the third aspect of the present invention.

[0041] Furthermore, the promoter described in the first aspect of the present invention, the DNA described in the second aspect of the present invention, or the gene expression unit described in the third aspect of the present invention can be introduced into the vector using a restriction enzyme recognition sequence. For example, the vector can be cut with a restriction enzyme, and a DNA fragment containing the promoter described in the first aspect of the present invention, the DNA described in the second aspect of the present invention, or the gene expression unit described in the third aspect of the present invention and having a restriction enzyme cutting sequence at the end can be added thereto, and then the promoter described in the first aspect of the present invention, the DNA described in the second aspect of the present invention, or the gene expression unit described in the third aspect of the present invention can be introduced into the vector using a ligase.

[0042] Furthermore, a plasmid vector, a phage vector or a virus vector, or a vector fragment constituting a part of the vector, can be used as the vector of the present invention. The vector or vector fragment can be appropriately selected according to the host cell to be used.

[0043] Furthermore, there is no particular limitation on the origin of the host cells.

[0044] Further, Gram-positive bacteria or Gram-negative bacteria can be used as host cells.

[0045] Further, Gram-positive bacteria include, but are not limited to, Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis, Bacillus brevis, or Bacillus species unidentified.

[0046] Further, Gram-negative bacteria include, but are not limited to, Escherichia coli.

[0047] Further, the E. coli strains include, but are not limited to, HB101, C600, JM109, DH5α, DH10B, XL-1BlueMRF' or TOP10F.

[0048] Furthermore, if a bacterium of the genus Bacillus is used as a host, the plasmid vector may include, but is not limited to, pHY, pUB110 or pE194.

[0049] Furthermore, if a bacterium of the genus Bacillus is used as a host, the phage vector that can be used includes, but is not limited to, 105 or SPβ.

[0050] Furthermore, if E. coli is used as a host, the plasmid vector may include but is not limited to pUC18, pUC19, pBluescript, pET or pGL3.

[0051] Furthermore, if Escherichia coli is used as a host, the phage vector that can be used includes but is not limited to λ phage vectors (eg λgt10, λgt11).

[0052] Furthermore, the expression vector can be introduced into the host cell using the spontaneous competence method, the calcium phosphate method, the electroporation method, the DEAE-dextran method or the liposome method.

[0053] Furthermore, the host cell is selected from Escherichia coli.

[0054] Furthermore, the Escherichia coli is selected from DH5α.

[0055] Furthermore, the vector is selected from a plasmid vector.

[0056] Furthermore, the plasmid vector is selected from pGL3.

[0057] The fifth aspect of the present invention provides a cell, which comprises the promoter described in the first aspect of the present invention or the DNA described in the second aspect of the present invention or the gene expression unit described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention.

[0058] Furthermore, the cells include eukaryotic cells and prokaryotic cells.

[0059] Furthermore, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0060] Furthermore, the mammalian cells include but are not limited to CHO cells, HEK cells, BHK cells or Sp2 / 0 cells.

[0061] Furthermore, the mammalian cells are selected from CHO cells and HEK cells.

[0062] Furthermore, the HEK cells include but are not limited to 293T cells and 293F cells.

[0063] Furthermore, the HEK cells are selected from 293T cells.

[0064] Further, prokaryotic cells include, but are not limited to, Gram-positive bacteria or Gram-negative bacteria.

[0065] Further, Gram-positive bacteria include, but are not limited to, Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis, Bacillus brevis, or Bacillus species unidentified.

[0066] Further, Gram-negative bacteria include, but are not limited to, Escherichia coli.

[0067] Further, the E. coli strains include, but are not limited to, HB101, C600, JM109, DH5α, DH10B, XL-1BlueMRF' or TOP10F.

[0068] Furthermore, the Escherichia coli strain is selected from DH5α.

[0069] Furthermore, the vector or vector fragment can be appropriately selected according to the cell to be used.

[0070] Furthermore, the vector or vector fragment may be appropriately selected according to the manner described in the fourth aspect of the present invention.

[0071] Furthermore, the host cell is selected from Escherichia coli, the Escherichia coli is selected from DH5α, and the vector is selected from a plasmid vector.

[0072] The sixth aspect of the present invention provides a method for selecting cells expressing a heterologous polypeptide, the method comprising transfecting cells with a nucleic acid comprising the promoter described in the first aspect of the present invention, and selecting the transfected cells under selective culture conditions.

[0073] In the present invention, a "polypeptide" is a polymer of amino acid residues linked by peptide bonds, which may be naturally occurring or synthetic. Polypeptides with less than about 20 amino acid residues may be referred to as "peptides". Polypeptides comprising two or more amino acid chains or amino acid chains with a length of 100 amino acids or more may be referred to as "proteins". Polypeptides or proteins may also contain non-peptide components, such as carbohydrate groups or metal ions.

[0074] Further, the nucleic acid comprises:

[0075] a) a first expression cassette encoding a heterologous polypeptide nucleic acid;

[0076] b) a second expression cassette comprising the promoter according to the first aspect of the invention and a second nucleic acid.

[0077] Furthermore, the second nucleic acid described in b) may also not exist, that is, the nucleic acid comprises a nucleic acid encoding a heterologous polypeptide and the promoter described in the first aspect of the present invention.

[0078] In the present invention, "heterologous polypeptide nucleic acid" or "heterologous polypeptide" refers to a nucleic acid molecule or polypeptide or a group of nucleic acid molecules or polypeptides that are not naturally present in a given host cell. In the present invention, heterologous includes any of the following situations: nucleic acid from a non-host cell source (i.e., exogenous nucleic acid); a combination of the host cell-derived nucleic acid and the exogenous nucleic acid.

[0079] Furthermore, the promoter described in the first aspect of the present invention is operably linked to a second nucleic acid.

[0080] In the present invention, "expression cassette" refers to a nucleic acid that contains at least the elements required for expression and secretion of a structural gene contained in a host cell. "Structural gene" refers to a gene region without a signal sequence, i.e., a coding region.

[0081] In the present invention, "operably linked" refers to the juxtaposition of two or more components. Operably linked DNA sequences may be adjacent or non-adjacent, and an operably linked promoter is usually located upstream of the coding sequence, but the promoter may not be adjacent to the coding sequence.

[0082] Furthermore, the second nucleic acid encodes a selectable marker.

[0083] In the present invention, "selective marker" means a nucleic acid that allows specific selection or non-selection of cells carrying the nucleic acid in the presence of a corresponding "selective agent". The selective marker allows selection of cells transformed with the selective marker in the presence of a corresponding selection agent. Selective markers can be positive, negative or bifunctional. Positive selective markers allow selection of cells carrying the marker, while negative selective markers allow specific exclusion of cells carrying the marker. Selective markers include but are not limited to aminoglycoside phosphotransferases, such as hygromycin phosphotransferase, neomycin phosphotransferase, dihydrofolate reductase, thymidine kinase, glutamine synthesis, asparagine synthetase, tryptophan synthetase, histidinol dehydrogenase, or genes that provide puromycin, bleomycin, phleomycin or chloramphenicol resistance, or include fluorescent proteins or luciferases.

[0084] Furthermore, the fluorescent protein includes green fluorescent protein, red fluorescent protein, yellow fluorescent protein and orange fluorescent protein.

[0085] Furthermore, the selectable marker is selected from luciferase.

[0086] Furthermore, the selection method includes flow cytometry, ELISA, immunoprecipitation, immunoaffinity column chromatography, magnetic bead immunoaffinity sorting, microscopy-based separation methods or immunobinding-based methods.

[0087] Furthermore, the cells include eukaryotic cells and prokaryotic cells.

[0088] Furthermore, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0089] Furthermore, the mammalian cells include CHO cells, HEK cells, BHK cells or Sp2 / 0 cells.

[0090] Furthermore, the mammalian cells are selected from CHO cells and HEK cells.

[0091] Furthermore, the HEK cells include 293T cells and 293F cells.

[0092] Furthermore, the HEK cells are selected from 293T cells.

[0093] The seventh aspect of the present invention provides use of the promoter described in the first aspect of the present invention in polypeptide expression.

[0094] The eighth aspect of the present invention provides a method for expressing a polypeptide, the method comprising culturing cells transfected with a nucleic acid containing the promoter and polypeptide described in the first aspect of the present invention, and collecting the polypeptide from the resulting culture.

[0095] Furthermore, the polypeptide can be purified by conventional protein purification methods, including but not limited to salting out, ion exchange chromatography, hydrophobic chromatography, affinity chromatography or gel filtration chromatography.

[0096] The ninth aspect of the present invention provides a kit, which includes the promoter described in the first aspect of the present invention or the DNA described in the second aspect of the present invention or the gene expression unit described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention or the cell described in the fifth aspect of the present invention.

[0097] In the present invention, the kit also includes other substances required by the promoter to initiate transcription or translation.

[0098] Furthermore, the kit includes at least one reactant of an enzyme for transcription or translation, dNTPs, a buffer solution, ribonucleotides, and amino acids.

[0099] Furthermore, the kit also includes a cell culture medium.

[0100] Furthermore, the kit also includes instructions.

[0101] The present invention has the following advantages and beneficial effects:

[0102] The present invention provides a novel promoter and application thereof. The present invention discovers the promoter P0 of EPAS1, and the promoter can effectively start the expression of genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 This is the peak graph of the CHIP-seq data of the H3K4me3 antibody upstream of the EPAS1 gene.

[0104] Figure 2 This is the peak graph of CHIP-seq data of H3K27ac antibody upstream of EPAS1 gene.

[0105] Figure 3 It is a vector plasmid structure diagram, among which Figure 3A is the PGL3-Basic vector plasmid structure diagram; Figure 3B is the PGL3-P0-S plasmid structure diagram inserted into EPAS1-P0-S; Figure 3C is the PGL3-P0-F plasmid structure diagram inserted into EPAS1-P0-F.

[0106] Figure 4 is a flow chart of the luciferase assay.

[0107] Figure 5 This is an analysis chart of luciferase assay results. DETAILED DESCRIPTION

[0108] The present invention is further described below in conjunction with specific examples, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0109] Example 1 CHIP-seq experiment

[0110] 1. Experimental methods:

[0111] (1) Wild-type HEK 293T cells and HEK 293T cells with TED sequence knocked out were inoculated into 10 cm cell culture dishes (Corning, 353803) and treated with hypoxia for 24 hours. When the cells grew to 70% to 90%, the culture medium was aspirated, an appropriate amount of trypsin was added to digest the cells, and the cells were resuspended in serum-containing culture medium and transferred to a 15 mL enzyme-free centrifuge tube.

[0112] (2) Centrifuge at 500 × g for 3 min at room temperature, discard the supernatant, recover the cells, and resuspend the cells in 10 mL of room temperature PBS.

[0113] (3) Add 280 μL of 37% formaldehyde and gently invert the tube to mix 10 times. Crosslink at room temperature for 10 min. At 2.5 min, 5 min, and 7.5 min, gently invert the tube to mix 10 times.

[0114] (4) Add 560 μL of 2.5 M glycine and mix well. Terminate cross-linking at room temperature for 5 min.

[0115] (5) Centrifuge at 1000 × g for 5 min, discard the supernatant, recover the cells, and add 5 mL of pre-chilled PBS to resuspend the cells.

[0116] (6) Centrifuge at 1000 × g and 4°C for 5 min, discard the supernatant, and recover the cells.

[0117] (7) After the cross-linked samples were ground with liquid nitrogen, 1 mL of cell lysis buffer (Cell lysis buffer: 10 mM Tris, 10 mM NaCl, 0.2% NP-40 (pH 8.0), 1× protease inhibitors) was added and centrifuged at 4°C to extract the cell nuclei.

[0118] (8) Add 200 μL of 1% SDS solution containing protease inhibitors, resuspend the nuclei by pipetting, and incubate on ice for 10 min.

[0119] (9) Ultrasonic fragmentation of DNA (8 pulses, 60 seconds on, 120 seconds off) was used to fragment the DNA into fragments of 200 to 1500 bp.

[0120] (10) Centrifuge at 13,000 rpm for 10 min at 4°C. Transfer the supernatant to a new 2 mL centrifuge tube and discard the precipitate.

[0121] (11) Dilute the supernatant after sonication into 10× ChIP diluent (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.01% SDS, 1% Triton X-100, 1× protease inhibitors). Add 200 μL of supernatant to 1.8 mL of ChIP diluent to make the final volume 2 mL.

[0122] (12) To remove nonspecificity, add 75 μL of Salmon Sperm DNA / Protein AAgarose-50% Slurry and incubate at 4°C for 60 min.

[0123] (13) Centrifuge at 1000 rpm for 3 min to precipitate Salmon Sperm DNA / Protein AAgarose-50% Slurry and collect the supernatant.

[0124] (14) 10 μg of antibodies H3K4me3 (CST, 5326) and H3K27ac (CST, 8173) were added to the supernatant and mixed at 4°C overnight.

[0125] (15) Add 60 μL of Salmon Sperm DNA / Protein AAgarose-50% Slurry to precipitate the antibody / antigen complex and rotate at 4°C for 60 min.

[0126] (16) Centrifuge at 1000 rpm for 3 min at 4°C to collect the precipitate, remove the supernatant, and start the elution process.

[0127] (17) Low-salt immune complex elution buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100 (pH 8.1)), rotate for 5 min, and centrifuge at 1000 rpm for 3 min to collect the precipitate.

[0128] (18) High-salt immune complex elution buffer (20 mM Tris, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100 (pH 8.1)), rotate for 5 min, and centrifuge at 1000 rpm for 3 min to collect the precipitate.

[0129] (19) Licl immune complex elution buffer (10 mM Tris, 0.25 M LiCl, 1 mM EDTA, 1% NP-40 (pH 8.1)), rotate for 5 min, and centrifuge at 1000 rpm for 3 min to collect the precipitate.

[0130] (20)TE Buffer, rotate for 5 min, and centrifuge at 1000 rpm for 3 min to collect the precipitate, twice.

[0131] (21) Now you have the protein A / antibody / histone / DNA complex. Prepare fresh elution buffer (1% SDS, 0.1M NaHCO3). Add 250 μL of elution buffer to the pellet, mix well, and rotate at room temperature for 15 min. Centrifuge at 1000 rpm for 3 min, transfer the supernatant to a new centrifuge tube, and repeat the above process. The final supernatant volume is about 500 μL.

[0132] (22) Add 20 μL of 5 M NaCl to dissolve the cross-linking and incubate at 65 °C overnight.

[0133] (23) Add 10 μL of 0.5 M EDTA, 20 μL of 1 M Tris-HCl, pH 6.5, and 2 μL of 10 mg / mL Proteinase K to the solution. Rotate at 55°C for 4 hours.

[0134] (24) Add an equal volume of phenol / chloroform to extract DNA, centrifuge at 14,000 g for 10 min, and collect the supernatant. Avoid aspirating filamentous proteins.

[0135] (25) Add 2.5 times the volume of pure ethanol and 1 / 10 volume of sodium acetate to precipitate the DNA. Centrifuge at 14000 g for 10 min, collect the precipitate, wash with 80% alcohol, and open the lid to dry.

[0136] (26) was dissolved in 50 μL of TE buffer.

[0137] (27) Sequencing was performed using the Illumina NovaSeq 6000 gene sequencer.

[0138] 2. Experimental results

[0139] (1) Bioinformatics analysis results of CHIP-seq detection data ( Figure 1 ) showed that there was a significant enrichment of H3K4me3 epigenetic modification signals upstream of the EPAS1 gene. The functional annotation results showed that this region was the promoter region of the EPAS1 gene (P0). The chromosome locations of the two repeated experiments were chr2:46297353-46299634 and chr2:46297423-46299684, respectively.

[0140] (2) Bioinformatics analysis results of CHIP-seq detection data ( Figure 2 ) showed that compared with TED sequence knockout cells, there was a significant enrichment of H3K27ac epigenetic modification signals upstream of the EPAS1 gene in wild-type cells under hypoxic conditions. Functional annotation results showed that this region was the promoter region of the EPAS1 gene (P0), and the chromosome positions of the two repeated experiments were chr2:46297470-46297793 and chr2:46297494-46298132, respectively.

[0141] (3) Based on the results of bioinformatics analysis, chr2:46297353-46299684 sequence (P0-F) and chr2:46297494-46297793 sequence (P0-S) were selected for promoter activity verification.

[0142] Example 2 Promoter Activity Verification

[0143] 1. Experimental methods

[0144] (1) Construction of PGL3-EPAS1-P0 luciferase reporter gene recombinant plasmid:

[0145] (a) Synthesized EPAS1-P0-S and EPAS1-P0-F sequences (Beijing Qingke Biotechnology Co., Ltd.) contain KpnI (GGTACC) and HindⅢ (AAGCTT) restriction sites at both ends of the sequences.

[0146] (b) EPAS1-P0 sequence and PGL3-basic vector plasmid (Promega, E1751) ( Figure 3 A) Double digestion was performed with restriction endonucleases KpnI (NEB, R3142V) and HindⅢ (NEB, R3104V), respectively. The reaction system is shown in Table 1:

[0147] Table 1 Enzyme digestion reaction system

[0148] Components 50μL reaction system DNA 1 μg 10×rCutSmartBuffer 5μL(1×) KpnI-HF 1.0μL HindⅢ-HF 1.0μL Nuclease-freeWater to50μL

[0149] After 1 hour of reaction at 37°C, the digestion product was subjected to 1% agarose gel electrophoresis, the digestion results were observed under UV light, and the double digestion bands were cut out with a surgical blade, and the DNA was recovered using a gel recovery kit (QIAGEN, 28704). The digestion-recovered purified product target DNA and the linear PGL3-basic vector plasmid were mixed in a ratio of 3:1.

[0150] (c) The EPAS1-P0 sequence after restriction digestion was ligated into the PGL3-Basic vector using T4 DNA ligase (NEB, M0202). The reaction system (10 μL) is shown in Table 2:

[0151] Table 2 Ligation reaction system

[0152] Components 20μl reaction system T4 DNA Ligase Buffer (10×)* 2μL VectorDNA(PGL3-basic) 0.020pmol InsertDNA(EPAS1-P0) 0.060pmol Nuclease-freewater to20μL T4DNALigase 1μL

[0153] Ligation was allowed to proceed overnight at 16°C.

[0154] (d) The ligation product was transformed into E. coli competent cells DH5α (TranGen, CD201), and a single clone was picked the next day. After amplification, the plasmid was extracted to obtain the structure as shown in Figure 3 PGL3-P0-S shown in B and Figure 3 The PGL3-P0-F recombinant plasmid shown in C was sequenced and identified.

[0155] (e) The monoclonal bacteria with correct identification results were amplified, a portion was frozen, and a portion was extracted to extract transfection-grade plasmid (QIAGEN, 12941) for activity verification.

[0156] (2) P0 promoter activity detection

[0157] (a) HEK293T cells were cultured in DMEM (Gibco, C11995500BT) containing 10% FBS (Gibco, 10091148) and 1% GlutaMAX (Gibco, 35050061). The cells were seeded into 24-well plates one day before transfection, with 8×10 cells per well. 5 cells.

[0158] (b) On the second day, the cell confluence reached 70%. Negative control (NC) PGL3-basic vector plasmid, positive control (PC) PGL3-promoter vector plasmid, PGL3-P0-F and PGL3-P0-S recombinant plasmids were transfected into cells respectively, and internal reference pRL-SV40 plasmid was added to each well.

[0159] Transfection method: Use opti-MEM (Gibco, 31985062) culture medium to dilute the plasmid and Lipofectamine 2000 (Invitrogen, 11668019) respectively, the ratio of plasmid to Lipofectamine 2000 is 1μg:2.5μL. After the opti-MEM diluted Lipofectamine 2000 is placed at room temperature for 5 minutes, add DNA diluent and gently mix to obtain the transfection solution.

[0160] (c) After the transfection solution was placed at room temperature for 20 minutes, it was added to the cultured cells. After the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 6 hours, the complete culture medium was replaced and the culture was continued.

[0161] (d) 24 hours after transfection, 80 μl of 1× passive lysis buffer (Promega, E194A) was added to each well and the cells were lysed at room temperature for 30 min.

[0162] (e) Take 50 μl of cell lysate and add it to a 96-well white plate (Corning, 3917). Follow the procedure below to use Dual- Detection was performed using Luciferase Assay System (Promega, E2920) and a multifunctional microplate reader (MD, M5).

[0163] Figure 4 Figure 2 is a flow chart of the above luciferase assay.

[0164] 2. Experimental results

[0165] (1) The sequencing results are shown in Table 3:

[0166] Table 3 Sequencing results

[0167]

[0168]

[0169]

[0170] (2) One-way ANOVA with multiple comparisons (ns, not significant, **p<0.01, ****p<0.0001, ***p<0.001).

[0171] result( Figure 5 ) showed that compared with the negative control (NC), the positive control (PC) and P0-S significantly promoted the expression of luciferase, while P0-F could not effectively promote the expression of luciferase. Combined with the results of CHIP-seq analysis, it was shown that P0-S could serve as the promoter of EPAS1 and promote the expression of EPAS1.

[0172] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A promoter, characterized in that The promoter comprises the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence of a variant, homologue, fragment or derivative thereof; Preferably, the promoter comprises a nucleotide sequence formed by deleting, replacing, adding and / or inserting one or more nucleotides into the nucleotide sequence of SEQ ID NO: 1; Preferably, the promoter comprises a nucleotide sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO:

1.

2. A DNA, characterized in that: The DNA comprises the promoter according to claim 1; Preferably, the DNA further comprises a target gene connected downstream of the promoter; Preferably, the target gene comprises a nucleic acid encoding a protein, a nucleic acid encoding an antisense RNA, a nucleic acid encoding a ribozyme and / or a nucleic acid encoding a selectable marker; Preferably, the target gene is selected from nucleic acids encoding proteins; Preferably, the target gene is a nucleic acid encoding EPAS1.

3. A gene expression unit, characterized in that The gene expression unit comprises the DNA according to claim 2 and a terminator.

4. A carrier, characterized in that The vector comprises the promoter according to claim 1 or the DNA according to claim 2 or the gene expression unit according to claim 3; Preferably, the vector comprises a plasmid vector, a phage vector or a viral vector; Preferably, the vector is selected from a plasmid vector; Preferably, the plasmid vector comprises pHY, pUB110, pE194, pUC18, pUC19, pBluescript, pET or pGL3; Preferably, the plasmid vector is selected from pGL3.

5. A cell, characterized in that The cell comprises the promoter according to claim 1 or the DNA according to claim 2 or the gene expression unit according to claim 3 or the vector according to claim 4; Preferably, the cells include eukaryotic cells and prokaryotic cells; Preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; Preferably, the mammalian cells include CHO cells, HEK cells, BHK cells or Sp2 / 0 cells; Preferably, the mammalian cells are selected from CHO cells and HEK cells; Preferably, the HEK cells include 293T cells and 293F cells; Preferably, the HEK cells are selected from 293T cells.

6. A method for selecting cells expressing exogenous polypeptides, characterized in that: The method comprises transfecting cells with a nucleic acid comprising the promoter of claim 1, selecting the transfected cells under selective culture conditions; Preferably, the cells include eukaryotic cells and prokaryotic cells; Preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells; Preferably, the mammalian cells include CHO cells, HEK cells, BHK cells or Sp2 / 0 cells; Preferably, the mammalian cells are selected from CHO cells and HEK cells; Preferably, the HEK cells include 293T cells and 293F cells; Preferably, the HEK cells are selected from 293T cells.

7. The method according to claim 6, characterized in that The nucleic acid comprises: a) a first expression cassette encoding an exogenous polypeptide nucleic acid; b) a second expression cassette consisting of the promoter of claim 1 and a second nucleic acid; Preferably, the promoter of claim 1 is operably linked to a second nucleic acid; Preferably, the second nucleic acid encodes a selectable marker; Preferably, the selectable marker comprises aminoglycoside phosphotransferase, an antibiotic resistance gene, a fluorescent protein or a luciferase; Preferably, the aminoglycoside phosphotransferase includes hygromycin phosphotransferase and neomycin phosphotransferase; Preferably, the antibiotic resistance gene comprises puromycin, bleomycin, phleomycin or chloramphenicol resistance gene; Preferably, the fluorescent protein includes green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and orange fluorescent protein; Preferably, the selectable marker is selected from luciferase; Preferably, the selection method comprises flow cytometry, ELISA, immunoprecipitation, immunoaffinity column chromatography, magnetic bead immunoaffinity sorting, microscopy-based separation methods or immunobinding-based methods.

8. Use of the promoter according to claim 1 in polypeptide expression.

9. A method for expressing a polypeptide, characterized in that: The method comprises culturing cells transfected with a nucleic acid comprising the promoter and polypeptide of claim 1, and collecting the polypeptide from the resulting culture. 10 . A kit comprising the promoter according to claim 1 , the DNA according to claim 2 , the gene expression unit according to claim 3 , the vector according to claim 4 , or the cell according to claim 5 .

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