A promoter and its application

Abstract

The present invention discloses a promoter and application thereof. The present invention discovers the promoter P0 of EPAS1, which can effectively start the expression of genes.

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 essential for the survival of most organisms. When exercising in a hypoxic environment or undergoing prolonged, high-intensity training, the internal environment of an organism changes, disrupting oxygen homeostasis and causing cells to become hypoxic. Hypoxia alters the physiological functions of the body and cells, and in severe cases can cause a range of injuries and diseases. Consequently, organisms have developed a comprehensive set of oxygen sensing mechanisms and mechanisms to regulate gene expression under varying oxygen conditions to adapt to low oxygen levels. Among these regulatory mechanisms, hypoxia-inducible factors (HIFs) are the most important transcription factor family, closely implicated in biological growth, development, and the pathogenesis of several diseases. HIFs are transcriptionally active heterodimers composed of an active regulatory subunit, α, and a constitutive expression subunit, β. Under aerobic conditions, HIFs are inactivated by post-transcriptional hydroxylation of specific amino acid residues in the α subunit. Currently, there are three types of α subunits: HIF-1α, HIF-2α, and HIF-3α. While these three α subunits differ in function, they share similar structures and are all regulated by oxygen.

[0003] Endothelial PAS1 protein (EPAS1), also known as hypoxia-inducible factor-2 (HIF-2), is a key transcription factor in response to hypoxia. Hypoxia-induced changes in enzymes and factors are mostly mediated through EPAS1. EPAS1 targets genes related to energy metabolism, iron metabolism, angiogenesis, 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 (HREs). EPAS1 recognizes the core sequence 5'-TACGTGCT-3' and binds to HREs, thereby inducing transcription of target genes and mediating the cellular response to hypoxia.

[0004] The regulation of gene expression mainly occurs at the transcriptional level, and promoters play a key role in the regulation of transcriptional level, and 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 accurately and effectively initiate transcription. Promoter can guide the correct combination of all templates, activate RNA polymerase, initiate gene transcription, thereby control the start time of gene expression (transcription) and the degree of expression. However, when different target genes are connected later in the current promoter, different starting effects can 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 gene expression.

[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 comprising the nucleotide sequence 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 conventional chemical synthesis or genetic engineering methods.

[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 mutagenesis.

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

[0015] Furthermore, mutation can also be performed using a commercially available site-specific mutagenesis introduction kit 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 obtained by deleting, replacing, adding and / or inserting one or several nucleotides into the nucleotide sequence of SEQ ID NO: 1.

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

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

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

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

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

[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 the nucleotide sequence of its variant, homologue, fragment or derivative.

[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 further 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 comprising 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 involved in its synthesis. The target gene may be a heterologous gene encoding a heterologous expression product, a gene of the same origin introduced from an external source, a gene encoding an expression product inherent to the host cell, or a gene encoding any other protein, peptide, nucleic acid, or the like.

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

[0031] Further, 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, but is 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 according to the second aspect of the present invention and a terminator.

[0038] Furthermore, the gene expression unit further comprises 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, 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 a vector using a restriction enzyme recognition sequence. For example, the vector can be cleaved 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 cleavage sequence at its end can be added thereto. The fragments can then be ligated using a ligase, thereby allowing 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 to be introduced into the vector.

[0042] Furthermore, a plasmid vector, a phage vector or a viral 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] Furthermore, Gram-positive bacteria or Gram-negative bacteria can be used as host cells.

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

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

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

[0048] Furthermore, if a bacterium of the genus Bacillus is used as a host, the plasmid vector that can be used includes, 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 Escherichia coli is used as the 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 host cells using the spontaneous competence method, calcium phosphate method, electroporation method, DEAE-dextran method or 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, the DNA described in the second aspect of the present invention, 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] Furthermore, Gram-positive bacteria include, but are not limited to, Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis, Bacillus brevis, or Bacillus species unidentified.

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

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

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

[0069] Furthermore, the vector or vector fragment can be appropriately selected depending on the cells 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] As used herein, a "polypeptide" is a polymer of amino acid residues linked by peptide bonds, which may be naturally occurring or synthetic. A polypeptide having less than about 20 amino acid residues may be referred to as a "peptide." A polypeptide comprising two or more amino acid chains or amino acid chains of 100 or more amino acids in length may be referred to as a "protein." Polypeptides or proteins may also contain non-peptide components, such as carbohydrate groups or metal ions.

[0074] Furthermore, 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 be absent, 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] As used herein, "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 does not naturally occur in a given host cell. As used herein, "heterologous" includes any of the following: nucleic acid derived from a non-host cell (i.e., exogenous nucleic acid); or a combination of a host cell-derived nucleic acid and an 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 in a host cell. "Structural gene" refers to the gene region without a signal sequence, i.e., the coding region.

[0081] In the present invention, "operably linked" refers to the juxtaposition of two or more components. The operably linked DNA sequences may be adjacent or non-adjacent. 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 selective 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 immune binding-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, which comprises culturing cells transfected with a nucleic acid containing the promoter and polypeptide according to the first aspect of the present invention, and collecting the polypeptide from the resulting culture.

[0095] Furthermore, the polypeptide can be purified using 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 comprising the promoter described in the first aspect of the present invention, the DNA described in the second aspect of the present invention, the gene expression unit described in the third aspect of the present invention, 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 for transcription or translation, including an enzyme, dNTPs, a buffer solution, ribonucleotides, and amino acids.

[0099] Furthermore, the kit also includes 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 discovered the promoter P0 of EPAS1, which can effectively start gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0106] Figure 4 Figure 1 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 will be further described below in conjunction with specific examples. The specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods in the following examples where specific conditions are not specified are generally tested under conventional conditions or according to the 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 knockout were seeded 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 and an appropriate amount of trypsin was added to digest the cells. 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 10 times to mix. Crosslink at room temperature for 10 minutes. At 2.5 minutes, 5 minutes, and 7.5 minutes, gently invert the tube 10 times to mix.

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

[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 for 5 min at 4°C, discard the supernatant, and recover the cells.

[0117] (7) After the cross-linked sample was 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 a final volume of 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 the 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 sediment, remove the supernatant, and start the elution process.

[0127] (17) Low-salt immune complex eluate (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)) was added, rotated for 5 min, and centrifuged at 1000 rpm for 3 min to collect the precipitate.

[0129] (19) Licl immune complex eluate (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, 0.1 M NaHCO3). Add 250 μL of elution buffer to the pellet, mix thoroughly, 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 until the supernatant volume is approximately 500 μL.

[0132] (22) Add 20 μL of 5 M sodium chloride to dissolve 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 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 positions 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, the 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) EPAS1-P0-S and EPAS1-P0-F sequences were synthesized (Beijing Qingke Biotechnology Co., Ltd.), containing KpnI (GGTACC) and HindIII (AAGCTT) restriction sites at both ends.

[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 50 μL reaction system DNA 1 μg 10×rCutSmartBuffer 5μL (1×) KpnI-HF 1.0μL HindⅢ-HF 1.0μL Nuclease-free Water to50μL

[0149] After incubation at 37°C for 1 hour, the digestion products were subjected to 1% agarose gel electrophoresis. The digestion results were observed under UV light, and the double-digested bands were excised with a scalpel blade. DNA was recovered using a gel extraction kit (QIAGEN, 28704). The target DNA was then purified and mixed with the linearized PGL3-basic vector plasmid at a ratio of 3:1.

[0150] (c) The digested EPAS1-P0 sequence 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 20 μl reaction system T4 DNA Ligase Buffer (10×)* 2μL VectorDNA(PGL3-basic) 0.020 pmol InsertDNA(EPAS1-P0) 0.060 pmol Nuclease-freewater to20μL T4DNALigase 1 μL

[0153] Ligation was carried out at 16°C overnight.

[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 FIG. 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 correctly identified monoclonal bacteria were amplified, a portion was frozen, and a portion was used 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 day before transfection, cells were seeded into 24-well plates at 8 × 10 cells per well. 5 cells.

[0158] (b) On the second day, the cell confluence reached 70%. The negative control (NC) PGL3-basic vector plasmid, the positive control (PC) PGL3-promoter vector plasmid, and the PGL3-P0-F and PGL3-P0-S recombinant plasmids were transfected into the cells, and the internal control 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) at a ratio of 1 μg plasmid to 2.5 μL. After the Lipofectamine 2000 diluted in Opti-MEM is allowed to stand at room temperature for 5 minutes, add the DNA diluent and gently mix to prepare 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 minutes.

[0162] (e) Take 50 μl of cell lysate and add it to a 96-well white plate (Corning, 3917). Follow the steps below to use 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 luciferase assay described above.

[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 can serve as the promoter of EPAS1 and promote the expression of EPAS1.

[0172] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A promoter, characterized in that The nucleotide sequence of the promoter is shown in SEQ ID NO:

1.

2. A DNA, characterized in that The DNA comprises the promoter according to claim 1.

3. The DNA according to claim 2, characterized in that The DNA also includes a target gene connected downstream of the promoter.

4. The DNA according to claim 3, characterized in that The target gene includes 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.

5. The DNA according to claim 4, characterized in that The target gene is selected from nucleic acids encoding proteins.

6. The DNA according to claim 5, characterized in that The target gene is a nucleic acid encoding EPAS1.

7. A gene expression unit, characterized in that The gene expression unit comprises the DNA according to any one of claims 2 to 6 and a terminator.

8. A carrier, characterized in that The vector comprises the promoter according to claim 1 or the DNA according to any one of claims 2 to 6 or the gene expression unit according to claim 7.

9. The carrier according to claim 8, characterized in that The vector includes a plasmid vector.

10. The carrier according to claim 9, characterized in that The plasmid vector includes pHY, pUB110, pE194, pUC18, pUC19, pBluescript, pET or pGL3.

11. The carrier according to claim 10, characterized in that The plasmid vector is selected from pGL3.

12. A cell, characterized in that The cell comprises the promoter according to claim 1, the DNA according to any one of claims 2 to 6, the gene expression unit according to claim 7, or the vector according to any one of claims 8 to 11, and the cell is a mammalian cell.

13. The cell according to claim 12, characterized in that The mammalian cells include CHO cells, HEK cells, BHK cells or Sp2 / 0 cells.

14. The cell according to claim 13, characterized in that The mammalian cells are selected from CHO cells or HEK cells.

15. The cell according to claim 14, characterized in that The HEK cells include 293T cells or 293F cells.

16. The cell according to claim 15, characterized in that The HEK cells are selected from 293T cells.

17. A kit comprising the promoter according to claim 1, the DNA according to any one of claims 2 to 6, the gene expression unit according to claim 7, the vector according to any one of claims 8 to 11, or the cell according to any one of claims 12 to 16.

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