A promoter element derived from porcine HSP40 gene and its application

Abstract

The present disclosure provides a promoter element derived from the porcine HSP40 gene. The promoter element of the present disclosure helps to reveal the structure of the porcine HSP40 / DNAJB6 gene core promoter and its regulatory mechanism in cellular response, which is of great significance for understanding the structure and function of the promoter and for gaining a deeper understanding of the molecular regulatory mechanism of pigs, and also provides a new perspective and tool for gene expression research in related fields.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a promoter element derived from a porcine HSP40 gene and an application thereof. Background Art

[0002] DNAJ (HSP40) is a highly conserved cross-species heat shock protein family that exists in various tissues of bacteria and humans. The HSP40 family may be the largest HSP family, and its conserved J domain is responsible for recruiting HSP70 and stimulating HSP70 ATPase activity. HSP40 family members can work synergistically or even independently with HSP70 to inhibit protein aggregation. DNAJ proteins can be divided into three subtypes: I, II, and III. Classes I and II are most common in most of almost all eukaryotic cells. Studies have shown that DNAJA1 / HSP40 is a member of the type I DNAJ / HSP40 family, which is selected by influenza A virus RNA polymerase to enhance its viral RNA synthesis in the infected cell nucleus and can serve as a positive regulator of influenza virus replication; at the same time, HSP40 / DNAJB1 promotes the nuclear import of incoming influenza A virus ribonucleoproteins (vRNPs), which can effectively promote the replication of influenza A virus. In addition, during HIV infection of T cells, several isoforms of HSP40 were significantly regulated at the mRNA level, among which DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22 and DNAJC30 all positively regulated viral replication, while DNAJB3, DNAJB6, DNAJB8 and DNAJC5 negatively regulated viral replication. Further identification of DNAJB8 negatively regulated the infectivity of progeny virions, which will provide a new strategy for DNAJ / HSP40-specific regulation of viruses.

[0003] With the continuous development of biotechnology, the study of porcine HSP40 gene has become increasingly important in understanding the key role of porcine physiology and stress response. However, to date, in-depth exploration of the core promoter of porcine HSP40 / DNAJB6 gene remains a challenge. Summary of the invention

[0004] In order to solve at least one of the above problems, the present disclosure provides a promoter element derived from porcine HSP40 gene and application thereof.

[0005] According to one aspect of the present disclosure, a promoter element derived from a porcine HSP40 gene is provided, the promoter element comprising a nucleotide sequence as shown in SEQ ID NO: 9, or a nucleotide sequence having at least 60% sequence identity thereto.

[0006] In some embodiments, the promoter element has a nucleotide sequence as set forth in any one or more of SEQ ID NO:9, or a nucleotide sequence having at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0007] In some embodiments, the promoter element comprises the nucleotide sequence shown in SEQ ID NO: 9 or multiple copies thereof having at least 60% sequence identity.

[0008] In some embodiments, the promoter element is regulated by a virus.

[0009] In some embodiments, the promoter element is positively regulated by a virus.

[0010] In some embodiments, the virus includes gastroenteritis virus TGEV, influenza virus, HIV, porcine epidemic diarrhea virus PEDV, encephalitis virus, porcine reproductive and respiratory syndrome virus PRRSV, porcine delta coronavirus PDCoV, porcine circovirus, Mycoplasma hyopneumoniae, pseudorabies virus PRV, swine eperythrozoon, and classical swine fever virus CSFV.

[0011] In some embodiments, the promoter element is positively regulated by a gastroenteritis virus.

[0012] In some embodiments, the promoter element is derived from the -2000th to -1000th nucleotides upstream of the transcription start site of the porcine HSP40 / DNAJB6 gene.

[0013] According to another aspect of the present disclosure, a transcription expression cassette is provided, comprising the promoter element of the first aspect and a target gene, wherein the promoter element is operably linked to the target gene for initiating transcription of the target gene.

[0014] According to another aspect of the present disclosure, an expression vector is provided, comprising the promoter element or the transcription expression cassette.

[0015] In some embodiments, the expression vector is selected from one or more of a prokaryotic expression vector, a eukaryotic expression vector, or a viral expression vector.

[0016] According to another aspect of the present disclosure, a host cell is provided, characterized in that the host cell comprises one or more of the promoter element, the transcription expression cassette or the expression vector.

[0017] In some embodiments, the host cell is selected from a prokaryotic cell and a eukaryotic cell.

[0018] In some embodiments, the prokaryotic cell comprises a bacterial cell, such as E. coli.

[0019] In some embodiments, the eukaryotic cell comprises a yeast cell, a mammalian cell, or an insect cell.

[0020] In some embodiments, the mammal is selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cows, pigs, dogs, and cats.

[0021] According to another aspect of the present disclosure, there is provided use of the promoter element, the transcription expression cassette, the expression vector or the host cell in any one or more of the following:

[0022] a) Transcription of the target gene,

[0023] b) Expression of target gene-encoded protein,

[0024] c) Screening for drugs to treat or prevent diseases.

[0025] Beneficial effects of the present invention:

[0026] The present disclosure provides a promoter element, which helps to reveal the structure of the core promoter of the porcine HSP40 / DNAJB6 gene and its regulatory mechanism in cellular response, which is of great significance for understanding the structure and function of the promoter and for gaining a deeper understanding of the molecular regulatory mechanism of pigs, and also provides a new perspective and a tool for efficient expression or transcription for gene expression research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It shows that the expression of HSP40 / DNAJB6 promoter (full length HSP40 / DNAJB6-QC) is regulated by the time and dose of TGEV infection, and the ordinate is the relative fluorescence activity of HSP40 promoter (HSP40 / DNAJB6-QC).

[0028] Figure 2 A schematic diagram of the HSP40 / DNAJB6 promoter region is shown.

[0029] Figure 3 The results of luciferase activity assay are shown. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0031] definition

[0032] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0033] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0034] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0035] The term "promoter" or "promoter element" as used herein is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcription start site of a polynucleotide to initiate transcription, the polynucleotide encoding a biologically active polypeptide. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region. The term "promoter" or "promoter element" should also be understood to include a 5' non-coding region (between the promoter and the translation start point) for translation after transcription into mRNA, cis-acting transcriptional regulatory elements such as enhancers, and / or other nucleotide sequences that can interact with transcription factors. A promoter or promoter element can be a wild-type promoter, a variant promoter, a hybrid promoter body, or a consensus promoter.

[0036] The term "core promoter" as used herein refers to the nucleic acid sequence contained by the promoter. The core promoter is usually the smallest part of the promoter required to correctly initiate transcription. The core promoter usually includes a transcription start site and a binding site for RNA polymerase.

[0037] As used herein, the term "sequence identity" refers to the "percentage of sequence identity" or "percent identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide, a gap present in the target sequence is not counted. Likewise, since the target sequence nucleotides are counted and the nucleotides from the reference sequence are not counted, gaps present in the reference sequence are not counted. At least 60% sequence identity includes a continuous segment having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity over the entire length of the sequence.

[0038] The term "MOI" or "multiplicity of infection" as used herein refers to the ratio of virus to cell number, and refers to the average number of virus particles that infect each cell. MOI = pfu / cell. PFU (Plaqueforming unit) is a plaque forming unit, which is used to describe the number of virus particles with infectious activity.

[0039] The term "expression vector" as used herein refers to the direct or indirect connection of the nucleic acid sequence with the regulatory elements on the vector when the above-mentioned isolated nucleic acid molecule is connected to the vector, as long as these regulatory elements can regulate the translation and expression of the nucleic acid molecule. Of course, these regulatory elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. In other words, the nucleic acid molecule is operably connected to the regulatory element. Commonly used expression vectors include prokaryotic expression vectors, eukaryotic expression vectors, or viral expression vectors, etc.

[0040] The term "operably linked" as used herein means that the exogenous gene is connected to the vector so that the regulatory elements in the vector, such as transcription regulatory sequences and translation regulatory sequences, etc., can play their intended function of regulating the transcription and translation of the exogenous gene.

[0041] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0042] Example

[0043] Example 1. Cloning of porcine HSP40 / DNAJB6 gene

[0044] The porcine HSP40 / DNAJB6 gene DNA sequence (NC_010460.4) and mRNA (XM_021079274.1) sequences were searched in the NCBI database, and the first base of the mRNA sequence (XM_021079274.1) was used as the transcription start site. The TaKaRaMiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 was used to extract DNA from porcine testicular cells (ST cells), and the extracted genomic DNA was used as a template, with the target being 2000 bp upstream of the transcription start site, and the primers HSP40 / DNAJB6-QC-F (upstream primer) and HSP40 / DNAJB6-QC-R (downstream primer) were used for polymerase chain reaction (PCR).

[0045] Each 50 μL of the PCR amplification reaction system contains Prime HSDNA Polymerase 25 μL, 10 μM upstream primer 1 μL, 10 μM downstream primer 1 μL, 500 ng / μL genomic template DNA 1 μL, and sterile water 22 μL.

[0046] The PCR amplification program was as follows: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds; annealing at 63°C for 15 seconds; extension at 72°C for 10 seconds, for a total of 35 cycles; and final extension at 72°C for 1 minute.

[0047] Subsequently, the PCR product was recovered using the PCR product recovery kit TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 to obtain the promoter fragment HSP40 / DNAJB6-QC. The primer sequences and amplification product sequences are shown in Table 1.

[0048] Table 1. Sequence Listing

[0049]

[0050]

[0051] Note: AGATCT is the restriction site of BGLII; AAGCTT is the restriction site of HindIII.

[0052] The pGL3-Basic plasmid and the amplified promoter fragment HSP40 / DNAJB6-QC were double-digested with restriction endonucleases BGLII and Hind III. T4 enzyme was then used for ligation. Each 10 μL of the ligation system contained 1 μg of double-digested HSP40 / DNAJB6 gene promoter, 100 μg of double-digested pGL3-Basic plasmid, 1 μL of T4 ligase buffer, and 1 μL of T4 ligase. The ligation conditions were set at 16°C for 4 hours.

[0053] The ligation product was transformed into E. coli Top10 competent cells, and the transformation steps were carried out according to the instructions of E. coli Top10 competent cells. After transformation, positive clones were selected and sent to Shanghai Shenggong Biotechnology Co., Ltd. for Sanger sequencing to verify the correct sequencing and obtain plasmid pGL3-HSP40-QC.

[0054] After the pig testicular cells were thawed and revived at 37°C, they were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. When the cell confluence reached 70% to 80%, they were passaged. First, the original culture medium was discarded, and the cells were rinsed twice with sterile DPBS buffer, and then placed in a 37°C incubator and digested with 0.25% EDTA trypsin for about 2 minutes. After that, the cells were taken out, added to 20% fetal bovine serum DMEM culture medium (purchased from Thermofisher), and 0.25% EDTA trypsin was added at the same time, and the cells were blown with a pipette until the cells fell off. The detached cells were inoculated in each well of the 6-well plate of the cell culture plate at a ratio of 1:3, 5ml of 10% fetal bovine serum DMEM culture medium was added, and then placed in a 37°C, 5% CO2 incubator for culture. Cells were passaged every 3 days, with a passage ratio of 1:3.

[0055] After the pig testicular cells were cultured to a confluence of 70-90%, Lipofectamine was diluted in Opti-MEM medium (purchased from Thermofisher). TM Next, dilute the endotoxin-free plasmid with Opti-MEM medium to prepare a plasmid premix.

[0056] The experimental group used pGL3-HSP40-QC plasmid and pRL-TK plasmid for co-transfection, while the negative control was co-transfected with two empty plasmids, pGL3-Basic (Biyuntian) and pRL-TK (Biyuntian). Add Lipofectamine TMTM3000 (purchased from Thermofisher) reagent and ensure that it is thoroughly mixed. TM The diluted DNA was added to the 3000 reagent at a ratio of 1:1, and after incubation at room temperature for 5 minutes, the DNA-liposome complex was added to the cells. After the cells were incubated at 37°C for 12 hours, the porcine transmissible gastroenteritis virus TGEV isolated and preserved in this laboratory was added at MOI (1, 2, 3 and 5), and samples were collected at (0, 12, 24 and 48 hours), and then the transfected cells were analyzed.

[0057] After transfected cells were collected, luciferase assays were performed using the Duo-Lite Luciferase Assay System kit. Untransfected cells were used as blank controls for background subtraction (including background Firefly and background Renilla). Experimental group cells were treated with experimental compounds (including experimental group Firefly and experimental group Renilla), while untreated transfected cells were used to normalize the results (including control group Firefly and control group Renilla).

[0058]

[0059] The results are as follows Figure 1 As shown, the relative promoter fluorescence activity of pGL3-HSP40-QC was enhanced after TGEV transfection, and the higher the MOI value, the stronger the relative promoter activity of pGL3-HSP40-QC.

[0060] Example 2. Identification of porcine HSP40 / DNAJB6 gene promoter activity using dual luciferase reporter gene

[0061] In order to determine the fragment with transcription initiation activity, according to the method described in Example 1, the primer pairs HSP40 / DNAJB6-P1-F and HSP40 / DNAJB6-P1-R, HSP40 / DNAJB6-P2-F and HSP40 / DNAJB6-P2-R were used to amplify the promoter fragments HSP40 / DNAJB6-P1 and HSP40 / DNAJB6-P2, as shown in FIG. Figure 2 The primer sequences and amplification sequences are shown in Table 2.

[0062] Table 2. Sequence Listing

[0063]

[0064] Note: AGATCT is the restriction site of BGLII; AAGCTT It is the restriction site of HindIII.

[0065] The amplified promoter fragments HSP40 / DNAJB6-P1 and HSP40 / DNAJB6-P2 were connected to the pGL3-Basic plasmid to obtain plasmids pGL3-HSP40-P1 and pGL3-HSP40-P.

[0066] According to the detection method of Example 1, luciferase detection was performed, and the results were as follows Figure 3 As shown, the relative activity of pGL3-HSP40-QC in the experimental group was 2.971±0.33; the relative activity of pGL3-HSP40-P1 was 1.34±0.032; the relative activity of pGL3-HSP40-P2 was 2.7233±0.731, and the relative activity of luciferase in the control group was 1.12147±0.042. The relative activity of luciferase in pGL3-HSP40-P2 in the experimental group was significantly higher than that in the control group (P<0.05), which indicated that the -2000bp to -1000bp upstream of the HSP40 promoter was the core region of the promoter.

Claims

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

9.

2. A transcription expression cassette, characterized in that: The transcription expression cassette comprises the promoter element of claim 1 and a target gene, wherein the promoter element is operably linked to the target gene and is used to initiate transcription of the target gene.

3. An expression vector, characterized in that: The expression vector comprises the promoter element of claim 1 or the transcription expression cassette of claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is selected from one or more of a prokaryotic expression vector, a eukaryotic expression vector or a viral expression vector.

5. A host cell, characterized in that The host cell comprises one or more of the promoter element according to claim 1, the transcription expression cassette according to claim 2, and the expression vector according to claim 3 or 4.

6. The host cell according to claim 5, characterized in that The host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells.

7. The host cell according to claim 6, characterized in that The prokaryotic cells include bacterial cells.

8. The host cell according to claim 6, characterized in that The prokaryotic cells include Escherichia coli.

9. The host cell according to claim 6, characterized in that The eukaryotic cells include yeast cells, mammalian cells or insect cells.

10. The host cell according to claim 9, characterized in that The mammal is selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cattle, pigs, dogs and cats.

11. Use of the promoter element of claim 1, the transcription expression cassette of claim 2, the expression vector of claim 3 or 4, or the host cell of any one of claims 5 to 10 in any one or more of the following: a) Transcription of the target gene, b) Expression of the protein encoded by the target gene.

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