Element for transcription initiation derived from porcine HSP40 gene and use thereof

Abstract

The present disclosure provides an element for transcription initiation derived from a porcine Hsp40 gene and use thereof. The promoter element of the present disclosure is helpful for revealing the structure of the porcine HSP40 / DNJB6 gene core promoter and the regulation mechanism thereof in cell response, and has important significance for understanding the structure and function of the promoter for deeply knowing the molecular regulation mechanism of pigs, and also provides a new perspective and tool for gene expression research in the related fields.

Description

An element for initiating transcription derived from the porcine HSP40 gene and its application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an element for initiating transcription derived from the porcine HSP40 gene and its application. Background Technology

[0002] DNAJ (HSP40) is a highly conserved family of heat shock proteins found in various tissues of bacteria and humans. The HSP40 family is likely the largest HSP family, with its conserved J domain responsible for recruiting HSP70 and stimulating HSP70 ATPase activity. Members of the HSP40 family can act synergistically with or independently of HSP70 to inhibit protein aggregation. DNAJ proteins can be classified into three subtypes: I, II, and III. Subtypes I and II are most prevalent in most eukaryotic cells. Studies have shown that DNAJA1 / HSP40, a member of the type I DNAJ / HSP40 family, is selected by influenza A virus RNA polymerase to enhance viral RNA synthesis in the nucleus of infected cells, acting as a positive regulator of influenza virus replication. Simultaneously, HSP40 / DNAJB1 promotes the nuclear importation of influenza A virus ribonucleoproteins (vRNPs), effectively promoting influenza A virus replication. Furthermore, during HIV infection of T cells, several HSP40 isoforms are significantly regulated at the mRNA level. Among them, DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22, and DNAJC30 positively regulate viral replication, while DNAJB3, DNAJB6, DNAJB8, and DNAJC5 negatively regulate viral replication. Further identification revealed that DNAJB8 negatively regulates the infectivity of progeny viral particles, which will provide a new strategy for DNAJ / HSP40-specific viral regulation.

[0003] With the continuous development of biotechnology, research on the porcine HSP40 gene has become increasingly important in understanding its crucial role in porcine physiology and stress response. However, to date, in-depth exploration of the core promoter of the porcine HSP40 / DNAJB6 gene remains challenging. Summary of the Invention

[0004] To address at least one of the above problems, this disclosure provides a promoter element derived from the porcine HSP40 gene and its application.

[0005] According to one aspect of this disclosure, a promoter element derived from the 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 with it.

[0006] In some embodiments, the promoter element has a nucleotide sequence as shown 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 with it.

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

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

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

[0010] In some embodiments, the viruses include 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, porcine mycoplasma pneumoniae, porcine pseudorabies virus (PRV), porcine eperythrozoonosis virus (PEV), and classical swine fever virus (CSFV).

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

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

[0013] According to another aspect of this disclosure, a transcription expression cassette is provided, the transcription expression cassette comprising the promoter element described in the first aspect and a target gene, the promoter element being operatively linked to the target gene for initiating transcription of the target gene.

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

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

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

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

[0018] In some embodiments, the prokaryotic cells include bacterial cells, such as Escherichia coli.

[0019] In some embodiments, the eukaryotic cells include yeast cells, mammalian cells, or insect cells.

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

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

[0022] a) Transcription of the target gene,

[0023] b) Expression of the protein encoded by the target gene.

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

[0025] The beneficial effects of this invention are:

[0026] This disclosure provides a promoter element that helps to reveal the structure of the core promoter of the porcine HSP40 / DNAJB6 gene and its regulatory mechanism in cellular response. Understanding the structure and function of this promoter is of great significance for a deeper understanding of the molecular regulatory mechanisms in pigs, and also provides a new perspective and a tool for efficient expression or transcription for gene expression research in related fields. Attached Figure Description

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

[0028] Figure 2 shows a schematic diagram of the HSP40 / DNAJB6 promoter region.

[0029] Figure 3 shows the results of luciferase activity assay. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0031] definition

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

[0033] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0034] The term "about" as used herein is as 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 unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0035] As used herein, the term "promoter" or "promoter element" is defined as a DNA sequence that binds to RNA polymerase and directs the polymerase to the correct downstream transcription start site of a polynucleotide to initiate transcription, said polynucleotide encoding a biologically active polypeptide. RNA polymerase efficiently 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 capable of interacting with transcription factors. A promoter or promoter element can be a wild-type promoter, a variant promoter, a heterozygous promoter body, or a consensus promoter.

[0036] As used in this article, the term "core promoter" refers to the nucleic acid sequence contained within the promoter. The core promoter is typically the smallest part of the promoter required for proper initiation of transcription. A core promoter usually includes a transcription start site and an RNA polymerase binding site.

[0037] As used herein, the term "sequence identity" refers to the "sequence identity percentage" or "identity percentage" between two polynucleotides, which is the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide is present in both the target and reference sequences. Since vacancies are not nucleotides, vacancies present in the target sequence are not counted. Similarly, vacancies present in the reference sequence are not counted because nucleotides from the target sequence are included but nucleotides from the reference sequence are excluded. At least 60% sequence identity includes 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%, up 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% of the total length of the sequence having sequence identity.

[0038] The term "MOI" or "Multiplicity of Infection" used in this article refers to the ratio of viruses to cells, which indicates the average number of viral particles infecting each cell. MOI = pfu / cell. PFU (Plaque forming unit) describes the number of infectious viral particles.

[0039] The term "expression vector" used in this article refers to the direct or indirect linking of the isolated nucleic acid molecules to regulatory elements on the vector when ligating them, as long as these regulatory elements can regulate the translation and expression of the nucleic acid molecule. These regulatory elements can originate directly from the vector itself or be exogenous, meaning they are not derived from the vector itself. In other words, the nucleic acid molecule and the regulatory element are operatively linked. Commonly used expression vectors include prokaryotic expression vectors, eukaryotic expression vectors, and viral expression vectors, etc.

[0040] The term "operably ligated" as used in this article refers to ligating a foreign gene to a vector so that regulatory elements within the vector, such as transcriptional and translational regulatory sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0041] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection 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 the porcine HSP40 / DNAJB6 gene

[0044] The porcine HSP40 / DNAJB6 gene DNA sequence (NC_010460.4) and mRNA sequence (XM_021079274.1) were located in the NCBI database, and the first base of the mRNA sequence (XM_021079274.1) was used as the transcription start site. DNA was extracted from porcine testicular cells (ST cells) using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0. Using the extracted genomic DNA as a template, polymerase chain reaction (PCR) was performed targeting 2000 bp upstream of the transcription start site using primers HSP40 / DNAJB6-QC-F (upstream primer) and HSP40 / DNAJB6-QC-R (downstream primer).

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

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

[0047] The PCR products were then recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver. 4.0. The promoter fragment HSP40 / DNAJB6-QC was obtained. Primer sequences and amplification product sequences are shown in Table 1.

[0048] Table 1. Sequence List

[0049] Note: AGATCT is the BGLII restriction site; AAGCT is the HindIII restriction site.

[0050] The pGL3-Basic plasmid and the amplified promoter fragment HSP40 / DNAJB6-QC were double-digested with restriction endonucleases BGLII and Hind III. Ligation was then performed using the T4 ligase. Each 10 μL 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. Ligation was performed at 16°C for 4 hours.

[0051] The ligation product was transformed into *E. coli* Top10 competent cells, following the instructions for use with *E. coli* Top10 competent cells. After transformation, positive clones were selected and sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing to verify the sequencing accuracy, yielding the plasmid pGL3-HSP40-QC.

[0052] After thawing and resuscitating porcine testicular cells at 37°C, they were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS). When the cells reached 70%-80% confluence, they were passaged. First, the original medium was discarded, and the cells were washed twice with sterile DPBS buffer. Then, the cells were placed in a 37°C incubator and digested with 0.25% EDTA trypsin for approximately 2 minutes. Afterward, the cells were removed and added to 20% FBS DMEM medium (purchased from Thermofisher), along with 0.25% EDTA trypsin. The cells were then pipetted until they detached. The detached cells were seeded at a 1:3 ratio into each well of a 6-well cell culture plate, with 5 ml of 10% FBS DMEM medium added. The plates were then incubated at 37°C in a 5% CO2 incubator. Cells were passaged every 3 days at a 1:3 ratio.

[0053] After porcine testicular cells were cultured to 70-90% confluence, Lipofectamine was diluted in Opti-MEM medium (purchased from Thermofisher). TM Use 3000 reagents to ensure thorough mixing. Next, dilute the endotoxin-free plasmid with Opti-MEM medium to prepare a plasmid premix.

[0054] The experimental group used pGL3-HSP40-QC plasmid and pRL-TK plasmid for co-transfection, while the negative control was co-transfection with two empty plasmids, pGL3-Basic (Beyotime) and pRL-TK (Beyotime). Lipofectamine was added to each tube. TM 3000 (purchased from Thermofisher) reagent and ensure thorough mixing. Add 3000 (purchased from Thermofisher) to each tube of diluted Lipofectamine. TM In reagent 3000, diluted DNA was added at a 1:1 ratio, and after incubation at room temperature for 5 minutes, the DNA-liposome complex was added to the cells. After incubating the cells at 37°C for 12 hours, porcine transmissible gastroenteritis virus (TGEV) isolated and preserved in our laboratory with MOIs (1, 2, 3, and 5) was added. Samples were collected at 0, 12, 24, and 48 hours, and then the transfected cells were analyzed.

[0055] After collecting transfected cells, luciferase assays were performed using the Duo-Lite Luciferase Assay System kit. Untransfected cells were used as a blank control for background subtraction (including background Firefly and background Renilla). Experimental cells were treated with the experimental compounds (including experimental group Firefly and experimental group Renilla), while untreated transfected cells were used to standardize the results (including control group Firefly and control group Renilla).

[0056] As shown in Figure 1, the relative promoter fluorescence activity of pGL3-HSP40-QC was enhanced after TGEV transfection. The higher the MOI value, the stronger the relative promoter activity of pGL3-HSP40-QC.

[0057] Example 2. Identification of porcine HSP40 / DNAJB6 gene promoter activity using dual-luciferase reporter gene assay.

[0058] To identify the fragments with transcription initiation activity, the promoter fragments HSP40 / DNAJB6-P1-F and HSP40 / DNAJB6-P1-R, and HSP40 / DNAJB6-P2-F and HSP40 / DNAJB6-P2-R were amplified using the method described in Example 1, as shown in Figure 2. The primer sequences and amplified sequences are shown in Table 2.

[0059] Table 2. Sequence List

[0060] Note: AGATCT is the BGLII restriction site; AAGCT is the HindIII restriction site.

[0061] The amplified promoter fragments HSP40 / DNAJB6-P1 and HSP40 / DNAJB6-P2 were ligated into the pGL3-Basic plasmid to obtain plasmids pGL3-HSP40-P1 and pGL3-HSP40-P, respectively.

[0062] The luciferase activity was measured according to the detection method in Example 1. The results are shown in Figure 3. The relative activities of pGL3-HSP40-QC in the experimental group were 2.971±0.33; pGL3-HSP40-P1 was 1.34±0.032; and pGL3-HSP40-P2 was 2.7233±0.731. The relative activity of luciferase in the control group was 1.12147±0.042. The relative activities of pGL3-HSP40-P2 in the experimental group were significantly higher than those in the control group (P<0.05). Therefore, it can be determined that the region from -2000bp to -1000bp upstream of the HSP40 promoter is the core region of the promoter.

Claims

1. A promoter element derived from the porcine HSP40 gene, characterized in that... The promoter element comprises a nucleotide sequence as shown in SEQ ID NO: 9, or a nucleotide sequence having at least 60% sequence identity with it.

2. The promoter element according to claim 1, characterized in that, The promoter element comprises 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 with the nucleotide sequence shown in SEQ ID NO:

9.

3. The promoter element according to claim 1, comprising a nucleotide sequence as shown in SEQ ID NO:9 or multiple copies having at least 60% sequence identity with it.

4. The promoter element according to claim 1, characterized in that, The promoter element is regulated by the virus.

5. The promoter element according to claim 1, characterized in that, The promoter element is positively regulated by the virus; Preferably, the viruses include 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, porcine mycoplasma pneumoniae, porcine pseudorabies virus (PRV), porcine eperythrozoonosis virus (PEV), and classical swine fever virus (CSFV). Preferably, the promoter element is positively regulated by the gastroenteritis virus.

6. A transcription expression cassette, characterized in that, The transcription expression cassette includes a promoter element as described in any one of claims 1 to 5 and a target gene, wherein the promoter element is operatively linked to the target gene for initiating transcription of the target gene.

7. An expression carrier, characterized in that, The expression vector comprises the promoter element of any one of claims 1 to 5 or the transcription expression cassette of claim 6.

8. The expression vector according to claim 7, characterized in that, The expression vector is selected from one or more of prokaryotic expression vectors, eukaryotic expression vectors, or viral expression vectors.

9. A host cell, characterized in that, The host cell includes one or more of the promoter element as described in any one of claims 1 to 5, the transcription expression cassette as described in claim 6, and the expression vector as described in claim 7 or 8; Preferably, the host cell is selected from prokaryotic cells and eukaryotic cells; Preferably, the prokaryotic cells include bacterial cells, more preferably Escherichia coli; Preferably, the eukaryotic cells include yeast cells, mammalian cells, or insect cells; More preferably, the mammal is selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cattle, pigs, dogs, and cats.

10. The use of the promoter element according to any one of claims 1 to 5, the transcription expression cassette according to claim 6, the expression vector according to claim 7 or 8, and the host cell according to claim 9 in any one or more of the following: a) Transcription of the target gene, b) Expression of the protein encoded by the target gene. c) Screening for drugs used to treat or prevent diseases.

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