Novel promoter

Abstract

Provided is a promoter comprising any one of the following DNAs (1) to (4): (1) DNA comprising a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3, (2) DNA comprising a base sequence having a substitution, addition or deletion of one or more bases in the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity, and (3) DNA comprising a base sequence having a substitution, addition or deletion of one or more bases in the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3, (3) DNA having a promoter activity and comprising a base sequence having 90% or more identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3, and (4) DNA having a promoter activity and comprising a base sequence capable of hybridizing under stringent conditions with a base sequence complementary to the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3.

Description

Technical Field

[0001] The present invention relates to a novel promoter. Background Art

[0002] Aquatic invertebrates, including shellfish, display extraordinary biological features in regeneration, adaptation, reproduction, aging, etc., and elucidating the underlying gene regulation is of great scientific interest. However, to date, the elucidation of gene function in aquatic invertebrates is very incomplete due to the lack of assay systems, such as using HeLa cell transfection or transgenic mice.

[0003] On the other hand, bivalves are classified as the second largest group among molluscs and are also an important food. For bivalves, early cytomegalovirus (CMV IE) promoters and the like (NPL1, NPL2, and NPL3) have been examined so far; however, no practical promoters have been found.

[0004] To further elucidate gene function in aquatic invertebrates such as bivalves, it is critical to establish cultured cell lines and discover strong gene promoters that highly express transgenes. There is a strong need to develop analytical methods that utilize these approaches to quantitatively assess gene action with high reproducibility.

[0005] Reference List

[0006] Non-patent literature

[0007] NPL 1: Front.Physiol.9,1-8(2018)

[0008] NPL 2: Mar.Biotechnol.3,322-335(2001)

[0009] NPL 3: Fish Shellfish Immunol.103,438-441(2020) Summary of the invention

[0010] Technical issues

[0011] The object of the present invention is to provide a novel promoter.

[0012] Solution

[0013] In this specification, the singular forms ("a", "an", "the", etc.) are intended to include both the singular and the plural, unless explicitly indicated otherwise herein or clearly contradicted by context.

[0014] To solve the above problems, the present inventors conducted extensive studies and found that the oshv117 promoter derived from oyster herpes virus 1 (OsHV-1) has strong promoter activity in various animal cells, including mollusk cells.

[0015] The present invention has been completed based on these findings, and includes the following broad aspects of the invention.

[0016] [Project 1]

[0017] A promoter consisting of any one of the following DNAs (1) to (4):

[0018] (1) a DNA comprising the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3,

[0019] (2) a DNA comprising a base sequence having one or more bases substituted, added or deleted in the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity,

[0020] (3) a DNA comprising a base sequence having 90% or more identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity, and

[0021] (4) A DNA comprising a base sequence that can hybridize under stringent conditions to a base sequence complementary to the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity.

[0022] [Project 2]

[0023] An expression vector comprising the promoter according to item 1.

[0024] [Item 3]

[0025] The expression vector according to item 2, wherein the target gene for expression is integrated under the control of the promoter according to item 1.

[0026] [Item 4]

[0027] A transformant comprising the expression vector according to item 2 or 3.

[0028] [Item 5]

[0029] The transformant according to item 4, which is an aquatic invertebrate.

[0030] [Item 6]

[0031] A method for preparing a target substance encoded by expressing a target gene, comprising culturing the transformant according to item 4 or item 5.

[0032] Beneficial effects of the present invention

[0033] The present invention can provide a novel promoter, an expression vector comprising the novel promoter, and a transformant comprising the expression vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown are the results of an examination of the optimization of the culture conditions for scallop heart cells.

[0035] Figure 2 An overview of electroporation of scallop heart cells is shown.

[0036] Figure 3 The results of the examination on the optimization of the electroporation conditions in Example 1 are shown.

[0037] Figure 4 The electroporation results of luciferase mRNA in Example 1 are shown. The state of cardiac cells 3 days after electroporation is shown.

[0038] Figure 5 The electroporation results of luciferase mRNA in Example 1 are shown. The luciferase expression level 3 days after electroporation is shown.

[0039] Figure 6 The electroporation results of luciferase mRNA in Example 1 are shown. The cell viability after electroporation is shown.

[0040] Figure 7 The luciferase vector in Example 2 is shown.

[0041] Figure 8 The correlation between the expression level of luciferase and the number of adherent cells 2 days after electroporation in Example 3 is shown.

[0042] Fig. 9 The results of the promoter activity evaluation in Example 4 are shown.

[0043] Fig.10 The results of the optimization of the electroporation conditions in Example 5 are shown. A: Expression level at different pore pulse voltages. B: Cell viability at different pore pulse voltages. C: Expression level at different impedances. D: Cell viability at different impedances. E: Expression level at different plasmid vector concentrations. F: Cell viability at different plasmid vector concentrations. G: Expression level at different culture periods after electroporation. H: Cell viability at different culture periods after electroporation.

[0044] Fig.11The EGFP vector in Example 6 is shown.

[0045] Fig.12 The results of GFP measurement of scallop heart cells in Example 6 are shown.

[0046] Fig.13 The results of GFP measurement of scallop blood cells in Example 6 are shown.

[0047] Fig.14 The results of the GFP assay of HEK293 in Example 6 are shown.

[0048] Fig.15 The results of GFP measurement of zebrafish embryos in Example 6 are shown.

[0049] Fig.16 The arrangement of oshv117 and its adjacent genes in Example 7 is shown.

[0050] Fig.17 The sequence analysis results of Poshv117 using NNPP and NSite in Example 7 are shown.

[0051] Fig.18 The results of promoter deletion analysis of Poshv117 in Example 8 are shown.

[0052] Fig.19 The sequences of SEQ ID NO: 1 to SEQ ID NO: 3 are shown.

[0053] Fig. 20 The sequences of promoter Nos. 1 to Nos. 5 in Example 8 are shown. DETAILED DESCRIPTION

[0054] In one embodiment, the present invention provides a promoter comprising any one of the following DNAs (1) to (4):

[0055] (1) a DNA comprising the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3,

[0056] (2) a DNA comprising a base sequence having one or more bases substituted, added or deleted in the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity,

[0057] (3) a DNA comprising a base sequence having 90% or more identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity, and

[0058] (4) A DNA comprising a base sequence that can hybridize under stringent conditions to a base sequence complementary to the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity.

[0059] In this specification, "promoter activity" refers to the activity of promoting the transcription of DNA (gene) into mRNA. Therefore, in the present invention, the phrase "having promoter activity" is intended to show the activity of promoting the transcription of DNA (gene) into mRNA in one or more experimental systems. In the present invention, if DNA shows this activity in at least one experimental system using cells belonging to molluscs, mammals, fish, etc. (preferably in a system using at least one type of aquatic invertebrate cells), then DNA is interpreted as having promoter activity.

[0060] The promoter activity can be confirmed by determining whether protein expression is observed using a suitable reporter gene. For example, the DNA (reporter gene) that is combined with the DNA encoding the detectable protein under the control of the promoter can be introduced into the cell, and the generation of the gene product from the reporter gene is measured to confirm the promoter activity. The example of the reporter gene includes a luciferase gene, a fluorescent protein gene, such as a green fluorescent protein (GFP) gene, a β-galactosidase (LacZ) gene, a β-glucuronidase (GUS) gene, a β-lactamase gene, etc. More specifically, the promoter activity can be measured, for example, by the method described in the embodiment of the application provided later.

[0061] In one aspect, the promoter activity of the promoter of the present invention is at least two times (more preferably at least three times, even more preferably at least five times, particularly preferably at least seven times) higher than that of a conventional promoter (early cytomegalovirus (CMV IE) promoter).

[0062] On the one hand, when the GFP gene is used as the reporter gene, the promoter of the present invention has a strong promoter activity, which allows fluorescence observation under a fluorescence microscope.

[0063] The promoter of the present invention may comprise a base sequence that is substantially the same as the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 in the sequence table, as long as it has promoter activity. Due to the "promoter activity" of the present invention, the gene controlled by the promoter (e.g., usually located downstream) is transcribed into mRNA.

[0064] (1) The above includes a DNA comprising a base sequence represented by SEQ ID NO:1 to SEQ ID NO:3 and having promoter activity. The DNA composed of the base sequence represented by SEQ ID NO:1 to SEQ ID NO:3 is a promoter derived from the family of Malacoherpesviridae. The DNA composed of the base sequence represented by SEQ ID NO:1 is the oshv117 promoter of oyster herpesvirus 1. The DNA composed of the base sequence represented by SEQ ID NO:2 is the oshv117 promoter of oyster herpesvirus 1 mutants (oyster herpesvirus 1 strain micromutant A and oyster herpesvirus 1 strain micromutant B). The DNA composed of the base sequence represented by SEQ ID NO:3 is the oshv117 promoter of scallop acute necrosis virus (AVNV). SEQ ID NO:2 is a sequence having two base substitutions in SEQ ID NO:1, and SEQ ID NO:3 is a sequence having five base substitutions and one base insertion in SEQ ID NO:1.

[0065] In the above (2), the number of one or more bases to be replaced, deleted, added or inserted is not particularly limited as long as it is an integer greater than 1. For example, the number of bases may be about 1 to several tens, preferably about 1 to 30, more preferably about 1 to 15, even more preferably about 1 to 10, and particularly preferably about 1 to 5. A nucleic acid molecule comprising a base sequence having a substitution, addition or deletion of one or more bases in a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 is included, wherein the nucleic acid molecule has promoter activity.

[0066] On the one hand, the number of one or more bases to be substituted, added or deleted can be about 1 to 107, so that the identity of the base sequence to the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 is 90% or higher, preferably about 1 to 54, so that the identity is 95% or higher, more preferably about 1 to 21, so that the identity is 98% or higher, and particularly preferably about 1 to 11, so that the identity is 99% or higher.

[0067] In the above (3), the base sequence identity can be 90% or higher, preferably 95% or higher, more preferably 98% or higher, and particularly preferably 99% or higher. The base sequence homology or identity can be set to less than 100%. The homology and identity between base sequences can be determined using known algorithms such as BLAST. A nucleic acid molecule comprising a base sequence having 90% or higher identity with a base sequence represented by any one of SEQ ID NO:1 to SEQ ID NO:3, wherein the nucleic acid molecule has promoter activity.

[0068] On the one hand, preferred is a base sequence having a TATA box sequence (TATATAA) and having 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more) identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3. In the above, the TATA box sequence (TATATAA) may have 1 (or 2 or 3) base sequence substitutions, deletions, additions or insertions.

[0069] On the one hand, preferred is a base sequence having a TACGTGGG sequence and having 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more) identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3. In the above, the TACGTGGG sequence may have 1 (or 2 or 3) base sequence substitutions, deletions, additions or insertions.

[0070] On the one hand, preferred is a base sequence having a GGATTGGC sequence and having 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more) identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3. In the above, the GGATTGGC sequence may have 1 (or 2 or 3) base sequence substitutions, deletions, additions or insertions.

[0071] On the one hand, preferred is a base sequence having a GAGGGAAGGT (SEQ ID NO: 36) sequence and having 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more) identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3. In the above, the GAGGGAAGGT sequence may have 1 (or 2 or 3) base sequence substitutions, deletions, additions or insertions.

[0072] On the one hand, preferred is a base sequence having a sequence of ACACCATTACATT (SEQ ID NO: 37) and having 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more) identity with a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3. In the above, the ACACCATTACATT sequence may have 1 (or 2 or 3) base sequence substitutions, deletions, additions or insertions.

[0073] On the one hand, preferred is a base sequence having a CAATT box consensus sequence (T / C)GATTGG(T / C)(T / C)(G / A) (SEQ ID NO: 38) and having 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more) identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3. In the above, the (T / C)GATTGG(T / C)(T / C)(G / A) sequence may have 1 (or 2 or 3) base sequence substitutions, deletions, additions or insertions.

[0074] On the one hand, preferred is a base sequence having a Kozak consensus sequence (A / G)NNATG(A / G) and having 90% or more (preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more) identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3. In the above, the (A / G)NNATG(A / G) sequence may have 1 (or 2 or 3) base sequence substitutions, deletions, additions or insertions.

[0075] In (4) above, "stringent conditions" refer to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Examples of such stringent conditions are, but are not limited to, "hybridization at 42°C in 1×SSC (0.9M NaCl, 0.09M trisodium citrate) or 6×SSPE (3M NaCl, 0.2M NaH2PO4, 20mM EDTA-2Na, pH 7.4), followed by washing with 0.5×SSC at 42°C." These conditions are described in, for example, J. Sambrook et al., Molecular Cloning, Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).

[0076] The method for obtaining the promoter of the present invention is not particularly limited, and the promoter can be obtained by conventional genetic engineering methods or chemical synthesis methods. For example, for a DNA comprising a base sequence represented by SEQ ID NO: 1, genomic DNA of oyster herpes virus 1 can be extracted from a shellfish infected by oyster herpes virus 1, such as the DNA of Pacific oysters (Crassostrea gigas), and a DNA consisting of a base sequence represented by SEQ ID NO: 1 can be cloned. For a DNA comprising a base sequence represented by SEQ ID NO: 2, genomic DNA of oyster herpes virus 1 strain microvariant A can be extracted from a shellfish infected by shellfish herpes virus 1 strain microvariant A, such as the DNA of Pacific oysters (Crassostrea gigas), or genomic DNA of oyster herpes virus 1 strain microvariant B can be extracted from a shellfish infected by shellfish herpes virus 1 strain microvariant B, such as Pacific oysters (Crassostrea gigas), and a DNA consisting of a base sequence represented by SEQ ID NO: 2 can be cloned. For the DNA containing the base sequence represented by SEQ ID NO: 3, the genomic DNA of scallop acute necrosis virus (AVNV) can be extracted from the DNA of shellfish infected by scallop acute necrosis virus (AVNV), such as Chlamys farreri, and the DNA consisting of the base sequence represented by SEQ ID NO: 3 can be cloned. The promoter DNA of the present invention can also be artificially synthesized based on the base sequences represented by SEQ ID NO: 1 to SEQ ID NO: 3.

[0077] The promoter of the present invention has much higher promoter activity in bivalve cells than conventional promoters. For example, when the luciferase gene is used as the reporter gene, the promoter comprising the base sequence represented by SEQ ID NO: 1 shows higher promoter activity than conventional promoters (early cytomegalovirus (CMV IE) promoter) ( Fig. 9 ) has a promoter activity 25 times higher than that of the promoter of SEQ ID NO: 1. In addition, when the GFP gene is used as the reporter gene, GFP fluorescence can be observed under a fluorescence microscope in the promoter comprising the base sequence represented by SEQ ID NO: 1. The promoter is particularly useful in bivalves.

[0078] In another embodiment, the present invention provides an expression vector comprising the above promoter.

[0079] The expression vector of the present invention can be constructed by combining the promoter of the present invention into an expression vector that allows the expression of the target gene. In the expression vector of the present invention, the target gene is integrated under the control of the DNA of the present invention. Usually, in the expression vector of the present invention, the target gene is operably connected to the downstream of the DNA of the present invention.

[0080] The expression vector to which the promoter of the present invention is to be incorporated is not particularly limited, as long as it can be stably maintained and propagated in the host cell. For example, plasmid vectors, viral vectors, cosmid vectors, pore vectors, artificial chromosome vectors, etc. can be appropriately selected according to the host cell. Among them, plasmid vectors are preferred.

[0081] As the expression vector, commercially available cloning vectors and expression vectors can be used. Examples include pNL1.1 / 1.2 / 1.3, pNL2.1 / 2.2 / 2.3, pNL3.1 / 3.2 / 3.3, pGL4, pGEM T / Teasy and pGEMEX-1 (all manufactured by Promega Corporation), T-Vector pMD20 / pMD19, pUC18 / 19, pUC118 / 119, pBR322 and pMW218 / 219 (all manufactured by Takara Bio Inc.), pCR4-TOPO, pCR-XL-2-TOPO, pCR-BluntII-TOPO pcDNA3.1 / V5-His-TOPO, pcDNA3.4-TOPO, pcDNA3.3-TOPO, pENTR / D-TOPO, pCR8 / GW / TOPO, pENTR / SD / D-TOPO, pcDNA3.1 V5-His A / B / C and pPICZ A / B / C (all manufactured by Thermo Fisher Scientific), pDRIVE and pQE (both manufactured by QIAGEN NV), pBluescript II SK(+ / -) (manufactured by Stratagene), pSC-A-amp / kan and pSC-A-amp / kan (both manufactured by Agilent Technologies, Inc.), pET and pGEX (manufactured by Merck KGaA), pACYC184 (manufactured by Nippon Gene Co., Ltd.), adeno-associated virus (AAV) vector, adenovirus vector, lentivirus vector, retrovirus vector, Sendai virus vector, etc.

[0082] In the expression vector, the expression target gene located downstream of the promoter of the present invention is not particularly limited. For example, the expression target gene is a gene encoding the target substance or an enzyme involved in its synthesis. The expression target gene may also be referred to as a nucleic acid molecule (e.g., DNA or RNA) encoding such a target substance, etc. The expression target gene may be a heterologous gene encoding a heterologous expression product, a gene encoding a host cell intrinsic expression product, or a gene encoding any other protein, peptide, nucleic acid, etc. The example of the target substance encoded by the expression target gene includes a reporter fluorescent protein, such as GFP, a growth promoting factor, a maturation promoting factor, a sterile factor, a disease resistance acquisition factor, a Cas9 protein, an enzyme, a hormone, a cytokine, a toxin, such as diphtheria, other physiologically active peptides, transporters, non-coding RNAs, and shRNAs (small hairpin RNAs or short hairpin RNAs) for RNA interference.

[0083] In another embodiment, the present invention provides a transformant comprising the above expression vector.

[0084] The host cell introduced into the expression vector of the present invention is not particularly limited, as long as the promoter of the present invention is used as a promoter. Examples include aquatic invertebrates (shellfish, squid, octopus, etc.), mammals (human, mouse, rat, rabbit, dog, monkey, orangutan, horse, donkey, hamster, guinea pig, etc.), fish (zebrafish, killifish, rainbow trout, salmon, red sea bream, yellowtail sea bream, tuna, etc.), annelids (earthworms, sea worms, leeches, etc.), flatworms (turbellarians, etc.), cnidarians (jellyfish, corals, sea anemones, etc.), echinoderms (sea urchins, starfish, sea cucumbers, etc.), arthropods (shrimp, crabs, insects, etc.), tunicates (sea squirts, etc.), etc. Among them, aquatic invertebrates are preferred, and aquatic molluscs are more preferred. Examples of aquatic molluscs include shellfish, such as gastropods and bivalves, cephalopods, such as octopus and squid, etc. Among them, shellfish are preferred, and bivalves are particularly preferred. Among shellfish, examples of gastropods include abalone, turban shells, etc. Examples of bivalves include scallops, oysters, mussels, Manila clams (little clams), basket clams, hard clams, red clams, surf clams, surf flaps, giant bamboo clams, barnacles, pearl oysters, etc.

[0085] The transformant can be obtained by introducing the expression vector containing the promoter of the present invention into the host cell by a general transformation method, such as electroporation, lipofection, microinjection, transformation, transfection, conjugation, protoplasts, particle guns and Agrobacterium. In this specification, "introducing into host cells" includes not only introducing cells obtained from living organisms or established cell lines, but also introducing cell clusters, tissues, etc. obtained from living organisms; introducing cell clusters, tissues, etc. constructed using cells obtained from living organisms; and introducing living organisms (e.g., non-human living organisms).

[0086] The conditions of electroporation vary depending on the host cell and the expression vector, and are not particularly limited. For example, the perforation pulse voltage is 100 to 250 V, preferably 175 to 200 V. The impedance is also not particularly limited, but is, for example, 30 to 100 Ω, preferably 50 to 70 Ω. The osmotic pressure of the expression vector solution is also not particularly limited, but is, for example, 300 to 1500 mOsm / kg, preferably 800 to 1200 mOsm / kg. For every 1×10 6 In the host cell, the amount of the expression vector to be added is, for example, 5 to 35 μg, preferably 10 to 30 μg.

[0087] In another embodiment, the present invention provides a method for preparing a target substance encoded by an expression target gene, which comprises culturing the above transformant.

[0088] The transformant (host cell) transformed with the expression vector is cultured in a suitable culture medium, wherein the expression target gene is connected to the downstream of the promoter of the present invention, and the expression target gene is expressed under the control of the promoter of the present invention to produce the target substance. The culture conditions of the host cell are not particularly limited, as long as the host cell can be propagated and the target substance can be produced. After the culture is completed, the target substance can be collected from the culture. Examples of the target substance include substances encoded by the above-mentioned target gene and products synthesized by the action of such substances.

[0089] By using the promoter of the present invention, gene recombination, gene editing, etc. can be effectively performed in aquatic invertebrates, such as bivalves, thereby promoting the elucidation of gene functions in aquatic invertebrates, such as bivalves. This is expected to have applications in ecosystem protection, such as genetic sterilization technology. The technology is also expected to be applied to technologies for producing excellent types of edible bivalves and other species by improving their taste and nutritional composition. In addition, it is expected to be applicable to adding water purification functions and producing functional proteins.

[0090] Example

[0091] The present invention is described in more detail using Preparation Examples and Examples; however, the present invention is not limited to these Examples.

[0092] Scallop cell culture

[0093] One to two year old scallops (Mizuhopecten yessoensis) were obtained from fish farms in Onagawa Bay (Miyagi Prefecture), Mutsu Bay (Aomori Prefecture), or Funka Bay (Hokkaido).

[0094] (1) Cardiac cell culture

[0095] The ventricles were isolated from the scallops and washed with artificial seawater (Instant Ocean, manufactured by Aquarium Systems) that had been filtered through a sterile filter with a pore size of 0.22 μm. After washing, the obtained heart tissue was finely cut using tweezers and scissors sterilized with ethanol, and then rinsed with DBSS (Table 1) at room temperature for 30 minutes at 50 rpm / min using an oscillator. After removing DBSS, the heart tissue fragments were enzymatically digested with trypsin-EDTA solution (Table 1) at 4°C for 18 to 21 hours. The enzymatic digestion was then stopped by adding an equal volume of growth medium (Table 1) supplemented with 10% fetal bovine serum (FBS), and small cells and debris were removed. The heart tissue fragments after enzymatic digestion were exocuted onto a multi-well plate (manufactured by Sumitomo Bakelite Co., Ltd.) or a T flask (Nunc, manufactured by Thermo Fisher Scientific) and cultured at 20°C. At the beginning of cell culture, the heart tissue fragments were first attached to the substrate with a small amount of growth medium (Table 1), and the next day, an appropriate amount of culture medium (for 75 cm 2 T flask, add 6ml at the time of inoculation and 9ml the next day). During this period, release cells from the excised cardiac tissue fragments attached and spread on the bottom of the T flask, thereby obtaining adherent primary culture cells. Replace the growth medium with fresh medium every 10 days or when the pH of the culture medium decreases. When the primary culture cells attached to the T flask reach confluence, use a cell scraper to separate the cells from the T flask and passage them in a new T flask with growth medium. Primary cardiac cells derived from these ventricular cells survive 3 to 4 weeks at 20°C, after which they tend to gradually degenerate, although some cells survive for 3 months or more. No additional supply of CO2 and humidity is required.

[0096] Table 1

[0097]

[0098] Before use in the experiments, all the various solutions listed in Table 1 were adjusted to pH 7.4 using hydrochloric acid (HCl) or sodium hydroxide solution (NaOH).

[0099] (2) Cultivation of blood cells

[0100] Scallop hemolymph was extracted directly from the ventricle using a 22-gauge needle and immediately mixed with modified Aldrich solution (MAS (Table 1)) at a 1:1 ratio. Blood cells in the hemolymph-MAS mixture were centrifuged at 2 × 10 5 Cells / ml were inoculated in growth medium and the final composition of the medium was adjusted to approximately 2% FBS and 10% scallop hemolymph-MAS mixture. To prevent cell aggregation, the collected blood cells were placed on ice before inoculation. Primary blood cells survived for 2 weeks at 20°C and denaturation was accelerated by changing the medium. No additional supply of CO2 and humidity was required.

[0101] (3) Optimization of scallop heart cell culture conditions

[0102] The culture conditions of scallop heart cells were examined. Heart tissue fragments enzymatically digested with trypsin-EDTA solution (Table 1) at 4°C for 18 hours were cultured under the following conditions (i) to (iv) for seeding density, culture temperature, osmotic pressure, and the presence of FBS. The number of adherent cells on the second day was then counted from five randomly taken micrographs (N=20) at 100x magnification. The results are shown in Figure 1 In addition, the FBS concentration was additionally checked and it was found that 2% FBS was sufficient. Based on these results, the optimal culture condition for subsequent experiments was determined to be a seeding density of 4 × 10 5 cells / ml, culture temperature was 10°C, osmotic pressure was 1100 mOsmol / kg, and 2% FBS.

[0103] (i) Seeding density: 2×10 5 cells / ml or 4×10 5 Cells / ml

[0104] (ii) Culture temperature: 10℃ or 20℃

[0105] (iii) Osmolality: 900mOsmol / kg or 1100mOsmol / kg

[0106] (iv) FBS: 0% or 10%

[0107] Culture of human HEK293 cells

[0108] The human HEK293 cells (JCRB9068) used were provided by JCRB Cell Bank. The cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco, manufactured by Thermo Fisher Scientific) supplemented with 10% FBS at 37°C in a humidified incubator supplied with 5% CO2.

[0109] Obtaining zebrafish embryos

[0110] Adult zebrafish were purchased from Meitosuien Co., Ltd. (Aichi Prefecture) or Masuyuki Co., Ltd. (Saitama Prefecture). The purchased adult fish were placed in a 60 cm water tank, fed with compound feed and raised. In addition, fertilized eggs were obtained by pairing mature male and female zebrafish.

[0111] Example 1: Examination of electroporation conditions

[0112] Scallop primary heart cells cultured for 1 to 2 weeks were used for electroporation. The growth medium was removed, and the heart cells cultured in the FBS-free growth medium were washed and collected with a cell scraper (manufactured by Sumitomo Bakelite Co., Ltd.). The cell suspension of the collected heart cells was filtered through a sterilized steel mesh with a pore size of 125 μm (manufactured by Yikai Metal Products Co., Ltd.) to remove heart tissue fragments during transplantation. After the obtained cultured cell suspension was centrifuged at 400 × g for 5 minutes at 20 ° C, the precipitated cultured cells were collected, and the cell number and viability were measured using a hemocytometer and trypan blue dye exclusion method.

[0113] Electroporation was performed using a NEPA21 electroporator and a 2 mm gap electroporation cuvette (manufactured by Nepa Gene Co., Ltd.). Cardiac cells (1×10 6 Cells) were resuspended in 100 μl Opti-MEM (Gibco, manufactured by Thermo Fisher Scientific) containing 10 μg of plasmid DNA or 10 μg of mRNA. Since the osmotic pressure of Opti-MEM was 300 mOsmol / kg when not adjusted, the osmotic pressure was adjusted to 1050 mOsmol / kg by adding 256.7 mg / ml sucrose. The impedance of the cell suspension was 50 to 60 Ω. The electroporation parameters were set as follows:

[0114] -For perforation pulse, variable pulse voltage, pulse length 2.5ms, pulse interval 50ms, pulse number 2, decay rate 10%, polarity switched to +.

[0115] -For transmission pulse, pulse voltage 20V, pulse length 50ms, pulse interval 50ms, pulse number 5, attenuation rate 40%, polarity switching is + / -.

[0116] (1) Testing electroporation conditions in the absence of mRNA

[0117] To find the optimal conditions for electroporation in scallop heart cells, the perforation pulse voltages from 75 to 200 V were examined at osmolarities of 300 mOsmol / kg and 1050 mOsmol / kg (examined in the absence of mRNA). Cells that were not electroporated or to which distilled water was added instead of DNA or mRNA were used as controls. The results are shown in Figure 3 At an osmotic pressure of 300 mOsmol / kg, cell viability was low even when the voltage was not high, whereas at an osmotic pressure of 1050 mOsmol / kg, cell viability increased by an average of 36.4%.

[0118] (2) Testing electroporation conditions in the presence of luciferase mRNA

[0119] Cells electroporated with luciferase mRNA at an osmotic pressure of 1050 mOsmol / kg and a perforation pulse voltage of 75 to 200 V were seeded in 24-well plates with 500 μl of growth medium and then examined in the presence of mRNA. Immediately after electroporation, cell viability was determined by trypan blue dye exclusion. In addition, luciferase assays were performed on mRNA-treated cells 3 days after electroporation. The results are shown in Figures 4 to 6 middle.

[0120] Example 2: Preparation of luciferase vector

[0121] (1) Selection of promoter

[0122] In order to identify a promoter with strong activity, a total of 11 promoters were selected and examined from the bivalve infectious virus oyster herpes virus-1 (OsHV-1) promoter, the endogenous promoter of the yesso scallop (M. yessoensis), the shrimp infectious virus white spot syndrome virus (WSSV) promoter, etc. The selected promoters are shown below. As a control, the early cytomegalovirus (CMV IE) promoter and no promoter, which are routinely used in bivalves, were also examined.

[0123] - Check the promoter

[0124] Oyster herpes virus-1 (OsHV-1) promoters Poshv027, Poshv029, Poshv072, Poshv080, Poshv088 and Poshv117

[0125] Endogenous promoters of the scallop (M. yessoensis): Pmy-actβ, Pmy-ef1α and Pmy-ef1β

[0126] White Spot Syndrome Virus (WSSV) promoter: Pwsv-ie1 and Pwsv465

[0127] (2) Preparation of luciferase vector

[0128] The above promoter was used to prepare a vector ( Figure 7 ).

[0129] The OsHV-1 promoter was extracted from Pacific oyster (Crassostrea gigas) larvae infected with OsHV-1, and the 5' upstream regions of the start codons -1201, -1162, -1017, -1038, -1101 and -1069 of six genes selected from OsHV-1 (Poshv027, Poshv029, Poshv072, Poshv080, Poshv088 and Poshv117, respectively) were cloned into the PCR linearized pNL1.1 vector.

[0130] For the endogenous promoter of the Yesso scallop, genomic DNA was extracted from the adductor muscle of the scallop from Mutsu Bay using the DNeasy Blood & Tissue Kit (manufactured by QIAGEN NV), and the 5' upstream region of the start codons -4706, -3007 and -2221 of the Yesso scallop actβ, ef1α and ef1β was cloned into the SpeedCut Hind III (manufactured by Rikaken Holdings Co., Ltd.) linearized pNL1.1 vector.

[0131] For the WSSV promoter, DNA sequences of wsv-ie1 promoter and wsv465 promoter were synthesized based on the NCBI database (commissioned to Eurofin Genomics), and the 5' upstream regions of the start codons -502 and -472 were cloned into the PCR linearized pNL1.1 vector.

[0132] For CMV-IE promoter as a control, pNL1.1.CMV[Nluc / CMV] vector (manufactured by Promega) was purchased.

[0133] Strain DH-5α (manufactured by Nippon Gene Co., Ltd.) and plasmid DNA extraction Mini / MIDI kit (manufactured by Favorgen Biotech Corp.) were used for amplification and extraction of vectors. For PCR cleanup and gel extraction, NucleoSpin Gel and PCR Cleanup Kit (manufactured by Takara Bio Inc.) and QIAquick Gel Extraction Kit (manufactured by Qiagen NV) were used. All molecular cloning procedures were performed using In-Fusion Snap Assembly (manufactured by Takara Bio Inc.).

[0134] The primers used to prepare the vector are shown in Table 2.

[0135] Table 2

[0136]

[0137] Example 3: Luminescence Normalization

[0138] To normalize the luminescence of cells into which the luciferase gene was introduced, the number of adherent cells was counted, and the luciferase luminescence value was divided by the number of adherent cells. The autofluorescence of scallop heart cells was used for counting. Scallop primary heart cells (1 × 10 6 The cardiac cells were then electroporated at 100 V (other conditions were the same as above). 4 Cells, 1×10 5 Cells, 2×10 5 Cells were seeded in 24-well plates and cultured in 500 μl of growth medium for 2 days. Each well was then washed twice with growth medium and observed under a GFP filter at 40x magnification with a long exposure time (Keyence BZ-X800, 0.67 seconds, manufactured by Keyence). The area of ​​autofluorescence emitted by adherent cells was 0.77 cm per well. 2 , and use the pixel classification and object classification pipeline in ilastik to count cells through guided machine learning (1.3.3). The results are shown in Figure 8 shown.

[0139] R by oshv088 and Poshv117 2 The values ​​were 0.95 and 0.54, respectively, indicating a positive correlation between the number of adherent cells and the luciferase expression level. 2 The value was 0.3; however, the luciferase expression levels (relative luminescence unit (RLU) values) were normalized to the cell number.

[0140] Example 4: Evaluation of promoter activity, luciferase assay

[0141] 10 μg of each luciferase vector prepared above was electroporated into scallop primary cardiac cells (1×10 6cells) (other conditions were the same as above). After culturing for 2 days, the growth medium was removed and the cells were washed twice with growth medium without FBS, leaving only the adherent cells. Then, 100 μl of 1% PBS-TX was added to each well, incubated for 10 minutes to lyse the cells, and the collected cell lysates were centrifuged at 12,000 × g for 5 minutes to remove unlysed substances. Then, luciferase assay was performed using the Nano-Glo luciferase assay system (manufactured by Promega). Luminescence was measured using a GloMax plate reader (manufactured by Promega) and normalized to the number of adherent cells in each well. The results are shown in Fig. 9 .

[0142] Poshv117 showed the highest expression, followed by Poshv088. Although CMV IE promoter and Pmy-ef1α showed some activity, the results were significantly lower than those of Poshv117 and Poshv088. The expression level of Poshv117 was 16.1 times that of Pmy-ef1α and 24.7 times that of CMV IE promoter (a promoter commonly used for bivalves).

[0143] Example 5: Further optimization of electroporation conditions

[0144] Using Poshv117, the electroporation conditions of scallop heart cells were further optimized. The optimization was checked under the following conditions: perforation pulse voltage (100V, 125V, 150V, 175V, 200V), impedance (60Ω, 54Ω, 49Ω, 42Ω, 38Ω), plasmid vector concentration (1μg, 2μg, 5μg, 10μg, 20μg) and culture period after electroporation (1 day, 2 days, 3 days, 4 days, 5 days, 6 days). Each condition was checked based on a perforation pulse voltage of 200V, an impedance of 60Ω, a plasmid vector concentration of 10μg and a culture period of 2 days after electroporation. The impedance of 60 to 38Ω was checked by adding +20μl, +40μl, +60μl, +80μl and +100μl of Opti-MEM adjusted to osmotic pressure of 1050mOsmol / kg. The results are shown in Fig.10 middle.

[0145] Example 6: Evaluation of promoter activity, GFP assay

[0146] (1) Preparation of EGFP vector

[0147] The EGFP vector was prepared by replacing the luciferase translation region of the above luciferase vector with the EGFP translation region (EGFPCDS) Fig.11 ). The amplification and extraction of the vector were performed in the same manner as described above.

[0148] The primers used to prepare the vector are shown in Table 3.

[0149] Table 3

[0150]

[0151] (2) GFP assay in scallop heart cells

[0152] 10 μg of EGFP vector was electroporated into scallop primary cardiac cells (1×10 6 cells) (other conditions were the same as above). The EGFP vectors used were Poshv117-EGFP vector, Poshv088-EGFP vector and Pmy-ef1α-EGFP vector, and the pCS2-GFP vector of the CMV IE promoter was used as a control. Two days after electroporation, the cells were observed under a fluorescence microscope (BZ-X800, manufactured by Keyence). The results are shown in Fig.12 Middle. Fluorescence was observed only under the condition where the Poshv117-EGFP vector was used.

[0153] Furthermore, in order to confirm that the fluorescence expressed by the electroporated scallop cells is a GFP signal, the scallop heart cells were stained with an immunofluorescent anti-GFP antibody. 10 μg of Poshv117-EGFP vector was electroporated into scallop primary heart cells (1×10 6 cells) (other conditions were the same as above). After 2 days, the cells were fixed with 4% formaldehyde for 15 minutes at room temperature. The cells were then permeabilized with 0.1% PBS-TX for 20 minutes and blocked with 3% BSA for 30 minutes at room temperature. Subsequently, the cells were reacted with anti-GFP antibody (rabbit IgG, Invitrogen, manufactured by Thermo Fisher Scientific) (10 μg / ml) diluted 200 times with 0.1% PBS-TX at 4°C overnight. The next day, the cells were reacted with a secondary antibody (Alexa Fluor Plus 594 donkey anti-rabbit IgG, Invitrogen, manufactured by Thermo Fisher Scientific) (10 μg / ml) diluted 200 times with 0.1% PBS-TX at room temperature for 1 hour. The cells were then observed under a fluorescence microscope (BZ-X800, manufactured by Keyence). The results are shown in Fig.12 In the figure, GFP and red fluorescent protein (RFP) signals were observed in the same scallop heart cells under a fluorescence microscope. From the results, it can be seen that the fluorescence expressed from the electroporated scallop cells is the GFP signal.

[0154] (3) Detection of GFP in scallop blood cells

[0155] Due to the high cell death rate after electroporation in scallop blood cells, the EGFP vector was introduced by fat transfection. Using Lipofectamine 3000 (Invitrogen, manufactured by Thermo Fisher Scientific), the blood cells were cultured in 500 μl growth medium containing 500 ng of EGFP vector for one day. The EGFP vectors used were Poshv117-EGFP vector, Poshv088-EGFP vector and Pmy-ef1α-EGFP vector, and the pCS2-GFP vector of the CMV IE promoter was used as a control. The medium containing the vector was then removed and the cells were observed under a fluorescence microscope (BZ-X800, manufactured by Keyence). The results are shown in Fig.13 GFP fluorescence was observed in scallop hemocytes only when the Poshv117-EGFP vector was used.

[0156] (4) GFP assay in HEK293 cells

[0157] HEK293 cells (3×10 5 Cells) were suspended in 100 μl Opti-MEM (300 mOsmol / kg) and electroporated with 10 μg of EGFP vector at a perforation pulse voltage of 155 V. The EGFP vectors used were Poshv117-EGFP vector and Poshv088-EGFP vector, and pCS2-GFP vector of CMV IE promoter was used as a control. Two days after electroporation, the cells were observed under a fluorescence microscope (BZ-X800, manufactured by Keyence). In addition, like scalloped cardiac cells, the cells were stained with anti-GFP antibody by immunofluorescence. The results are shown in Fig.14 GFP fluorescence was observed in HEK293 cells only when the Poshv117-EGFP vector was used.

[0158] (5) GFP assay in zebrafish embryos

[0159] The EGFP vector adjusted to 100 ng / μl was microinjected into the zebrafish blastoderm (cell body) at the one-cell stage or the cell stage. The EGFP vectors used were Poshv117-EGFP vector and Poshv088-EGFP vector, and the pCS2-GFP vector of the CMV IE promoter was used as a control. The embryos were cultured at 28°C for 24 hours after microinjection and observed under a fluorescence microscope (BZ-X800, manufactured by Keyence). The results are shown in Fig.15 GFP fluorescence was observed in zebrafish embryos only when the Poshv117-EGFP vector was used.

[0160] Example 7: Sequence Analysis

[0161] The DNA sequences of the poshv117 and oshv117 translated regions were analyzed manually using the NNPP, NSite, and CDD programs.

[0162] Fig.16 The arrangement of oshv117 and its adjacent genes is shown. The OsHV-1 gene contains 4600-bp overlapping regions, each of which includes oshv116, oshv117, oshv118, and oshv119. The regions are 100% identical in DNA sequence and complementary to each other (i.e., one in the 5'-3' direction and the other in the 3'-5' direction).

[0163] Poshv117 contains the 5'-UTR of oshv117 (233 bp), the entire translation region of oshv118 (669 bp), and a partial 5'-UTR of oshv118 (167 bp).

[0164] Fig.17 Sequence analysis of Poshv117 using NNPP and NSite is shown (SEQ ID NO: 1). The neural network-based promoter prediction program (NNPP) predicted that the transcription start site is located at -58 and -30 upstream of the start codon of oshv117 and oshv118. In addition, a TATA box (TATATAA) was detected at -30 upstream of the transcription start site of oshv117.

[0165] In addition, the following binding motifs were predicted by NSite (a consensus-based search tool for putative functional motifs) (the lowercase letters below indicate the parts that differ from the consensus sequence):

[0166] -TACGTGGG, a conserved sequence that binds to mammalian hypoxia-inducible factor 1 (HIF-1) (Homo sapiens, Mus musculus, Rattus norvegicus);

[0167] - GGATTGGC, putative binding site of mammalian dihydrofolate reductase (DHFR) (M. musculus);

[0168] -GAGGGAagGT, putative binding site for mammalian mammary cell activating factor (MAF) (Homo sapiens, Mus musculus);

[0169] -ACACCatTACATT, a RPG box-like sequence near the TATA box of oshv117 that is putatively bound to Ras-associated protein 1 (RAP1) of Saccharomyces cerevisiae.

[0170] In addition, a CAATT box consensus sequence (T / C)GATTGG(T / C)(T / C)(G / A) was found at position -91 5' upstream of the transcription start site of oshv117. The Kozak consensus sequence (A / G)NNATG(A / B) was conserved around the start codon of oshv117. In addition, continuous and discrete palindromic sequences (TTCCCTGGT and GCCAGGGAA, AATGCGT and ACGCATT; AACATGTT, AAATATTT, TCCATATGGA, TGATATCA) and three direct repeat sequences (CAACAACAA, CTGTATCTGTAT, AACAACAACAAC) were found.

[0171] Example 8: Promoter deletion analysis

[0172] In order to identify the core promoter region controlling the expression of Poshv117, promoter deletion analysis was performed. The mutant promoter used was as follows. Fig.17 No. 1 to No. 5 in the figure correspond to the deletion sites in the promoter region.

[0173] -Promoter No. 1: 1 to 1069. Full-length region (about 1 kbp upstream of ORF117, Poshv117). (SEQ ID NO: 1)

[0174] -Promoter No. 2: 1 to 902. The upstream region of ORF118 is deleted (SEQ ID NO: 32)

[0175] - Promoter No. 3: 1 to 587. About half of the translated region of ORF118 (including the HIF binding motif) is missing (SEQ ID NO: 33)

[0176] -Promoter No.4: 1 to 233. The entire translation region of ORF118 (including the HIF binding motif) is missing, leaving only the upstream region of ORF117. (SEQ ID NO: 34)

[0177] - Promoter No. 5: 1 to 151. Only the region upstream of ORF117 includes a CCAAT box (SEQ ID NO: 35)

[0178] - Promoter No. 6: no promoter insertion sequence.

[0179] After cloning the above promoter into the pNL1.1 vector to prepare the vector, luciferase assay was performed in the same manner as in Example 4. The results are shown in Fig.18 middle.

[0180] The full-length sequence had the highest promoter activity, and shortening the length of the sequence from the upstream side decreased the promoter activity in proportion to the length.

Claims

1. A promoter comprising any one of the following DNAs (1) to (4): (1) a DNA comprising the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3, (2) a DNA comprising a base sequence having one or more bases substituted, added or deleted in the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity, (3) a DNA comprising a base sequence having 90% or more identity with the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity, and (4) A DNA comprising a base sequence that can hybridize under stringent conditions to a base sequence complementary to the base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 3 and having promoter activity. An expression vector comprising the promoter according to claim 1.

3. The expression vector according to claim 2, wherein The target gene is integrated and expressed under the control of the promoter according to claim 1. A transformant comprising the expression vector according to claim 2 or claim 3. The transformant according to claim 4 , which is an aquatic invertebrate.

6. A method for preparing a target substance encoded by an expression target gene, comprising culturing the transformant according to claim 4.

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