JP2014233223A - Transcriptional regulatory polynucleotide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transcriptional regulatory polynucleotide which enables large quantities of recombinant proteins to be expressed using amphibian oocytes.SOLUTION: This invention provides a transcriptional regulatory polynucleotide having an identity of at least 80% or more to a base sequence composed of a specific sequence of Xenopus laevis and for allowing recombinant proteins to be expressed in amphibian oocytes.

Description

  The present invention relates to a transcription control polynucleotide.

As a method for mass-producing useful proteins, a method using microorganisms such as E. coli and yeast as a host is common.
On the other hand, in the production of antibody drugs, vaccines, etc., a method using a large mammal as a host has been attempted in order to maintain the physiological activity of the protein. However, when using large mammals as hosts, there are technical problems such as the management of genetically modified large mammals, the elimination of pathogens common to humans, and the resistance to using mammals as laboratory animals. There are ethical issues.

On the other hand, amphibians are prolific, and adult females have an extremely large number of eggs. Moreover, it has the advantage that it is easy to breed.
For example, Xenopus can be raised at high density in the laboratory. In addition, a technique for producing a transgenic frog obtained by introducing a foreign gene into Xenopus was established in the 1990s (Non-patent Document 1). A method of expressing the target protein in Xenopus oocytes using this technique is also known (Patent Document 1).
In addition, the base sequence of the genome DNA of Nettles megael was elucidated in the 2000s (Non-patent Document 2).

JP 2003-135065 A

Kroll and Amaya, Development. 122, 3173-3183 (1996) Bowes et al. Nucleic Acid Res. 36 (Data base issue), D716-717 (2008)

  However, no method has been known so far that allows recombinant proteins to be expressed in large quantities in amphibian oocytes.

  An object of the present invention is to provide a transcription control polynucleotide that enables a recombinant protein to be expressed in large quantities using amphibian oocytes.

The present invention is as follows.
<1> A transcription control polynucleotide for expressing a recombinant protein in amphibian oocytes having at least 80% identity to the base sequence shown in SEQ ID NO: 1 in the Sequence Listing.
<2> The transcription control polynucleotide according to <1>, wherein the amphibian is a frog.
<3> The transcription control polynucleotide according to <1> or <2>, wherein the amphibian is Xenopus laevis.
<4> A recombinant vector in which the transcription control polynucleotide according to any one of <1> to <3> is incorporated.
<5> A transformant having a recombinant vector in which the transcription control polynucleotide according to any one of <1> to <3> is incorporated.
<6> A method for producing a physiologically active substance from the transformant according to <5>.
<7> A transgenic organism into which the transcription control polynucleotide according to any one of <1> to <3> is incorporated.
<8> A method for producing a physiologically active substance from the transgenic organism according to <7>.

  ADVANTAGE OF THE INVENTION According to this invention, the transcription | transfer control polynucleotide which makes it possible to express a recombinant protein in large quantities using an amphibian oocyte can be provided.

1 is a schematic diagram of a primer according to Example 1. FIG. 2 is a photograph showing a result of observation with a fluorescence microscope according to Example 1. FIG.

  The transcription control polynucleotide according to the present invention is a polynucleotide having at least 80% identity to the base sequence shown in SEQ ID NO: 1 in the sequence listing and for expressing a recombinant protein in amphibian oocytes. .

  The base sequence shown in SEQ ID NO: 1 in the sequence listing of the present invention is a base sequence contained in the flanking region of the linker histone B4 gene in the nettle megal genome, and is represented by JGI Xenopus tropicalis genome database (ver. 4.1, Scaffold 196: 1642104-1645183 sequence of http://genome.jgi-psf.org/Xentr4/Xentr4.home.html) and a base sequence composed of base numbers 1 to 3080 with 1642104 as base number 1 It is.

The polynucleotide contained in the flanking region of the linker histone B4 gene in the nettle megael genome functions as an enhancer sequence for expressing the recombinant protein in amphibian oocytes in amphibians other than nettle megael. Can be expressed in large quantities in amphibian oocytes.
Although this mechanism is not clear, linker histone B4 is a protein that accumulates in large amounts only in the oocyte, so it needs to be produced in large quantities in frog eggs with a large size, and an expression control system to guarantee it Is presumed to be present in the linker histone B4 gene.

In the present specification, a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In the present invention, the amount of each component in the composition is such that when there are a plurality of substances corresponding to each component in the composition, the plurality of substances present in the composition unless otherwise specified. It means the total amount.
The present invention will be described below.

<Transcriptional control polynucleotide>
The transcription control polynucleotide according to the present invention is a polynucleotide having at least 80% identity to the base sequence shown in SEQ ID NO: 1 in the sequence listing and for expressing a recombinant protein in amphibian oocytes. .

  A transcription control polynucleotide is a polynucleotide that can function as an enhancer sequence for expressing a recombinant protein in amphibian oocytes. Therefore, a larger amount of recombinant protein can be expressed in amphibian oocytes by using a transcription control polynucleotide than when not using a transcription control polynucleotide.

The transcription control polynucleotide is 85% or more identity, 90% or more identity, 95% or more with respect to the base sequence shown in SEQ ID NO: 1 in the sequence listing from the viewpoint of the expression efficiency of the recombinant protein in amphibian oocytes. , Identity of 96% or more, identity of 97% or more, identity of 98% or more, or identity of 99% or more.
When the identity between the transcription control polynucleotide and the nucleotide sequence in SEQ ID NO: 1 in the sequence listing is less than 80%, the expression efficiency of the recombinant protein in the amphibian oocyte is lowered.

  The transcription control polynucleotide also includes a transcription control polynucleotide that hybridizes under stringent conditions to a complementary strand of a base sequence having the same base sequence as SEQ ID NO: 1 in the sequence listing.

Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). This document is hereby incorporated by reference.
Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. Typical stringent conditions include, for example, conditions for performing hybridization in a potassium concentration of about 25 mM to about 50 mM and a magnesium concentration of about 1.0 mM to about 5.0 mM. Examples of the conditions of the present invention include, but are not limited to, conditions for performing hybridization in Tris-HCl (pH 8.6), 25 mM KCl, and 1.5 mM MgCl ₂ . Other stringent conditions are described in Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). This document is hereby incorporated by reference. Those skilled in the art can easily select such conditions by changing the hybridization reaction, the salt concentration of the hybridization reaction solution, and the like.

Furthermore, the transcription control polynucleotide also includes a transcription control polynucleotide in which a base is inserted, deleted or substituted into the base sequence shown in SEQ ID NO: 1 in the Sequence Listing.
A transcription control polynucleotide into which a base has been inserted, deleted or substituted may exhibit an effect equivalent to that of the transcription control polynucleotide according to the present invention. The position of loss or substitution is not particularly limited.
Examples of the number of inserted, deleted or substituted bases include 1 base or 2 bases or more, and for example, 1 base to 10 bases or 1 base to 5 bases may be mentioned depending on the total length of the transcription control polynucleotide.

A transcription control polynucleotide is a polynucleotide that can function as an enhancer sequence for expressing a gene encoding a protein located at a controllable position in amphibian oocytes.
The controllable position includes the 3 ′ end or 5 ′ end of the transcription control polynucleotide. Further, an embodiment in which a gene encoding a protein is bound to the 3 ′ end of the transcription control polynucleotide is more preferable. Thereby, it is speculated that the transcription control polynucleotide can act more functionally.
The gene encoding the protein is not particularly limited. For example, genes encoding proteins such as albumin, blood coagulation factor, virus coat protein and the like can be mentioned.
In the present specification, the recombinant protein means a protein artificially produced by a gene recombination technique.

  There is no particular limitation on the method for binding a gene encoding a protein to the 3 'end of the transcription control polynucleotide. For example, there is a method in which a transcription control polynucleotide and a gene encoding a protein are digested with a restriction enzyme and then bound.

  Amphibians include frogs, newts, salamanders and the like. Among these, Xenopus is preferable in terms of easy breeding and breeding, and there are fewer problems in the management of genetically modified individuals than in so-called domestic animals. The size of the amphibian is not the largest among the frogs, but compared to Japanese native species whose breeding season is decided once a year, Africa that produces eggs regardless of the season Xenopus is preferable because a large number of eggs can be efficiently collected.

Since amphibians are vertebrates, for example, they have the same protein synthesis control mechanism as humans. Therefore, human proteins expressed in amphibian oocytes are presumed to have physiological activity that can function in humans without any modification even when administered to humans.
Amphibians are also very unlikely to have pathogens peculiar to mammals.

<Recombinant vector>
The recombinant vector in the present invention has at least 80% identity to the base sequence shown in SEQ ID NO: 1 in the sequence listing, and incorporates a transcription control polynucleotide for expressing the recombinant protein in amphibian oocytes. Vector.

Moreover, it is preferable that the recombinant vector in the present invention incorporates a transcription control polynucleotide and a gene encoding a protein.
The vector may be a vector that can be expressed in prokaryotic cells such as Escherichia coli (eg, pBR322, pUC119, or derivatives thereof), or a vector that can be expressed in eukaryotes, A vector that can be expressed in a eukaryote is preferred. The vector is preferably a vector that can be expressed in vertebrates, and more preferably a vector that can be expressed in amphibians. It is also preferable to use a vector that can transform amphibian oocytes.

  Examples of vectors that can be expressed in eukaryotes include plasmid vectors such as pBluescript (Stratagene) and pcDNA (Invitrogen), and viral vectors such as pDON-5 (Takara).

The recombinant vector may have a promoter, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), a terminator such as NOS, and the like as necessary.
The promoter is not particularly limited as long as the transcription control polynucleotide can function. For example, an actin promoter, cytokeratin promoter, globin promoter and the like can be mentioned. The origin of the promoter is not particularly limited, but when a viral promoter such as SV40 is used, tissue-specific expression is lost, and non-specific expression in organs and tissues other than the ovary may be observed. Therefore, in the present invention, in order to efficiently confirm the function of the transcription control polynucleotide of the present invention, it is more preferable to use a chick triactin promoter consisting of only a minimal element.
Examples of the selection marker include kanamycin, ampicillin, tetracycline and the like.
The recombinant vector may contain a reporter gene for confirming the introduction of the target gene. Examples of such reporter genes include GUS (β-glucuronidase) gene, luciferase gene, GFP (green fluorescent protein) gene and the like.

  As a method for incorporating the transcription control polynucleotide and the gene encoding the protein into the vector, a known method can be used. For example, a DNA fragment containing a transcription control polynucleotide and a gene encoding a protein is cleaved with an appropriate restriction enzyme and inserted into an appropriate restriction enzyme site downstream of the overexpression promoter in the vector so as to be in-frame. Methods and the like.

<Transformant>
The transformant in the present invention has at least 80% or more identity to the base sequence shown in SEQ ID NO: 1 in the sequence listing, and incorporates a transcription control polynucleotide for expressing the recombinant protein in amphibian oocytes. Possessing the recombinant vector.

  In addition, the transformant in the present invention preferably has a recombinant vector in which a transcription control polynucleotide and a gene encoding a protein are incorporated in a host.

  The host is not particularly limited, but amphibian oocytes are preferably used. When somatic cells (cells other than eggs) are used, it is presumed that the transcription control polynucleotide of the present invention may not function. Therefore, in the present invention, it is more preferable to use an oocyte obtained by surgically removing a stage XI (with a maximum diameter) obtained from an adult female ovary and removing a follicular cell by collagenase treatment. Among them, it is preferable to use Xenopus oocytes.

A transformant can be obtained by introducing a recombinant vector into a host so that a transcription control polynucleotide incorporated in the recombinant vector can function.
The method for introducing the recombinant vector into the host is not particularly limited. In general, a method of injecting 1.0 ng to 5.0 ng of a recombinant vector with a microinjector per amphibian egg can be mentioned.

  Whether or not the recombinant vector has been incorporated into the host can be confirmed by the presence or absence of expression of the reporter gene described in the section of the recombinant vector.

<Transgenic organism>
The transgenic organism has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, and has incorporated therein a transcription control polynucleotide for expressing the recombinant protein in amphibian oocytes It is.

The transgenic organism in the present invention is preferably an organism in which a transcription control polynucleotide and a gene encoding a protein are inserted into a chromosome.
More preferably, the transgenic organism is an amphibian in which a transcription control polynucleotide and a protein-encoding gene are inserted into the chromosome, and the transcription control polynucleotide and the protein-encoding gene are inserted into the chromosome. More preferably, it is Xenopus laevis, in which a transcription control polynucleotide and a gene encoding a protein are inserted into the chromosome.

  In transgenic organisms, a transcriptional regulatory polynucleotide and a protein-encoding gene are inserted into the chromosome, so that a gene encoding a protein bound downstream of the transcriptional regulatory polynucleotide is expressed only in the ovary. Can do.

  Transgenic organisms can be produced by known methods. For example, it can be produced by a method of injecting vector DNA together with a meganuclease into an egg or a method of injecting a sperm nucleus to which vector DNA has been added into an egg. Specifically, Pan et al. (2006) I-SceI meganuclease-mediated transgenesis in Xenopus. Dev. Dyn. 235, 247-252. And the like.

<Method for producing physiologically active substance>
The production method of the present invention is a method for producing a physiologically active substance from a transformant or a transgenic organism.
The production method of the physiologically active substance may include extracting a component containing the physiologically active substance from the cultured transformant or the reared transgenic organism. Furthermore, the method for producing a physiologically active substance preferably includes purifying the objective physiologically active substance from the extracted components.

The culture method or culture conditions for the transformant can be appropriately set according to the type of host.
The medium can be appropriately selected according to the type of host, but it is preferable to use an MMR medium or an MBS medium.
The culture temperature can be appropriately set according to the type of host, but is preferably 18 ° C to 23 ° C.
The culture time can be appropriately set according to the type of host, but is preferably 12 hours to 72 hours.
When Xenopus oocytes are used as the host, they can be cultured at 18 ° C. to 23 ° C. for 12 hours to 72 hours using MMR medium or MBS medium.

The breeding method or breeding conditions of the transgenic organism can be appropriately set according to the type of the transgenic organism.
The breeding temperature can be appropriately set according to the type of the target organism, but is preferably 18 ° C to 23 ° C, more preferably 20 ° C to 23 ° C.
When Xenopus laevis is used as the target organism, it can be fed at 18 ° C. to 23 ° C. with solid food for fish farming or frozen pig liver as food. In addition, for raising larvae, plant food such as green peas and pumpkins can be fed and reared under water circulation by an air pump.

  The method for extracting a component containing a physiologically active substance is not particularly limited, and can be performed by methods known to those skilled in the art. For example, when an oocyte is used as a host for transformation, Evans and Kay (1994) Biochemical fractionation of ootyes. Methods Cell Biol. 36, 133-148. Extraction may be performed according to the method described in the above.

  The method for purifying the target physiologically active substance from the extracted components is not particularly limited, and can be performed by methods known to those skilled in the art. For example, solvent extraction method, ion exchange resin method, adsorption or distribution column chromatography method, gel filtration method, dialysis method, precipitation method, crystallization method, etc. alone or in appropriate combination to purify the desired physiologically active substance can do.

In the present specification, the physiologically active substance specifically means a protein derived from a gene encoding a protein bound to the 3 ′ end of a transcription control polynucleotide.
As the protein, the matters described in the section of the transcription control polynucleotide are applied.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them.

[Example 1]
Using the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing, the flanking region of the B4 gene in the nettle Xenopus genome (JGI Xenopus tropicalis genome database (ver. 4.1, http: //genome.jgi) -The sequence of Scaffold 196: 1642104-1645183 of psf.org/Xentr4/Xentr4.home.html) was obtained by the PCR method.
In the PCR method, DNA amplification by a high-temperature DNA polymerase (Tks Gflex (registered trademark) DNA polymerase, Takara) was performed using a PCR reaction solution described below (composition of 50 μl of PCR reaction solution). Company).

Composition of PCR reaction solution;
Tsk Gflex DNA polymerase 1 μl
2x PCR buffer 25 μl
X. tropicalis genomic DNA 1 μl (300 ng)
Primer 1 (SEQ ID NO: 2) 1 μl (20 pmol)
Primer 2 (SEQ ID NO: 3) 1 μl (20 pmol)
21 μl of H ₂ O

  PCR was performed at 94 ° C. for 60 seconds, followed by 30 cycles of 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 68 ° C. for 90 seconds.

The base sequence described in SEQ ID NO: 1, the chicken triactin promoter (sequence including Accession No. DI075036, Matsuo et al. (1991) Binding of a factor to an enhancer, the first, and the second, the second, the second, and the second. First, a comparative plasmid (promoter / GFP / pBluescript) was prepared using the GFP gene (trade name pEGFP vector, Invitrogen), and a-crystallin gene.Development, 113, 539-550. Subsequently, the plasmid was linearized using the restriction enzyme SmaI site upstream of the promoter, and the B4-3k sequence amplified by PCR was inserted (B4-3k / promoter / GFP / pBluescript). FIG. 1 shows a schematic diagram of the prepared plasmid construct (B4-3k / promoter / GFP / pBluescript).
B4-3k / promoter / GFP and promoter / GFP were each injected into the nuclei of Xenopus laevis oocytes (Watanabe Culture Co., Ltd.) according to a conventional method and cultured for 24 hours using MMR medium.
Then, it observed under the fluorescence microscope (M205FA, Leica company).
The results are shown in FIG. As is clear from FIG. 2, strong expression of GFP protein was confirmed in the oocytes injected with B4-3k / promoter / GFP. In addition, the upper right side of FIG. 2 shows how many oocytes are colored, and the lower right side shows an enlarged photograph of one oocyte.
From the above, it has been clarified that the transcription control polynucleotide can function as an enhancer sequence for expressing a gene encoding a protein linked to the 3 ′ end in amphibian oocytes.

[Example 2]
Instead of GFP used in Example 1, a gene encoding protein A is used and inserted into amphibian oocytes in the same manner as described in Example 1. As a result, protein A can be expressed in amphibian oocytes.

[Example 3]
A transgenic frog in which the transcriptional regulatory polynucleotide set forth in SEQ ID NO: 1 and a gene encoding protein A have been inserted into the chromosome is described in Pan et al. (2006) I-SceI meganuclease-mediated transgenesis in Xenopus. Dev. Dyn. 235, 247-252. It is produced according to the method described in 1. As a result, a female individual expressing protein A in the ovary of a transgenic frog can be produced. The offspring of the female individual can similarly express protein A in the ovary.

Claims (8)

  A transcription control polynucleotide for expressing a recombinant protein in amphibian oocytes having at least 80% identity to the base sequence shown in SEQ ID NO: 1 in the Sequence Listing.   The transcriptional regulatory polynucleotide according to claim 1, wherein the amphibian is a frog.   The transcriptional regulatory polynucleotide according to claim 1 or 2, wherein the amphibian is Xenopus laevis.   A recombinant vector in which the transcription control polynucleotide according to any one of claims 1 to 3 is incorporated.   A transformant having a recombinant vector in which the transcription control polynucleotide according to any one of claims 1 to 3 is incorporated.   A method for producing a physiologically active substance from the transformant according to claim 5.   A transgenic organism into which the transcription control polynucleotide according to any one of claims 1 to 3 is incorporated.   A method for producing a physiologically active substance from the transgenic organism according to claim 7.