JP2021040506A - Expression vectors and methods for producing proteins of interest - Google Patents

Abstract

To provide a vector that enables the production of a protein of interest in a practical amount using a small amount of fertilized fish eggs or embryos in a short period of time, as well as a method for producing a protein of interest using such a vector.SOLUTION: Disclosed is a vector having a structure comprising a promoter sequence to which two or more copies of a gene expression cassette comprising the gene of interest are linked in tandem downstream the promoter sequence. Also disclosed is a method for producing a protein of interest comprising a step of introducing the vector into a fertilized fish egg or embryo.SELECTED DRAWING: None

Description

The present invention relates to a vector for expressing a target protein in fish. Furthermore, the present invention relates to a method for producing a target protein using this vector.

As a method for expressing a recombinant protein, a method for expressing it by microorganisms such as Escherichia coli and yeast, and cultured cells of insects and mammals is known. However, expression methods by microorganisms and insects do not allow accurate folding and sugar chain modification of heterologous proteins, and biologically inactive proteins may be obtained. Since the desired activity of such an inactive protein cannot be obtained, its function and structure cannot be analyzed. In addition, although the expression method using cultured mammalian cells can perform accurate folding and sugar chain modification, it takes time to culture the cells, so that the cost is very high to express a large amount of protein. was there.

It is known that the use of small roe such as zebrafish is useful in the technique for producing a target recombinant protein or the like by inserting a needle into a fertilized egg and injecting a gene or the like.
For example, Patent Document 1 discloses a method for expressing a polypeptide such as factor VII and interleukin in a cell of fish ectoderm tissue or a cell of embryo tissue using an expression construct for expressing a polypeptide in fish. Has been done. Further, Patent Document 2 discloses a method for producing a recombinant protein by introducing a vector having a specific promoter sequence into a fertilized egg or embryo of a fish.
However, although the desired recombinant protein can be produced by these methods, the expression level per fertilized egg is not sufficient, and a large number of fertilized eggs are required to obtain a practical amount. There was a problem.

As a method for highly expressing a recombinant protein, in Patent Documents 3 and 4, a method in which a DNA containing a construct in which a fragment containing a specific sequence is ligated at least twice in tandem is incorporated into a vector is used, or a series including a gene expression cassette is used. A method of preparing a repetitive sequence and introducing a gene into the repetitive sequence is disclosed. However, all of these methods targeted mammalian cells, not fish.
Therefore, the present inventors have developed a vector capable of producing a practical amount of the target protein in a short period of time using a small amount of fertilized fish eggs, embryos, etc., and a method for producing the target protein using this vector. I tried.

Special Table 2001-501482 Gazette JP-A-2007-143497 International Publication No. 2014/045674 Pamphlet Japanese Unexamined Patent Publication No. 2012-157293

An object of the present invention is to provide a vector capable of producing a practical amount of a target protein in a short period of time using a small amount of fertilized eggs or embryos of fish, and a method for producing the target protein using this vector. To do.

As a result of diligent research to solve the above problems, the present inventors have obtained a vector containing a promoter sequence and a structure in which the same gene expression cassette containing the target gene is tandemly linked twice or more downstream thereof. It was produced to complete the present invention.
In fertilized eggs, embryos, etc. into which the vector prepared in the present invention has been introduced, the expression level of the target protein per one increases, so that a sufficiently practical amount of the target protein can be obtained even with the use of a small amount of fertilized eggs, embryos, etc. Is possible.
In the method for producing the target protein of the present invention using this vector, the number of fertilized eggs and the like required for producing the target protein is reduced, so that the amount of genes to be injected can be reduced as compared with the conventional method. There is an advantage that the time required for the injection process can be shortened accordingly.

That is, the present invention relates to the following vectors (1) to (7), a method for producing a target protein, and the like.
(1) A vector for expressing a target protein in fish, which contains a structure in which a promoter sequence and the same gene expression cassette containing the target gene are tandemly linked twice or more downstream thereof.
(2) The vector according to (1) above, wherein the gene expression cassette contains a tag cleavage sequence and a tag sequence.
(3) The vector according to (1) or (2) above, wherein the gene expression cassette further contains a signal sequence.
(4) The vector according to any one of (1) to (3) above, wherein the fish is a zebrafish.
(5) A method for producing a target protein, which comprises a step of introducing the vector according to any one of (1) to (4) above into a fertilized egg or embryo of a fish.
(6) The production method according to (5) above, wherein the fish is zebrafish.
(7) Transgenic fish into which the vector according to any one of (1) to (4) above has been introduced.

The vector of the present invention makes it possible to produce a practical amount of the target protein in a short period of time using a small amount of fertilized fish eggs, embryos, or the like.
Further, since the production method of the present invention can produce a biologically active (native) protein, the base sequence is known in a genomic library or the like by the production method of the present invention, but the structure and function are unknown. By producing and analyzing a protein as a target protein, it becomes easy to screen for useful proteins.

It is a figure which showed the outline of pUpZef-EGFP-pZef-Feutin-A-HRV3C-3xFLAG (registered trademark) vector construction (Example 2). It is a figure which showed the outline of the pUpZef-EGFP-pZef-Feutin-A-HRV3C-3xFLAG (registered trademark) vector (Example 2). ~ It is a figure which showed the outline of the pUpZef-EGFP-pZef-Feutin-A-HRV3C-3xFLAG (registered trademark) vector (Example 2). It is a figure which showed the schematic diagram of the vector (triple tandem vector) containing the structure concatenated in tandem three times (Example 2). In the figure, Amp indicates an ampicillin (antibiotic) resistance gene, pZef indicates a promoter sequence, SV40PA indicates a signal sequence, HRV3C indicates a tag cleavage sequence, and 3 × FLAG® indicates a tag sequence. It is a figure which showed the standard curve used for the quantification of Fetuin-A (Example 3).

The "vector" of the present invention is a vector capable of expressing a target protein in fish, and contains a "gene expression cassette" containing a promoter sequence and the target gene downstream thereof, and the same "gene expression cassette" is used twice. The above refers to a vector containing a structure linked in tandem.
The "same gene expression cassette" refers to a gene expression cassette containing the same promoter sequence and the same target gene. Here, the same promoter sequence is preferably a sequence that completely matches the sequence specified as the base sequence encoding the promoter, but is preferably a sequence having 90% or more identity, and more preferably 95% or more. Any sequence having the same identity may be used, and even if a sequence is partially deleted or substituted, a sequence capable of exerting the same promoter function may be included.
Further, the same target gene is preferably a sequence that completely matches the sequence specified as the base sequence encoding the target protein, but a sequence having 90% or more identity, more preferably 95% or more identity. Any sequence may be used, and even if a sequence is partially deleted, substituted, or the like, a sequence capable of encoding a protein having similar properties may be included.

The "promoter sequence" refers to a sequence that controls the expression of the "target gene", and such a control sequence may be any one that can control the expression of the "target gene". For example, a base sequence encoding all or part of Ef1α can be mentioned, and it may be a base sequence encoding all or part of CMV, and other conventionally known control sequences. It may be.
The "target gene" is a gene encoding the "target protein" to be expressed and produced, and is in the complete biologically active (native) form of the protein to be expressed and produced. It may be a base sequence encoding all that can be expressed, or it may be a base sequence encoding a part thereof. Further, it may encode a mutant such as a derivative, and may encode a polypeptide different in one or more amino acids from the native one. As such a "target gene", for example, a base sequence encoding all or a part of Feutin-A can be mentioned.
These "promoter sequence" and "target gene" may be contained in the "gene expression cassette" in either form of DNA or RNA, but are preferably DNA, particularly genomic DNA or cDNA. Is preferable.

The "gene expression cassette" of the present invention preferably further contains a tag cleavage sequence and a tag sequence, and more preferably contains a signal sequence. Further, in addition to these sequences, other sequences useful for producing the target protein may be contained.
Here, the "tag sequence" refers to a base sequence for detecting, recovering and / or purifying the expression of a target protein. For this "tag sequence", any protein encoding any of the conventionally known proteins can be used. For example, GST (Glutathione S-transferase), GFP (Green Fluorescent Protein) can be used. ), EGFP (Enhanced Green Fluorescent Protein), His-tag, FLAG-tag sugar-binding domain, polysaccharide-binding domain, lectin, protein interaction domain, nucleic acid-binding domain, etc. it can.
Further, the "tag cleaving sequence" refers to a base sequence encoding all or a part of a protein for cleaving a tag from a target protein produced in a state where the tag is bound. As the protein encoded by such a "tag cleavage sequence", any protein encoding any conventionally known protein can be used, and examples thereof include HRV3C and the like.

The "signal sequence" refers to a base sequence encoding all or part of a protein that can increase the translation efficiency of the target gene encoding the target protein. Any of the conventionally known "signal sequences" can be used, for example, a polyadenylation (poly A) signal operably linked from 3'to the nucleic acid coding sequence, eg. Examples thereof include SV40 poly A signal or chinook salmon poly A signal.

Such a "vector" of the present invention is preferably a replicable vector, for example, a replicable expression vector. By replicating the vector designed by the present invention in a host cell such as a bacterial host cell or a yeast host cell, stable introduction into fish becomes possible.
Furthermore, the "vector" of the present invention is a marker that enables selection of, for example, an ampicillin resistance gene, a neomycin resistance gene, or a yeast transformant as a marker gene in order to identify a transformant into which the vector has been introduced. It preferably contains a gene.

In the production of the "vector containing a structure in which the same gene expression cassette is linked twice or more in tandem" of the present invention, the vector used as a base for inserting the "gene expression cassette" produces a target protein in fish. Any vector can be used as long as it can be used. The vector may be in a linear form or in a circular form. Examples of such a vector include the pUC19 vector (Takara Bio Inc.) and the like.
When the vector is defined as "a structure in which the same gene expression cassette is linked in tandem more than once", there is no limit to the length of the base sequence between the inserted "gene expression cassettes", and the inserted "gene expression". It suffices if a "cassette" can be expressed. In addition, any technique such as In-Fusion technique or Ligation technique can be used for inserting the "gene expression cassette".
The "vector" of the present invention may have a structure in which the gene expression cassette is tandemly linked twice or more, and is tandemly linked three or more times, four times or more, five times or more, and even six times or more. Is preferable.

As the "fish" of the present invention, any fish corresponding to fish can be used, but teleost fish are preferable, and the classification is as follows: teleost fish such as Cyprinidae, Cichlidae, Salmonidae, Claridae, Siluridae and Ictaluridae. Can be mentioned.
Particularly preferred fish species are carp (eg Cyprinus carpio), zebrafish (Danino rerio), African catfish (Clarias gariepinus), terapia (Oreochronis niloticus), Atlantic salmon (Salmo salar), rainbow trout (Oncorhyncus mykiss), and medaka. (Oryzias la tips).

The "method for producing a target protein" of the present invention refers to a "vector" of the present invention incorporating all or part of a base sequence encoding this protein, with the protein to be expressed and produced as the "target protein". It refers to expression, recovery and / or purification by introducing into fertilized eggs or embryos of fish.
Such a "method for producing a target protein" of the present invention may be any production method including a "step of introducing the" vector "of the present invention into a fertilized egg or embryo of a fish", and is used for producing the target protein. It may further include useful steps.
Any of the commonly used introduction methods can be used to introduce the vector. For example, the vector can be packaged into retrovirus particles or lambda virus particles and transferred into cells. It can also be introduced by microinjection, electroporation, calcium phosphate precipitation, biological methods (eg, tungsten bombardment), or by contacting a naked nucleic acid vector or construct with cells in solution. Of these, introduction by microinjection is particularly preferable.
The fertilized egg or embryo used in the production method of the present invention can be any fish as long as it is a fish. When the vector of the present invention is introduced by microinjection, it can be carried out in the two-cell, four-cell, or eight-cell stage before fertilization, after fertilization, and after cleavage.

For the recovery of the target protein obtained by the production method of the present invention, any commonly used recovery method can be used. For example, a method of cutting out a tissue or the like in which protein expression has been confirmed and recovering from the tissue, or a method of cutting out a region in which protein expression has been confirmed by Western blotting and extracting from the region can be mentioned.
Further, the recovered protein can be purified by any conventionally known purification method. Conventionally known purification methods include column chromatography and affinity chromatography, and proteins can be purified by using these. Examples of commercially available affinity chromatography include GSTtrap HP and HisTrap HP (both manufactured by Amersham Bioscience).
The produced target protein can be recovered from F0 generation embryos, but it may also be recovered from adult fish.

The "transgenic fish" of the present invention refers to a fish into which a vector containing a structure in which the gene expression cassette of the present invention is tandemly linked two or more times is introduced.

Hereinafter, examples of the present invention will be shown, but the present invention is not limited thereto.

[Example 1]
Preparation of gene expression cassette 1. Gene expression cassette A
The efla1 gene (SEQ ID NO: 1) of zebrafish was used as a promoter sequence, and the Feutin-A gene (SEQ ID NO: 2) was integrated downstream of this as a gene expression cassette A.
2. Gene expression cassette B
In the gene expression cassette A, the HRV3C gene (SEQ ID NO: 3) is incorporated as a tag cleavage sequence downstream of the target gene, and the 3xFLAG® gene (SEQ ID NO: 4) is incorporated as a tag sequence downstream thereof, and the gene expression cassette B is incorporated. And said.
3. 3. Gene expression cassette C
In the gene expression cassette A, the SV40PA gene (SEQ ID NO: 5) was incorporated as a signal sequence downstream of the target gene to form the gene expression cassette C1.
In addition, in the gene expression cassette B, the SV40PA gene (SEQ ID NO: 5) was integrated downstream of the tag sequence to form the gene expression cassette C2.

[Example 2]
Construction of expression plasmid vector 1. Construction of pUpZef-EGFP-pZef-Feutin-A-HRV3C-3xFLAG® vector
Using the pUC Origin and Ampicillin resistance genes of the pUC19 vector (Takara Bio Co., Ltd.) and the primers shown in Table 1, pUpZef-EGFP-pZef-Feutin-A-HRV3C-3xFLAG was used as an expression plasmid vector by In-Fusion technology. A (registered trademark) vector (hereinafter sometimes referred to as vector A) was constructed.
Vector A was able to transcribe Fetuin-A (target protein), which is a fusion of EGFP and 3xFLAG® at the C-terminus, by two ef1a1 (zEF1A) promoter regions. The Fetuin-A gene (target gene) in which the 3xFLAG® gene is fused to the C-terminal can be excised by the restriction enzyme SfiI, and the target gene encoding another protein can be incorporated in place of the Fetuin-A gene. Was designed to. It does not include components of transposable factors such as Transposable elements.
Fig. 1 shows the outline of vector A construction, and Fig. 2 and Fig. 3-1 to 3-8 show the outline of vector A. In addition, the entire base sequence of vector A is shown in SEQ ID NO: 6.

2. Preparation of tandem vector According to the following procedure, a vector containing a structure in which the gene expression cassette is linked twice in tandem (double tandem vector) and a vector containing a structure in which the gene expression cassette is linked three times in tandem (triple tandem vector) are prepared. did. For reference, a schematic diagram of the triple tandem vector is shown in FIG.
In each prepared vector, whether the gene expression cassette is linked twice or three times is confirmed by the length of the gene by re-cutting at the restriction enzyme sites Pac1 and Nde1 or Pac1 and Not1 used for gene insertion. , The presence or absence of mutation at the 1-base level was confirmed by a DNA sequencing service.

1) Dual tandem vector (1) Preparation of expression cassette From vector A, efla1 gene (SEQ ID NO: 1), Feutin-A gene (SEQ ID NO: 2), HRV3C gene (SEQ ID NO: 3), 3xFLAG (registered trademark) gene (sequence) The region containing the No. 4) and SV40PA gene (SEQ ID NO: 5) was excised at the restriction enzyme site Bgl2.
Using this cleaved fragment as a template, Pac1 and the sequence for In-Fusion (SEQ ID NO: 11) are added to the 5'end, and Nde1 and the sequence for In-Fusion (SEQ ID NO: 12) are added to the 3'end to add the expression cassette C2 for insertion. Was prepared.
(2) Preparation of double tandem vector Vector A was cleaved with restriction enzyme sites Pac1 and Nde1, and the expression cassette C2 prepared in (1) above was subjected to In-Fusion® HD Cloning Kit (Takara Bio Inc.). A vector in which the expression cassettes C2 are lined up in two rows (hereinafter, may be referred to as vector B) was prepared by joining them together.

2) Triple tandem vector (1) Preparation of expression cassette Similar to 1) and (1) above, the region containing each gene was excised with the restriction enzyme site Bgl2, and then this cleavage fragment was used as a template for the 5'end and 3'. An expression cassette C2 for insertion was prepared by adding Pac1 and an In-Fusion sequence (SEQ ID NO: 11) to the ends.
(2) Preparation of triple tandem vector The vector B prepared in 1) and (2) above was cleaved with the restriction enzyme site Not1, and the expression cassette C2 prepared in (1) above was used as an In-Fusion® HD Cloning Kit. A vector in which expression cassettes C2 are lined up in triplets (hereinafter, may be referred to as vector C) was prepared by joining them together using (Takara Bio Inc.).

[Example 3]
Production of target protein 1. Growth of zebrafish Zebrafish were bred at a water temperature of 28 ° C, 14 hours in the light period, and 10 hours in the dark period. Fertilized eggs should be collected immediately after birth by an arbitrarily selected healthy male and female pair, and the breeding water should be Holtfreter's Solution (HS) (CaCl ₂ 0.1 g, NaCl 3.5 g, KCl 0.05 g, _{LVDS 3} 0.2 g). It was replaced with (1 L) by adding _{dH 2 O and bred at 28 ° C.}
Unfertilized eggs were defined as pre-spawning embryos, and female ovaries that entered the spawning stage were washed with HS and collected. In addition, embryos of 12, 24 and 48 hpf were defined as embryos after each hour when fertilized eggs collected within 30 minutes including 15 minutes before and after the collection time were set as 0 hpf and bred at 28 ° C in HS.

2. Introduction of expression plasmid vector Each expression plasmid vector (vectors A, B, C) prepared in Example 2 above was introduced into the fertilized egg of 0hpf of zebrafish obtained in 1 above by microinjection. The concentration of the vector at the time of introduction was adjusted to 0.02 μg / μl, and the concentration was approximately 50-200 eggs / μl per fertilized egg. The introduced fertilized zebrafish eggs are bred in HS at 28 ° C, and FLAG® is used by FACS (Light-CaptureII (Ato Co., Ltd.)) using an anti-FLAG® antibody (SIGMA-ALDRICH). Expression was confirmed.

3. 3. Target protein 1) Confirmation of expression level
Fetuin-A contained in EGFP-expressing eggs was quantified by the ELISA method using HUMAN FETUIN-A ELISA (UNITECH). Figure 5 shows the standard curve. The OD was obtained by subtracting the wavelength of 630 nm from the wavelength of 450 nm.

2) Results When Vector A containing only one gene expression cassette was introduced, the expression level of the target protein (Fetuin-A) per fertilized egg was 1.6 ng, but it was tandem more than once. It was 4.9 ng when the vector (vector B) containing the structure linked to was introduced, and 16.4 ng when the vector (vector C) containing the structure linked to the tandem was introduced three or more times.
Therefore, from this result, it was shown that the more the gene expression cassette was included in the vector more than once, the more synergistically the protein expression level increased.

With the vector of the present invention, a practical amount of the target protein can be produced in a short period of time using a small amount of fertilized fish eggs, embryos, or the like. Further, by the production method of the present invention, a useful protein can be screened by producing and analyzing a protein whose base sequence is known in a genome library or the like but whose structure and function are unknown as a target protein. It will be easy.

Claims (7)

A vector for expressing a target protein in fish, which contains a structure in which a promoter sequence and the same gene expression cassette containing the target gene are tandemly linked two or more times downstream thereof. The vector according to claim 1, wherein the gene expression cassette contains a tag cleavage sequence and a tag sequence. The vector according to claim 1 or 2, wherein the gene expression cassette further contains a signal sequence. The vector according to any one of claims 1 to 3, wherein the fish is a zebrafish. A method for producing a target protein, which comprises the step of introducing the vector according to any one of claims 1 to 4 into a fertilized egg or embryo of a fish. The production method according to claim 5, wherein the fish is zebrafish. A transgenic fish into which the vector according to any one of claims 1 to 4 has been introduced.

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