JP2012019719A - MODIFIED Stx2e PROTEIN SUPPRESSED OF SUGAR CHAIN MODIFICATION - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for producing Stx2e B subunit protein using a plant of immunogenic protein usable as a vaccine for porcine edema disease caused by Shiga toxin (Stx)2e, concretely, provide the technique for producing Stx2e B subunit protein suppressed of sugar chain modification.SOLUTION: DNA encoding modified Stx2e B subunit protein prepared by modifying a partial amino acid sequence consisting of Asn-X-Cys at 55-57th positions of an amino acid sequence of Stx2e B subunit protein having a specific sequence is brought to be expressed in plant cells.

Description

  The present invention relates to a technique for producing a plant using an immunogenic protein that can be used in a vaccine for swine edema disease caused by Shiga toxin (Stx) 2e.

  Stx2e is a protein exotoxin produced by enterohemorrhagic Escherichia coli and causes porcine edema disease. Porcine edema disease is highly occurring in piglets 1 to 2 weeks after weaning, and the mortality rate is extremely high, 50 to 90%. It is known that Stx2e consists of a B subunit pentamer involved in cell attachment and a toxic A subunit monomer.

Conventionally, medical proteins used for vaccines and the like have been produced using microorganisms, insect cells, animal cells and the like. As for the Stx2e protein, for example, conventionally, a technique is known in which a detoxified Stx2e protein is produced using recombinant Escherichia coli and administered to a pig by injection (Non-patent Document 1).
However, since such a method is costly because it requires labor for injection, a technique for substituting and supplementing this method has been demanded. As one of such techniques, it has been studied to produce a vaccine using a plant and orally administer the vaccine-producing plant to livestock.

In such a background, the present inventors have developed a technique for causing a plant to efficiently produce a bacterial toxin protein such as Stx2e using a transgenic technique (Patent Documents 1 and 2).
In Patent Document 1, Stx2e B subunit protein to which a plant-derived secretory signal peptide is added at the amino terminus is expressed using a 5′-untranslated region (ADH5′UTR) of a plant-derived alcohol dehydrogenase gene. Discloses a technique for efficiently producing Stx2e B subunit protein in a plant such as lettuce and accumulating it in a plant body at a high concentration.
Patent Document 2 discloses a technique for producing in a plant a protein in which two or three Stx2e B subunit proteins are linked in tandem via a specific peptide, and accumulating the Stx2e B subunit protein in the plant body at a high concentration. Is disclosed.

By the way, in eukaryotes, glycosylation (glycosylation, glycosylation) is one of the important processes of post-translational modification of proteins. As sugar chains added to proteins by sugar chain modification, O-linked sugar chains and N-linked sugar chains are known. Among these, N-linked sugar chains have a common trimannosyl core structure consisting of Man ₃ GlcNAc _2, but the core sugar chain portion of N-linked sugar chains derived from plants is recognized in animal types. There are α-1,3-fucose modifications and β-1,2-xylose modifications unique to plants. It has been pointed out that such plant-specific modifications may become allergens when administered in vivo in animals. In such a background, as a method for reducing or suppressing fucose modification of proteins produced by plants, a plant transformation vector into which a gene fragment that suppresses gene expression encoding an enzyme involved in the synthesis of GDP-fucose is inserted. A technique has been reported in which a plant is introduced into a plant body or a plant cell to transform the plant (Patent Document 3). In addition, as a technique for suppressing α-1,3-fucose modification and β-1,2-xylose modification of proteins produced by plants, a transfer reaction of a galactose residue to a non-reducing terminal acetylglucosamine residue can be performed. A technique for transforming plant cells by introducing an enzyme gene and a heterologous glycoprotein gene has been reported (Patent Document 4).

In general, it is said that an N-linked sugar chain is added to an Asn residue in the primary sequence Asn-Y-Ser / Thr (Y represents an amino acid other than Pro) (Non-patent Document 2).
In yeast and insect cells, there is a report that an N-linked sugar chain is added to an Asn residue in an Asn-X-Cys (X represents an arbitrary amino acid) sequence (non-patent literature). 3-5).

International Publication No. 2009/004842 Pamphlet International Publication No. 2009/133882 Pamphlet JP 2010-51297 A JP 2008-212161 A

Makino et al., Microbial Pathogenesis, vol. 31, Number 1, pp. 1-8 (08), 2001 Lerouge et al., Plant Molecular Biology, vol. 38, pp. 31-48, 1998 Sato et al., J. Biochem. Vol. 127, pp. 65-72, 2000 Wang et al., Yang P, J. Dairy Sci. Vol. 91, pp. 4466-4476, 2008 Borisov et al., Anal. Chem., Vol. 81, pp. 9744-9754, 2009

In the previous research (see Patent Documents 1 and 2), the present inventors have made Stx2e a highly efficient plant.
Although the B subunit protein was successfully produced, it was discovered that a protein having a slightly higher molecular weight was produced together with the Stx2e B subunit protein which is the production target.
As a result of analyzing the protein having a large molecular weight, the present inventors have found that the protein has an N-linked sugar chain. Furthermore, as a result of further searching for the sugar chain modification site of the protein, the present inventors have been reported as a sugar chain modification site in yeast and insect cells, and have been reported as a sugar chain modification site in plants. It was discovered that a sugar chain was bound to the Asn residue of the partial amino acid sequence Asn (asparagine) -X-Cys (cysteine) (X represents any amino acid).

Considering the application of Stx2e protein produced in plants to oral vaccines for domestic animals, the protein produced by plants has a sugar chain added, which is close to the original antigen, ie, the protein produced by E. coli. It is considered desirable to have a structure that does not.
In particular, sugar chains having α-1,3-fucose and β-1,2-xylose peculiar to plants have the possibility of becoming allergens in livestock as described above. When the proteins possessed are mixed, the originally expected Stx2e immunogenicity and quality uniformity may be reduced, so it is desirable to avoid sugar chain modification.

Accordingly, the present invention provides a method for producing a protein in which sugar chain modification is suppressed in a technique for producing a Stx2e B subunit protein using a plant, as well as DNA and recombinants used in the method. The challenge is to provide vectors.
Moreover, this invention makes it a subject to provide the novel protein produced by the said method, the plant containing this, and the pig edema disease vaccine using this novel protein and plant.

  The present inventors modify a partial sequence composed of Asn-X-Cys (X represents an arbitrary amino acid) contained in a Stx2e B subunit protein (also referred to herein as “Stx2eB”). Thus, it was clarified that the sugar chain modification of the Stx2e B subunit protein can be suppressed, and the present invention was completed.

That is, the present invention that solves the above problems is a DNA that encodes a modified Stx2e B subunit protein shown in (A) or (B) below.
(A) Modified Stx2e B subunit protein Asn-X-Cys (X is an arbitrary amino acid sequence) having an amino acid sequence obtained by modifying the following partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 Amino acids are shown.)
(B) an amino acid in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence obtained by modifying the partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 Modified Stx2e B subunit protein Asn-X1-Cys having the sequence and not including the following partial amino acid sequence (X1 represents an amino acid other than Pro or Asp)

  By expressing the DNA in a plant cell, it is possible to cause a plant to produce a protein in which sugar chain modification is suppressed.

  In a preferred form of the invention, the modification of the partial amino acid sequence is a substitution of the amino acid at position 55 of the amino acid sequence represented by SEQ ID NO: 1 with Ser. In another preferred embodiment, the modification of the partial amino acid sequence is substitution of the amino acid at position 57 of the amino acid sequence represented by SEQ ID NO: 1 with Ala.

The preferable form of the DNA of the present invention is that the modified Stx2e B subunit protein shown in (A) and / or (B) above has the following characteristics (x) and (y), respectively. A DNA encoding a hybrid protein linked in tandem via a peptide having a peptide.
(X) 12-30 amino acids (y) Pro content of 20-35%
A preferred form of modification of the partial amino acid sequence is as described above.

  As shown in Patent Document 2, the Stx2e B subunit protein is efficiently expressed in plant cells by expressing DNA encoding a hybrid protein in which Stx2e B subunit protein is linked in tandem with a specific peptide. It is possible to accumulate well. This means that the amount of accumulated Stx2e B protein to which a sugar chain has been added increases. Therefore, in the above technique, if the Stx2e B subunit protein is replaced with the modified Stx2e B subunit protein of the present invention, it is possible to allow the plant to efficiently produce a protein in which sugar chain modification is suppressed.

In a preferred embodiment of the present invention, the DNA comprises the modified Stx2e B subunit protein of (A) or (B), wherein a plant-derived secretory signal peptide (SP) is added to the amino terminus, or the hybrid protein. Code. In another preferred embodiment, the DNA encodes the protein having an endoplasmic reticulum residue peptide or a vacuolar translocation signal peptide added to the carboxyl terminus.
With this form, it is possible to achieve efficient accumulation of the protein in the plant.

  The present invention provides a recombinant vector containing the above-described DNA, and a transformed plant cell or transformed plant transformed with the recombinant vector. The present invention also provides seeds obtained from the transformed plant.

  The present invention also provides a swine edema disease vaccine using the above-described transformed plant cell or transformed plant as a raw material.

The present invention provides a method for producing a protein having Stx2e immunogenicity, which comprises expressing the above DNA in a plant cell.
By this method, it is possible to suppress sugar chain modification, which is a particular problem in the production of Stx2e B subunit protein using plants.

  The present invention also provides the modified Stx2e B subunit protein of the above (A) or (B), or the above hybrid protein, and a plant containing the protein.

  Since the modified plant cell or the transformed plant of the present invention and the protein contained in the plant of the present invention are suppressed in sugar chain modification, allergic reactions in livestock when the plant is used as a raw material for oral vaccine of animals. It is expected to be possible to suppress this.

The present inventors have discovered a sugar chain modification site (partial amino acid sequence) of a type that has not been reported for plants so far for the Stx2e B subunit protein produced in plant cells. And it succeeded in controlling protein not to receive a sugar chain modification by artificially changing the amino acid sequence of this sugar chain modification site.
That is, by expressing the DNA of the present invention in a plant cell, it becomes possible to cause a plant to produce a modified Stx2e B subunit protein in which sugar chain modification is suppressed. The modified Stx2e B subunit protein thus obtained contributes to suppression of allergenicity. Such proteins are also expected to contribute to the expected immunogenicity or quality uniformity.

  Further, in a preferred embodiment of the present invention, which has been developed by the present inventors and applied the technology for producing a Stx2e B subunit protein in plant cells at a high production rate, efficient accumulation of the target protein in the plant and allergenicity are achieved. It is possible to achieve both suppression.

It is a figure which shows the design of the recombinant vector used by the test example. Vector Type 1 contains DNA encoding a protein (2 × Stx2eB-PG12) in which two Stx2e B subunit proteins are linked in tandem via a PG12 spacer (a), and Vector Type 2 contains two Stx2e B subs It contains DNA encoding a protein (2 × Stx2eB-PG12ver2) in which a unit protein is linked in tandem via a PG12ver2 spacer (b), and the vector Type 3 contains two modified Stx2e B subunit proteins in tandem via a PG12ver2 spacer. A DNA encoding a protein (2 × Stx2eBN55S-PG12ver2) linked to (c). Vector Type 4 contains DNA encoding a protein (2 × Stx2eBC57A-PG12ver2) in which two modified Stx2e B subunit proteins are linked in tandem via a PG12ver2 spacer (d). The arrow indicates the translation start point. It is a photograph which shows the detection result of the protein accumulate | stored in the Arabidopsis cultured cell transformant and the lettuce stable transformant obtained by the transformation experiment using the vector Type 1 shown in FIG. 1 (GPF-). Moreover, the detection result when the protein accumulated in the transformant is treated with GPF is also shown (GPF +). Processing time (hour) shows the time processed by GPF. It is a photograph which shows the detection result of the protein accumulate | stored in the Arabidopsis cultured cell protoplast obtained by the transient expression experiment using the vector Type1, Type2, Type3 shown in FIG. 1 (GPF-). Moreover, the detection result when the protein accumulated in the protoplast is treated with GPF is also shown (GPF +). It is a photograph which shows the detection result of the protein accumulate | stored in the Arabidopsis cultured cell protoplast obtained by the transient expression experiment using vector Type2 and Type4 shown in FIG. It is a photograph which shows the detection result of the protein accumulate | stored in each protoplast of rice obtained by the transient expression experiment using vector Type2 and Type3 shown in FIG. 2 is a photograph showing detection results of proteins accumulated in Arabidopsis cultured cell transformants and lettuce stable transformants obtained by transformation experiments using vectors Type 1 and Type 3 shown in FIG. 1. a and b show different clones.

Hereinafter, the form for implementing this invention is demonstrated in detail.
The DNA of the present invention encodes a protein having Stx2e immunogenicity.
In the present invention, “having Stx2e immunogenicity” means inducing an anti-Stx2e antibody when administered to pigs.
In one form of the present invention, the protein encoded by the DNA of the present invention has (A) an amino acid sequence in which the following partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 is modified, It is a modified Stx2e B subunit protein.
Asn-X-Cys (X represents any amino acid)

SEQ ID NO: 1 shows the amino acid sequence of the mature region of the Stx2e B subunit protein (GenBank Accession No. AAQ63639) (Ala19 to Asn87, excluding the secretion signal peptide for the periplasm). Note that positions 55 to 57 of the amino acid sequence shown in SEQ ID NO: 1 correspond to positions 73 to 75 of the amino acid sequence shown in GenBank Accession No. AAQ63639. SEQ ID NO: 2 shows an example of the base sequence of the DNA encoding the Stx2e B subunit protein shown in SEQ ID NO: 1.
In the present specification, “Stx2e B subunit protein” and “Stx2eB” simply refer to the Stx2e B subunit protein consisting of the amino acid sequence of the mature region.

  The modification of the partial amino acid sequence at positions 55 to 57 may be any modification such that the sequence is lost, for example, substitution of amino acids at positions 55 and / or 57, or any of positions 55 to 57. Amino acid deletion, insertion of an amino acid between amino acids at positions 55 to 57, and the like.

The modification is preferably performed so that the change in the three-dimensional structure of the protein due to the modification is small.
For example, when substituting Asn at position 55 with another amino acid, it may be substituted with Ser, Gln, Thr, Asp, and Glu, which are the same hydrophilic amino acids as Asn. Of these, substitution with Ser, Gln, and Thr is preferred, and substitution with Ser is more preferred. The reason why Ser is particularly preferable is that the Stx2 B subunit similar to the Stx2e B subunit has Ser at the position corresponding to the 55th position, so that the change in the three-dimensional structure due to substitution can be reduced. Because it is. SEQ ID NO: 3 shows an amino acid sequence in which Asn at position 55 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with Ser.
In addition, for example, when Cys at position 57 is substituted with another amino acid, the same hydrophilic amino acids as Cys, such as Tyr, Asn, and Gln, and Gly and Ala having a small side chain among hydrophobic amino acids can be mentioned. . SEQ ID NO: 4 shows an amino acid sequence in which Cys at position 57 of the amino acid sequence represented by SEQ ID NO: 1 is substituted with Ala.

In another form of the present invention, the protein encoded by the DNA of the present invention is (B) the partial amino acid sequence (Asn-X-Cys (X is the position of amino acid sequence 55 to 57) of the amino acid sequence represented by SEQ ID NO: 1. And any amino acid.))) Is a modified amino acid sequence having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, and includes the following partial amino acid sequences: There is no modified Stx2e B subunit protein with Stx2e immunogenicity.
Asn-X1-Cys (X1 represents an amino acid other than Pro or Asp)

  That is, even if the modified Stx2e B subunit protein mutant protein shown in (A) above does not contain the sugar chain modification site consisting of the partial amino acid sequence in the other part, the DNA of the present invention Can be a protein encoded by Here, in Kornfeld et al., Ann. Rev. Biochem., 1985, 54: 631-664, when X in Asn-X-Ser / Thr is Pro or Asp, the glycosylation efficiency is low. Is described. Similarly, in Asn-X1-Cys, when X1 is Pro or Asp, it is predicted that sugar chain addition efficiency is low. Therefore, the mutant protein may contain a partial amino acid sequence of Asn-Pro-Cys or Asn-Asp-Cys.

In the description of the modified Stx2e B subunit protein shown in (B) above, “several” is preferably 2 to 10, more preferably 2 to 5, and more preferably 2 to 3.
The modified Stx2e B subunit protein shown in (B) is preferably an amino acid sequence in which the amino acid at position 55 or 57 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with another amino acid, preferably 85% or more, It may be a protein having an identity of 90% or more, more preferably 95% or more.

  The protein encoded by the DNA of the present invention may contain a structure other than the modified Stx2e B subunit protein described above. That is, proteins obtained by adding various peptides to the modified Stx2e B subunit protein are also included in the protein encoded by the DNA of the present invention as long as the addition does not lose Stx2e immunogenicity.

  In addition, the protein encoded by the DNA of the present invention is from Asn-Y-Ser / Thr (Y represents an amino acid other than Pro), which is reported to be a general N-linked sugar chain modification site. It is preferable that the amino acid sequence is not included. However, as shown in the examples described later, in a hybrid protein in which two or three Stx2e B subunit proteins described below are linked in tandem via a specific peptide, Asn-Thr-Ser is N- It has been found that at least the sequence may be included because it does not serve as a modification site for the conjugated sugar chain.

For example, as a protein encoded by the DNA of the present invention, in a hybrid protein in which two or three Stx2e B subunit proteins described in Patent Document 2 are linked in tandem via the following specific peptide, Stx2e B Examples include hybrid proteins having a structure in which at least one of the subunit proteins is replaced with the above-described modified Stx2e B subunit protein.
In a preferred embodiment of the present invention, the DNA of the present invention encodes a hybrid protein having a structure in which two or three Stx2e B subunit proteins are all replaced with modified Stx2e B subunit proteins.

That is, in a preferred embodiment of the present invention, two or three proteins encoded by the DNA of the present invention, each of the above-described modified Stx2e B subunit proteins (A) and / or (B), have the following characteristics: It is a hybrid protein linked in tandem via a peptide having (x) and (y).
(X) The number of amino acids is 12-30 (y) The content of Pro (proline) is 20-35%

The number of amino acids in the peptide is preferably 12-25, more preferably 12-22. The proline content of the peptide is preferably 20 to 27%, more preferably 20 to 25%.
In the peptide, proline is preferably arranged every two or every third. However, even in this case, at the terminal of the peptide, amino acids other than proline may be continuous within 5 or less, preferably 4 or less.

  In the peptide, among amino acids other than proline, the total content of serine, glycine, arginine, lysine, threonine, glutamine, asparagine, histidine and aspartic acid is preferably 70% or more, more preferably 80% or more. More preferably, it is 90% or more. In the peptide, among the amino acids other than proline, the total content of serine, glycine and asparagine is preferably 70% or more, more preferably 80% or more, more preferably 90% or more. In the peptide, the total content of serine and glycine among amino acids other than proline is preferably 70% or more, more preferably 80% or more, and more preferably 90% or more. This is because peptides containing a large amount of these amino acids are unlikely to have a secondary structure (beta sheet structure or helix structure).

On the other hand, in the peptide, among the amino acids other than proline, the total content of alanine, methionine and glutamic acid is preferably 30% or less, more preferably 20% or less, and more preferably 10% or less. This is because a peptide containing many of these amino acids tends to have a helix structure. In the peptide, among amino acids other than proline, the total content of tryptophan, leucine, isoloincin, tyrosine, phenylalanine and valine is preferably 20% or less, more preferably 10% or less, more preferably 5% or less. It is. This is because a peptide containing a large amount of these amino acids tends to take a beta sheet structure and a helix structure.
The peptide is preferably a peptide consisting of an amino acid sequence represented by SEQ ID NO: 5 (PG12 spacer), a peptide consisting of an amino acid sequence represented by SEQ ID NO: 6 (PG12ver2 spacer), an amino acid sequence represented by SEQ ID NO: 7 Or a peptide consisting of the amino acid sequence represented by SEQ ID NO: 8 (PG22 spacer).

The hybrid protein preferably further has the peptide added to the C-terminus.
The hybrid protein encoded by the DNA of the present invention has an amino acid sequence represented by SEQ ID NOs: 9 and 10, for example. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 9, two modified Stx2e B subunit proteins (substitution at position 55) are linked in tandem via a PG12 spacer. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 10, two modified Stx2e B subunit proteins (substitution at position 55) are tandemly linked via a PG12 spacer, and further a PG12 spacer is linked to the C-terminus thereof Has been. Such a hybrid protein may be a modified Stx2e B subunit protein in which position 57 in the amino acid sequence shown in SEQ ID NO: 1 is substituted.
By using the peptide such as the PG12 spacer as a linker for linking the modified Stx2eB protein, the accumulation level of the modified Stx2eB protein in plant cells is increased (see Patent Document 2).

  In addition to the structure of the modified Stx2e B subunit protein, the protein encoded by the DNA of the present invention may include, for example, the structure of a Stx2e A subunit protein. That is, the protein encoded by the DNA of the present invention may be a hybrid protein of a modified Stx2e B subunit protein and a Stx2e A subunit protein. However, when the Stx2e A subunit protein is used, it is preferably detoxified.

  In addition, the protein encoded by the DNA of the present invention may be added with a signal peptide such as a secretory signal peptide endoplasmic reticulum residual signal peptide or a vacuolar translocation signal peptide at the end. Thereby, it becomes possible to optimize the accumulation level of protein according to the plant species which express protein (refer patent document 1).

The protein encoded by the DNA of the present invention has, for example, a plant-derived secretory signal peptide added to the amino terminus.
The secretory signal peptide is preferably a plant belonging to the family of Solanaceae, Brassicaceae, Asteraceae, Onagraceae, more preferably Nicotiana, Arabidopsis, Akinonogesi It is derived from a plant belonging to the genus (Lactuca), Oenothera, etc., more preferably from tobacco (Nicotiana tabacum), Arabidopsis thaliana, lettuce (Lactuca sativa), Oenothera laciniata and the like.
Preferably, it is derived from tobacco β-D-glucan exohydrolase and tobacco 38 kDa peroxidase (GenBank Accession D42064).
Examples of the secretory signal peptide include a peptide having the amino acid sequence represented by SEQ ID NO: 11 derived from tobacco β-D glucan exohydrolase.

Furthermore, the protein encoded by the DNA of the present invention may have a signal peptide such as an endoplasmic reticulum residual signal peptide or a vacuolar transit signal peptide added to the carboxyl terminus. Here, the “addition” is a concept including a case where the signal peptide is directly bonded to the carboxyl terminus of the hybrid protein and a case where it is bonded via another peptide.
The protein encoded by the DNA of the present invention preferably has an endoplasmic reticulum residual signal peptide added to its carboxyl terminus. Examples of the endoplasmic reticulum residual signal peptide include an endoplasmic reticulum residual signal peptide containing the HDEL sequence (SEQ ID NO: 12).

The vacuolar translocation signal peptide is preferably a plant belonging to the family Solanaceae, Brassicaceae, Asteraceae, Onagraceae, more preferably Nicotiana, Arabidopsis. ), Plants belonging to the genus Lactuca, horseradish (Armoracia), Oenothera, etc., more preferably tobacco (Nicotiana tabacum), Arabidopsis thaliana, lettuce (Lactuca sativa), horseradish (Armora) rusticana), Oenothera laciniata, etc. Also preferably derived from chitinase. Also preferably derived from horseradish peroxidase C1a isozyme.

In the base sequence of the DNA of the present invention, the codon indicating the amino acid constituting the protein may be appropriately modified so that the translation amount of the modified Stx2e B subunit protein increases according to the host cell that produces the protein. preferable.
As a codon modification method, for example, the method of Kang et al. (2004) can be referred to. In addition, a method of selecting a codon that is frequently used in the host cell, selecting a codon having a high GC content, or selecting a codon that is frequently used in the housekeeping gene of the host cell.
For example, a DNA having the base sequence represented by SEQ ID NO: 13 in which a codon having a high GC content is selected can be used as the DNA of the present invention.

In a preferred form of the DNA of the present invention, the modified Stx2e protein or the coding region DNA of the hybrid protein is linked to an enhancer so that it can be expressed. Here, “expressible” means that the protein of the present invention is produced in a host cell when the DNA of the present invention is inserted into a vector containing an appropriate promoter and the vector is introduced into an appropriate host cell. Say. In addition, the term “linkage” is a concept that includes a case where two DNAs are directly bonded and a case where they are bonded via another base sequence.
Examples of the enhancer include a Kozak sequence and a 5′-untranslated region of a plant-derived alcohol dehydrogenase gene. Particularly preferably, the DNA encoding the protein is operably linked to the 5′-untranslated region of a plant-derived alcohol dehydrogenase gene.

The 5′-untranslated region of the alcohol dehydrogenase gene refers to a region including the base sequence from the transcription start point of the gene encoding alcohol dehydrogenase to the translation start point (ATG, methionine). This region has a function of increasing the translation amount. The “translation amount increasing function” refers to a function of increasing the amount of protein produced by translation when the information encoded by the structural gene is translated and translated to produce a protein. The region may be derived from a plant, preferably a plant belonging to the family Solanaceae, Brassicaceae, Asteraceae, Onagraceae, more preferably Nicotiana, Plants belonging to the genus Arabidopsis, Lactuca, Oenothera and the like, more preferably tobacco (Nicotiana tabacum), Arabidopsis thaliana, lettuce (Lactuca sativa), Koenyo lac Derived from.
As the 5′-untranslated region of the alcohol dehydrogenase gene, for example, the 5′-untranslated region (NtADH5′UTR) of the alcohol dehydrogenase gene derived from tobacco (Nicotiana tabacum), the nucleotide sequence represented by SEQ ID NO: 14 The area which becomes is mentioned.

  The 5'-untranslated region of a plant-derived alcohol dehydrogenase gene can be isolated from, for example, the alcohol dehydrogenase gene of a plant cultured cell that highly expresses alcohol dehydrogenase (see JP-A-2003-79372). In addition, as for the 5'-untranslated region of tobacco alcohol dehydrogenase gene, etc. whose nucleotide sequence has been determined, genomic DNA is used as a primer for the chemical synthesis or the 5 'and 3' terminal nucleotide sequences of the region. It can also be synthesized by PCR or the like using as a template. Further, by using a part of the region whose base sequence is determined as a probe, it is possible to search for and isolate the 5'-untranslated region of the alcohol dehydrogenase gene of another plant.

Further, the 5′-untranslated region of the alcohol dehydrogenase gene represented by the nucleotide sequence of SEQ ID NO: 14 is substituted or deleted with one or several bases as long as it retains the function of increasing the translation amount. You may have insertions or additions. The “several” is preferably 2 to 10
The number is more preferably 2 to 5, particularly preferably 2 to 3.
Further, DNA having the identity of the 5′-untranslated region of the alcohol dehydrogenase gene, preferably 85% or more, particularly preferably 90% or more, and having the function of increasing the translation amount may be used. .

  Whether or not the region has the desired function of increasing the amount of translation can be determined by, for example, a transient assay using a GUS (β-glucuronidase) gene or a luciferase gene as a reporter gene in a cultured tobacco cell, or a transformed cell integrated into a chromosome. This can be confirmed by the assay.

  As such a base sequence, the 5'-untranslated region (SEQ ID NO: 15) of the ATG neighborhood sequence modified tobacco alcohol dehydrogenase gene can be used.

The DNA of the present invention has a base sequence represented by SEQ ID NOs: 16 to 18, for example.
The DNA having the base sequence represented by SEQ ID NO: 16 contains two modified Stx2e B subunits in the 5′-untranslated region (NtADH5′UTRmod, SEQ ID NO: 15) of the alcohol dehydrogenase gene derived from the ATG neighborhood modified tobacco. This is DNA obtained by ligating DNA encoding a hybrid protein in which a protein (substitution at position 55) is tandemly linked through a PG12 spacer.

  DNA having the nucleotide sequence represented by SEQ ID NO: 17 is linked to NtADH5'UTRmod with two modified Stx2e B subunit proteins (substitution at position 55) in tandem via a PG12 spacer, and a secretory signal at the amino terminus The peptide is a DNA in which a DNA encoding a hybrid protein in which an endoplasmic reticulum residual signal peptide is added to the carboxyl terminus is linked.

  The DNA having the nucleotide sequence represented by SEQ ID NO: 18 is obtained by linking two Stx2eB subunit proteins (substitution at position 55) to NtADH5'UTRmod in tandem via a PG12 spacer, and a secretory signal peptide at the amino terminus. It is a DNA in which a PG12 spacer is added to the carboxyl terminus and a DNA encoding a hybrid protein in which an endoplasmic reticulum residual signal peptide is added to the carboxy terminus.

The DNA of the present invention described above can be prepared by a general genetic engineering technique. If necessary, the DNA encoding the modified Stx2e B subunit protein is added to the plant-derived alcohol dehydrogenase gene 5. '-DNA such as DNA encoding non-translated region, plant-derived secretory signal peptide, DNA encoding endoplasmic reticulum residual signal peptide may be cleaved with an appropriate restriction enzyme and ligated with an appropriate ligase.
In addition, when synthesizing a DNA encoding a hybrid protein in which a modified Stx2e B subunit protein and a hybrid protein of another protein or a modified Stx2e B subunit protein are linked in tandem via the specific peptide, each protein is synthesized. The DNA encoding can be ligated with the reading frame aligned except for the stop codon. For example, a DNA encoding a hybrid protein in which two modified Stx2e B subunit proteins are linked in tandem via the specific peptide, for example, a DNA encoding the modified Stx2e B subunit protein shown in SEQ ID NO: 13; It can be synthesized by ligating the PG12 spacer shown in SEQ ID NO: 5 with the reading frame aligned except for the stop codon.

DNA encoding the modified Stx2e B subunit protein can be synthesized by PCR using appropriate primers. For example, Stx2 described in the embodiment
e In the synthesis of DNA encoding the B subunit protein, the base sequence of the primer used may be changed so as to encode the target amino acid sequence.
In addition, as shown in the Examples, a plasmid containing DNA encoding Stx2e B subunit protein is used as a template, and an inverse PCR is performed using appropriate primers to define a codon corresponding to the target amino acid. Thus, the base substitution can be performed.

The recombinant vector of the present invention is characterized by containing the DNA of the present invention. The recombinant vector of the present invention may be inserted into the vector so that the DNA of the present invention can be expressed in plant cells into which the vector is introduced. Examples of the vector include binary plasmid DNA and viral DNA for stably incorporating a gene into a plant genome. The former vector preferably contains a selection marker such as a drug resistance gene, and commercially available plasmids such as pRI909, pBI121, pBI101, and pIG121Hm can also be used. As the viral DNA, for example, pTB2 (Donson et al., 1991) can be used (Donson J., Kerney CM., Hilf ME., Dawson WO. Systemic expression of
See Proc. Natl. Acad. Sci. (1991) 88: 7204-7208. a bacterial gene by a tobacco mosaic virus-based vector. )

  The promoter used in the recombinant vector can be appropriately selected according to the plant cell into which the vector is introduced. For example, cauliflower mosaic virus 35S promoter (Odell et al. 1985 Nature 313: 810), rice actin promoter (Zhang et al. 1991 Plant Cell 3: 1155), maize ubiquitin promoter (Cornejo et al. 1993 Plant Mol. Biol . 23: 567) is preferably used. Similarly, the terminator used in the vector can be appropriately selected according to the host cell into which the vector is introduced. For example, nopaline synthase gene transcription terminator, cauliflower mosaic virus 35S terminator, Arabidopsis heat shock protein 18.2 gene terminator (HSP-T) and the like are preferably used.

The recombinant vector of the present invention can be prepared, for example, as follows.
First, the DNA of the present invention is cleaved with an appropriate restriction enzyme or a restriction enzyme site is added by PCR and inserted into a restriction enzyme site or a multiple cloning site of a vector.

The transformed plant cell or transformed plant of the present invention is characterized by being transformed with the recombinant vector of the present invention.
Plant cells and plant types to be transformed are not particularly limited, but belong to the family Solanaceae, Brassicaceae, Asteraceae, Onagraceae, Chenopodiaceae Plants, more preferably plants belonging to the genus Tobacco (Nicotiana), Arabidopsis (Arabidopsis), Lactuca, Oenothera, etc., more preferably Nicotiana tabacum, Arabidopsis thaliana, lettuce ( Lactuca sativa), Oenothera laciniata, etc. are preferably used.

The transformed plant cell of the present invention can be prepared by introducing the recombinant vector of the present invention into a plant cell using a general genetic engineering technique. For example, the introduction method using Acrobacterium (Hood, et al., 1993, Transgenic, Res. 2: 218, Hiei, et al., 1994 Plant J. 6: 271), electroporation method (Tada, et al. al., 1990, Theor.Appl.Genet, 80: 475), polyethylene glycol method (Lazzeri, et al., 1991, Theor. Appl. Genet. 81: 437), particle gun method (Sanford, et al., 1987) , J. Part. Sci.
tech. 5:27), polycation method (Ohtsuki) and the like can be used.
After introducing the recombinant vector of the present invention into a plant cell, the transformed plant cell of the present invention can be selected according to the phenotype of the selection marker. Moreover, the target protein can be produced by culturing the selected transformed plant cells. The medium and conditions used for the culture can be appropriately selected according to the species of the transformed plant cell.
In addition, the transformed plant cell of the present invention can be obtained by regenerating the plant body by culturing the selected transformed plant cell according to a conventional method, and the intended purpose is inside the plant cell or outside the cell membrane of the plant cell. Protein can be accumulated. As a method of regenerating a plant body, for example, the method of Visser et al. (Theor. Appl. Genet 78: 594 (1989)) can be cited for potato, and Nagata for tobacco can be used. Takebe (Planta 99:12 (1971)) is the method.

  In the case of lettuce, shoot regeneration is possible with MS medium containing 0.1 mg / l NAA (naphthalene acetic acid), 0.05 mg / l BA (benzyladenine) and 0.5 g / l polyvinylpyrrolidone. Rooting is possible by culturing in 1/2 MS medium containing g / l polyvinylpyrrolidone.

  The seeds of the present invention can be obtained by collecting seeds from plants regenerated as described above. The seed of the present invention can be sown and cultivated by an appropriate method to produce a plant that produces the modified Stx2e B subunit protein or hybrid protein. Included in convertible plants.

  The porcine edema disease vaccine of the present invention is characterized by using the transformed plant cell or transformed plant of the present invention as a raw material. The pig edema disease vaccine of the present invention can be a transformed plant itself that produces the above-described modified Stx2e B subunit protein or hybrid protein. However, the transformed plant cell or transformed plant is dried, pulverized, or the like. It can also be produced by processing together with other components. Moreover, the pig edema disease vaccine of this invention can also be mix | blended with the adjuvant which raises the immunogenicity of the said protein. In general, aluminum hydroxide or the like is used as an adjuvant in consideration of safety.

The method for controlling porcine edema disease of the present invention is characterized by administering the porcine edema disease vaccine of the present invention to pigs. Immunization with the pig edema disease vaccine of the present invention is preferably performed on piglets with a high incidence of edema disease. As a method of immunization, the pig edema disease vaccine of the present invention is administered to a mother pig, and the antibody produced by the mother pig is given to the piglet by milk, the pig edema disease vaccine of the present invention is administered to the piglet, A method of directly immunizing a piglet is mentioned.
Examples of the method for administering the pig edema disease vaccine of the present invention to pigs include a method in which the pig edema disease vaccine of the present invention is mixed with pig feed and given to pigs, and a method of nasally irrigating pigs.

Hereinafter, the present invention will be described in more detail with reference to examples.
[Test Example 1] Analysis of post-translational modification of Stx2e B subunit protein In order to analyze post-translational modification of Stx2e B subunit protein (Stx2eB, SEQ ID NO: 1), Arabidopsis Cultured cells and lettuce cotyledon fragments were transformed, and proteins accumulated in the transformants were analyzed.
(1) Construction of Vector Type 1 Vector Type 1 containing DNA in which DNAs encoding two Stx2eBs were linked via DNA encoding PG12 spacer (SEQ ID NO: 5) was prepared as follows. The design of the DNA is shown in FIG.

A method for producing a vector is shown below.
First, based on the base sequence shown in SEQ ID NO: 2, a codon-modified DNA encoding Stx2eB was designed so as to increase the GC content (SEQ ID NO: 19). Next, primers having the following six types of base sequences were prepared based on the base sequence shown in SEQ ID NO: 19.
Stx2eB A: SEQ ID NO: 20
Stx2eB B: SEQ ID NO: 21
Stx2eB C: SEQ ID NO: 22
Stx2eB D: SEQ ID NO: 23
Stx2eB E: SEQ ID NO: 24
Stx2eB F: SEQ ID NO: 25

  Using the primers prepared above, PCR was performed according to the following procedure to synthesize codon-modified DNA encoding Stx2eB. That is, 1st PCR was performed using a combination of primers A and B, C and D, and E and F to synthesize gene fragments of 92 bp (A + B), 78 bp (C + D), and 86 bp (E + F), respectively. Next, 2nd PCR was performed using a combination of A + B and C + D, and a gene fragment of 153 bp (A + B + C + D) was synthesized. Finally, 3rd PCR was performed with a combination of A + B + C + D and E + F, and finally a 210 bp gene was synthesized. The reaction conditions were such that 10 pmol oligonucleotide, 0.2 mM dNTP, pfu DNA polymerase buffer, 0.3 U pfu DNA polymerase were added to a final volume of 25 μl, heated at 94 ° C. for 5 minutes, then at 94 ° C. for 1 minute, 56 The heat treatment was carried out at 25 ° C. or 30 ° C. for 1 minute, 72 ° C. for 1 minute, and finally heated at 72 ° C. for 5 minutes. For 2nd PCR and 3rd PCR, 1 μl of the reaction solution after 1st PCR and 2nd PCR was used as a template, respectively. The obtained DNA fragment was introduced into the HincII site of pUC118 (plasmid 1).

The 5 ′ untranslated region (NtADH 5 ′ UTRmod, SEQ ID NO: 15) of the ATG neighborhood sequence modified tobacco alcohol dehydrogenase gene was transferred to ADH-221 (Sato et. Al., J. Biosci. Bioeng., Vol. 98, pp. 1-8, 2004) was used as a template and amplified by PCR using ADH XbaIKpnI-F primer (SEQ ID NO: 26) and ADHmod NsiI-R primer (SEQ ID NO: 27). βD NsiI-F primer (SEQ ID NO: 28) encoding a secretory signal peptide (SP, SEQ ID NO: 11) of β-D glucan exohydrolase (GenBank Accession No. AB017502) using tobacco genomic DNA as a template No. 29) and βD BamHI-R primer (SEQ ID NO: 30). The obtained NtADH 5'UTRmod and each of the secretory signal peptide DNA fragments were treated with NsiI (TOYOBO), ligated using ligation high (TOYOBO), the ends were blunted, and pBluescript II SK (Stratagene) In the EcoRV gap (plasmid 2).
Subsequently, plasmid 2 was treated with NsiI, the ends were blunted with T4 DNA polymerase (manufactured by TOYOBO), self-ligated, and the start codon (atg) of NtADH 5'UTRmod and the start codon of the secretory signal peptide were matched. Were ligated (plasmid 3).

  Next, the ligated DNA of NtADH 5′UTRmod fragment and secretory signal peptide was amplified using plasmid 3 as a template and using ADH XbaIKpnI-F primer (SEQ ID NO: 26) and βD BamHI-R primer (SEQ ID NO: 30). . The obtained DNA fragment was treated with KpnI and BamHI and inserted into the KpnI-BamHI gap of plasmid 1 (plasmid 4).

  Subsequently, in order to add an endoplasmic reticulum residual signal (SEQ ID NO: 12), the HDEL-F primer (SEQ ID NO: 31) and the HDEL-R primer (SEQ ID NO: 32) were annealed, phosphorylated with T4 PNK, AP) (TaKaRa) was inserted into the BglII gap of plasmid 4 dephosphorylated (plasmid 5).

  An HA tag was added as a peptide tag for detecting Stx2eB. For addition of the HA tag, the HA-F primer (SEQ ID NO: 33) and the HA-R primer (SEQ ID NO: 34) were annealed and phosphorylated with T4 PNK. The obtained phosphorylated HA fragment was inserted into the BglII gap of plasmid 5 (plasmid 6).

  In order to insert a PG12 spacer (SEQ ID NO: 5) between Stx2eB and the HA tag, PG12-F primer (SEQ ID NO: 35) and PG12-R primer (SEQ ID NO: 36) were annealed and phosphorylated with T4 PNK. The obtained phosphorylated DNA fragment was inserted into the BglII gap of plasmid 6 (plasmid 7).

  A DNA fragment (1 × Stx2eB-PG12) in which the DNA encoding Stx2eB and the DNA encoding the PG12 spacer were ligated was excised from plasmid 7 using BamHI-BglII and inserted into the BglII gap of plasmid 7 (plasmid 8).

The cauliflower mosaic virus 35S RNA promoter (35S pro.) Fragment was PCR amplified using 35S XbaI-F primer (SEQ ID NO: 37) and 35S SmaIKpnI-R primer (SEQ ID NO: 38), and pRI909 (TaKaRa Bio Inc.) It inserted into the XbaI-KpnI site (plasmid 9).
The Arabidopsis heat shock protein 18.2 gene terminator region (HSP-T) fragment was PCR amplified using HSPT-SacI-F primer (SEQ ID NO: 39) and HSPT-SpeIEcoRI-R primer (SEQ ID NO: 40), and plasmid 9 It was inserted into the SacI-EcoRI gap (plasmid 10).
A DNA fragment corresponding to NtADH5'UTRmod-SP-2 × (Stx2eB-PG12) -HA-HDEL was excised from plasmid 8 using KpnI-SacI, inserted into the KpnI-SacI gap of plasmid 10, and two Stx2eB were added to PG12 Vector Type 1 containing DNA encoding the hybrid protein 2 × Stx2eB-PG12 linked in tandem via a spacer was obtained (FIG. 1 (a)).

(2) Transformation experiment (i) Production of transformed Agrobacterium Using the produced vector Type 1, Agrobacterium tumefacience EHA105 (Hood et al., Transgenic Res., Vol. 2, pp. 208-218, 1993).
A single colony of EHA105 was added to 5 ml of YEB medium (Bacto-peptone 5 g / l, Beaf extract 5 g / l, Yeast extract 1 g / l, sucrose 5 g / l, MgSO ₄・ 7H ₂ O 0.5 g / l ) And cultured with shaking at 28 ° C. overnight. This culture was inoculated into 500 ml of YEP medium and cultured with shaking at 28 ° C. until the turbidity at 600 nm reached 0.5. The culture is collected by centrifugation (5000 rpm, 10 minutes, 4 ° C; BECKMAN JLA-10,500 rotor), the supernatant is discarded, 500 ml of sterile water is added to suspend the cells, and the suspension is centrifuged again. The cells were collected by separation (5000 rpm, 10 minutes, 4 ° C; BECKMAN JLA-10,500 rotor) and the supernatant was discarded. After this operation was repeated twice, 20 ml of chilled sterilized 10% glycerol was added to the precipitate and suspended. The cells were transferred to a Nalgen tube, collected by centrifugation (5000 rpm, 10 min, 4 ° C. BECKMAN JLA-10,500 rotor), and the supernatant was discarded. The precipitate was suspended in 3 ml of chilled sterilized 10% Glycerol, dispensed 40 μl each into a 1.5 ml microcentrifuge tube, frozen in liquid nitrogen, and stored at −80 ° C.
After thawing competent cells in ice, 1-2 μl of Vector Type 1 solution was added and transferred to an iced 2 mm cuvette. An electric pulse (2.5 KV, 25 μF, 400 Ω) was applied by an electroporator (BIO RAD, Gene Pulser), and the recombinant vector was introduced. Add 1 ml of SOC medium and shake culture at 28 ° C for 1 hour. Spin down and remove most of the supernatant. Suspend the cells in the remaining medium and place on an LB agar medium containing appropriate antibiotics. Spread and incubate at 30 ° C for 2 nights.

(Ii) Preparation of stable lettuce transformant The seeds of lettuce (Lactuca sativa L. cv. Green wave) were surface sterilized and then seeded in 1/2 MS medium containing 3% sucrose solidified with 0.8% agar. The cells were cultured and germinated at 25 ° C. for 16 hours in the light period to 8 hours in the dark period. A cotyledon was cut into a size of about 0.5 cm square to prepare a fragment, which was floated on 12 ml of MS liquid medium containing 1% (w / v) Tween 20. 3 ml of an Agrobacterium culture solution that had been cultured until the optical density (OD) reached about 0.1 and acetosyringone with a final concentration of 100 μM were added and allowed to stand for 10 minutes. The cotyledons from which excess liquid has been wiped off are added to the selection medium (0.05 mg / l
MS medium containing BA, 0.1 mg / l NAA, 0.5 g / l PVP, 50 mg / l kanamycin, 250 mg / l cefotaxime was allowed to stand in a medium solidified with 0.8% agar). The cells were cultured under conditions of 25 ° C., 16 hours light period to 8 hours dark period, and replaced with a new selection medium every week. 4-8 weeks later, the redifferentiated individuals were transferred to a rooting medium (MS medium containing 0.5 g / l PVP, 50 mg / l kanamycin, 250 mg / l cefotaxime solidified with 0.8% agar). did.

(Iii) Transformation of Arabidopsis cultured cells The Arabidopsis cultured cells were cultured in a 300 ml flask containing 100 ml of modified LS medium at 22 ° C. with continuous light period. Every week, 2 ml of suspension culture was inoculated into fresh modified LS medium. 500 μl of agrobacterium cultured in 5 ml of LB medium containing 100 mg / l of kanamycin at 28 ° C. overnight and 100 μM of acetosyringone at a final concentration of 100 μM of Arabidopsis cultured cell suspension on day 4 of culture 100 Added to ml and shake cultured for 2 days. In order to remove Agrobacterium, the culture solution was transferred to a 50 ml centrifuge tube and centrifuged (500 rpm, 5 minutes, 25 ° C.), and the supernatant was removed. 250
Modified LS medium containing mg / l carbenicillin was added and centrifuged (500 rpm, 5 minutes, 25 ° C.) to wash the cells. This operation was repeated 4 times, and cultured cells from which Agrobacterium had been removed were added to 100 ml of a modified LS medium containing 250 mg / l carbenicillin and cultured with shaking for 2 days. The culture solution was transferred to a 50 ml centrifuge tube and centrifuged (500 rpm, 5 minutes, 25 ° C.), and a part of the supernatant was removed to adjust the cell concentration to about 1/2 v / v. Modifications containing 250 mg / l carbenicillin and 40 mg / l kanamycin
The cells were seeded on LS agar medium and allowed to stand at 22 ° C. under continuous light period and cultured. About 2-3 weeks later, the callus cells were transplanted to a new plate, and proliferating clones were selected. The cells were transferred to a modified LS medium containing 250 mg / l carbenicillin and 40 mg / l kanamycin and subcultured.

(3) GPF treatment The transformed Arabidopsis cultured cells and lettuce cotyledon fragments obtained above were crushed in liquid nitrogen and mixed with 1 ml of 10 mM Tris-HCl pH 8.0 per 1 g of wet weight. The obtained mixed solution was centrifuged (15,000 rpm, 20 minutes, 4 ° C.), and the supernatant was collected (GPF untreated sample). A part of the collected supernatant was treated with glycopeptidase F (GPF, TaKaRa Bio Inc.), an enzyme that cleaves N-linked sugar chains from proteins fundamentally (GPF-treated sample).

(4) Western analysis SDS-sample buffer was added to the GPF untreated sample and GPF-treated sample obtained above so that the cell wet weight would be 5 μl per mg, and heat-denatured at 95 ° C for 10 minutes for analysis. A sample was used. After separating the protein using a 15% acrylamide gel, the protein was blotted onto a PVDF membrane (Hybond-P; Amersham) using an electrotransfer apparatus. Stx2eB was detected using an anti-HA antibody (No. 11 867 423 001, Roche).

The results are shown in FIG. Signals were detected at positions of 17 kDa (band 1), 20 kDa (band 2), and 23 kDa (band 3) using both Arabidopsis cultured cells and lettuce stable transformants.
When the protein sample was treated with GPF and then Western analysis was performed, the signal intensity of bands 2 and 3 decreased and the signal intensity of band 1 increased.
Therefore, band 2 corresponds to a protein in which an N-linked sugar chain is added to one of two Stx2eB-PG12 in 2 × Stx2eB-PG12, and band 3 corresponds to two Stx2eB-PG12 in 2 × Stx2eB-PG12. The band 1 was considered to be 2 × Stx2eB-PG12 with no N-linked sugar chain added.

[Test Example 2] Search for sugar chain modification site Next, the N-linked sugar chain modification site of Stx2eB-PG12 was analyzed.
(1) Construction of Stx2e B recombinant vector (a) Construction of vector Type 1 Vector Type 1 was constructed by the method of Test Example 1 (FIG. 1 (a)).
(B) Construction of Vector Type 2 Generally, N-linked sugar chains are said to be added to Asn residues in the sequence Asn-Y-Ser / Thr (Y is an amino acid other than Pro). (Non-patent document 2). This sequence does not exist in the amino acid sequence of Stx2eB, but the sequence of Asn-Arg-Ser (Asn is the C-terminal amino acid of Stx2eB, followed by the amino acid sequence of PG12 spacer) at the junction of PG12 spacer and Stx2eB Exists. In order to verify whether an N-linked sugar chain is added to this Asn residue, a PG12ver2 spacer (sequence) in which Ser (position 2 of the amino acid sequence shown in SEQ ID NO: 5) is replaced with Ala is present at this junction. A vector Type 2 containing DNA encoding a hybrid protein in which two Stx2eBs were linked via No. 6) was prepared by the following method (FIG. 1 (b)).

  Using the vector Type 1 obtained above as a template, PCR was performed using the ADH KpnI-F primer (SEQ ID NO: 26) and the PG12ver2-R primer (SEQ ID NO: 41), and NtADH5′UTRmod-SP-2 × Stx2eB-PG12ver2 The corresponding DNA fragment was amplified. DNA obtained by treating the obtained DNA fragment with KpnI and BglII, introducing it into the KpnI-BglII site of vector Type 1, and encoding the hybrid protein 2 × Stx2eB-PG12ver2 in which two Stx2eBs are linked in tandem via the PG12ver2 spacer A recombinant vector containing (vector Type 2) was obtained.

(C) Construction of Vector Type 3 In yeast and insect cells, Asn-X-CysX represents any amino acid. There is a report that an N-linked sugar chain is added to the Asn residue in the sequence (Non-Patent Documents 3 to 5). The primary sequence of Stx2eB contains the sequence Asn-Thr-Cys.
In order to verify the presence or absence of N-linked sugar chain addition to this Asn-Thr-Cys, in the vector Type 2 obtained above, the Asn-Thr-Cys sequence present in Stx2eB (55 to 57 of SEQ ID NO: 1) A vector Type 3 containing DNA in which Asn of the sequence corresponding to the position was substituted with Ser was prepared by the following method (FIG. 1 (c)).

In the following manner, a vector containing a DNA (SEQ ID NO: 13) encoding a modified Stx2eB (hereinafter, Stx2eBN55S) in which Asn at position 55 of the amino acid sequence of Stx2eB shown in SEQ ID NO: 1 was replaced with Ser was prepared.
First, using the above plasmid 2 as a template, inverse PCR is performed using the Stx2eBN55S-F primer (SEQ ID NO: 42) and the Stx2eBN55S-R primer (SEQ ID NO: 43) to define Asn at position 55 of the amino acid sequence of Stx2eB. The base constituting the codon was replaced (plasmid 11).
Subsequently, in order to insert a PG12 spacer downstream of Stx2eBN55S, PG12-F primer (SEQ ID NO: 35) and PG12-R primer (SEQ ID NO: 36) were annealed and phosphorylated with T4 PNK. The obtained phosphorylated DNA fragment was inserted into the BglII gap of plasmid 11 (plasmid
12). Perform PCR using plasmid 12 as a template and Stx2eB A primer (SEQ ID NO: 20) and PG12ver2-R primer (SEQ ID NO: 41) to define the Ser of Asn-Arg-Ser sequence at the junction of Stx2eBN55S and PG12 spacer The base sequence was replaced with a base sequence defining Ala, and the obtained DNA fragment was introduced into the HincII site of pUC118 (plasmid 13).
A DNA fragment corresponding to Stx2eBN55S-PG12ver2 was excised from plasmid 13 using BamHI and BglII, and this DNA fragment was inserted into the BamHI site of plasmid 13 to join two DNA fragments corresponding to Stx2eBN55S-PG12ver2 (plasmid 14 ). A DNA fragment corresponding to 2 × Stx2eBN55S-PG12ver2 was excised from plasmid 14 using BamHI and BglII, and inserted into the BamHI-BglII gap of vector Type 1 (vector Type 3).

(D) Construction of Vector Type 4 In Asn-Y-Ser / Thr (Y is an amino acid other than Pro), which is a typical N-linked glycosylation sequence, the third Ser or Thr is important. Has been shown to work. The third Cys is also expected to be important in N-linked glycosylation of the Asx-Thr-Cys sequence of Stx2eB. In order to verify this, the vector Type 2 obtained above contains DNA in which Cys of Asn-Thr-Cys (sequence corresponding to positions 55 to 57 of SEQ ID NO: 1) present in Stx2eB is replaced with Ala. Vector Type 4 was produced by the following method (FIG. 1 (d)).
In the following manner, a vector containing a DNA encoding a modified Stx2eB (hereinafter, Stx2eBC57A) in which Cys at position 57 in the amino acid sequence of Stx2eB shown in SEQ ID NO: 1 was replaced with Ala was prepared. Inverse PCR was performed using plasmid 2 as a template and Stx2eBC57A-F primer (SEQ ID NO: 44) and Stx2eBC57A-R primer (SEQ ID NO: 45) to replace the base corresponding to Cys at position 57 (plasmid 15). Subsequently, in order to insert a PG12 spacer downstream of Stx2eB5C57A, the PG12-F primer (SEQ ID NO: 35) and the PG12-R primer (SEQ ID NO: 36) were annealed and phosphorylated with T4 PNK. The obtained phosphorylated DNA fragment was inserted into the BglII gap of plasmid 15 (plasmid 16). Using Plasmid 16 as a template, PCR was performed using Stx2eB A primer (SEQ ID NO: 20) and PG12ver2-R primer (SEQ ID NO: 41), and the Asn-Arg-Ser sequence in the linker region was replaced with Asn-Arg-Ala. The obtained DNA fragment was introduced into the HincII site of pUC118 (plasmid 17).
A DNA fragment corresponding to Stx2eBC57A-PG12ver2 was excised from plasmid 17 using BamHI and BglII, and this DNA fragment was inserted into the BamHI site of Plasmid 17 to connect two DNA fragments corresponding to Stx2eBC57A-PG12ver2 (plasmid 18). A DNA fragment corresponding to 2 × Stx2eBC57A-PG12ver2 was excised from plasmid 18 using BamHI and BglII and inserted into the BamHI-BglII gap of vector Type 1 (vector Type 4).

(2) Transient expression experiment (i) Preparation of protoplasts A cultured cell suspension of Arabidopsis thaliana was centrifuged at 40 g for 5 minutes to recover the cultured cells. The collected cultured cells are immersed in a 50 ml protoplastase solution (1.0% cellulose RS (Yakult Honsha), 0.25% macerozyme R-10 (Yakult Honsha), 400 mM mannitol, 8 mM CaCl ₂ , and 5 mM Mes-KOH, pH 5.6). And shaken at room temperature for about an hour and a half. Subsequently, the protoplast suspension was centrifuged at 60 g for 5 minutes to precipitate the protoplast. Protoplasts were resuspended in an aqueous solution containing 167 mM mannitol and 133 mM CaCl ₂ and centrifuged at 40 g for 5 minutes. Protoplasts were resuspended in an aqueous solution containing 333 mM mannitol and 66.7 mM CaCl ₂ and centrifuged at 40 g for 5 minutes. Protoplasts were suspended in W5 solution (154 mM NaCl, 125 mM CaCl ₂ , 5 mM KCl, 2 mM Mes-KOH, pH 5.6) and left on ice for 1 hour. The protoplast suspension was centrifuged at 40 g for 5 minutes and suspended in MaMg solution (400 mM mannitol, 15 mM MgCl ₂ , and 4 mM Mes-KOH, pH 5.6) so that the protoplast concentration was 2 × 10 ⁶ cells / ml. .

(Ii) Introduction of Recombinant Vector After the vectors Type 1, Type 2, Type 3 and Type 4 prepared above were mixed with 120 μl of protoplast suspension, 140 μl of PEG solution (400 mM mannitol, 100 mM Ca (NO ₃ ) ₂ , and 40% PEG) were added and mixed gently and incubated for 7 minutes. Over about 20 minutes, 1 ml of W5 solution was added to the protoplast suspension. To the protoplasts precipitated by centrifugation, 1 ml of a solution prepared by mixing 400 mM mannitol and W5 solution in a ratio of 4: 1 was added. 0.5 ml of LS medium containing 1% sucrose, 400 mM mannitol and 0.3 mM carbenicillin was added to the protoplasts precipitated by centrifugation, and cultured at 25 ° C. in the dark for 24 hours.

(3) GPF treatment The cultured cells obtained above were crushed using liquid nitrogen and subjected to GPF treatment in the same manner as in Example 1.

(4) Western analysis Western analysis was performed in the same manner as in Example 1. The results are shown in FIGS.
When vector Type 1 was introduced, two bands of 17 kDa (band 1) and 20 kDa (band 2) were detected, and the 23 kDa band was below the detection limit (FIG. 3). GP protein sample
When Western analysis was performed after F treatment, band 2 disappeared and only band 1 was detected. From this, it was found that N-linked sugar chains were added to 2 × Stx2eB-PG12 even in transient expression experiments. Even when vector Type 2 was expressed, two bands of band 1 and band 2 were detected (FIG. 3). Therefore, it is considered that Asn at the Stx2eB C-terminal is not the main modification site. On the other hand, when vector Type 3 was expressed, only band 1 was detected (FIG. 3). From this, it is considered that an N-linked sugar chain is added to Asn (position 55 of SEQ ID NO: 1) in the Asn-Thr-Cys sequence of Stx2eB. At the same time, it was found that N-linked glycosylation can be avoided by substituting Asn in the Asn-Thr-Cys sequence with Ser. In addition, when vector Type 4 was expressed, only band 1 was detected (FIG. 4). From this, it was found that the third Cys residue is important in N-linked glycosylation, and at the same time, introduction of mutation into this Cys can avoid the addition of N-linked glycosylation.

[Test Example 3] Transient expression experiment using various plant cells Transient expression of vector type 2 and vector type 3 using protoplasts made from cultured rice cells, tobacco cultured cells, and leaves of Japanese evening primrose The experiment was conducted.

(1) Transient expression experiment (i) Preparation of protoplast A protoplast suspension was prepared in the same manner as in Test Example 2 using rice cultured cells, tobacco cultured cells, and leaf of primrose.

(Ii) Introduction of Recombinant Vector After the vectors Type 2 and Type 3 prepared above were mixed with 120 μl of protoplast suspension, 140 μl of PEG solution (400 mM mannitol, 100 mM Ca (NO ₃ ) ₂ , and 40 % PEG) and gently mixed and incubated for 7 minutes. Over about 20 minutes, 1 ml of W5 solution was added to the protoplast suspension. To the protoplasts precipitated by centrifugation, 1 ml of a solution prepared by mixing 400 mM mannitol and W5 solution in a ratio of 4: 1 was added. 0.5 ml of LS medium containing 1% sucrose, 400 mM mannitol and 0.3 mM carbenicillin was added to the protoplasts precipitated by centrifugation, and cultured at 25 ° C. in the dark for 24 hours.

(2) Western analysis Western analysis was performed in the same manner as in Test Example 1. The results are shown in FIG.
In both cases, when vector Type 2 was introduced, two bands of 17 kDa (band 1) and 20 kDa (band 2) were detected, but when vector type 3 was expressed. Only band 1 was detected. Therefore, in various plant species, N-linked sugar chains are added to Asn in the Asn-Thr-Cys sequence, and at the same time, by replacing Asn in the Asn-Thr-Cys sequence with Ser, N -It was found that conjugated glycosylation can be avoided.

[Test Example 4] Transformation experiment using various plant cells The usefulness of Vector Type 3 in which addition of N-linked sugar chains was not observed in transient expression experiments was demonstrated using Arabidopsis cultured cells and lettuce cotyledon fragments. This was confirmed by the transformation experiment used.

(1) Transformation In the same manner as in Test Example 1, Arabidopsis cultured cells and lettuce cotyledon fragments were transformed.

(2) Western analysis Western analysis was performed in the same manner as in Test Example 1. The results are shown in FIG.
In the transformant of vector Type 3, only a signal corresponding to band 1 of 17 kDa was detected. From this, it was found that N-linked glycosylation can also be avoided in the transformant by substituting Asn (position 55 of SEQ ID NO: 1) in the Asn-Thr-Cys sequence of Stx2eB with Ser.

  The present invention is useful for developing a porcine edema disease vaccine.

Claims (14)

DNA encoding the modified Stx2e B subunit protein shown in (A) or (B) below.
(A) Modified Stx2e B subunit protein Asn-X-Cys (X is an arbitrary amino acid sequence) having an amino acid sequence obtained by modifying the following partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 (Indicates amino acid)
(B) an amino acid in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence obtained by modifying the partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 Modified Stx2e B subunit protein Asn-X1-Cys having a sequence and not including the following partial amino acid sequence (X1 represents an amino acid other than Pro or Asp) Two or three modified Stx2e B subunit proteins shown in (A) and / or (B) below are linked in tandem via peptides having the following characteristics (x) and (y), respectively: DNA encoding a hybrid protein.
(A) Modified Stx2e B subunit protein Asn-X-Cys (X is an arbitrary amino acid sequence) having an amino acid sequence obtained by modifying the following partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 (Indicates amino acid)
(B) an amino acid in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence obtained by modifying the partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 Modified Stx2e B subunit protein Asn-X1-Cys having a sequence and not including the following partial amino acid sequence (X1 represents an amino acid other than Pro or Asp)
(X) 12-30 amino acids (y) Pro content of 20-35%   The DNA according to claim 1 or 2, wherein the modification of the partial amino acid sequence is substitution of the amino acid at position 55 with Ser.   The DNA according to any one of claims 1 to 3, wherein the modification of the partial amino acid sequence is substitution of the 57th amino acid with Ala.   The DNA according to any one of claims 2 to 4, which encodes the hybrid protein having a plant-derived secretory signal peptide added to the amino terminus.   The DNA according to any one of claims 2 to 5, which encodes the hybrid protein having an endoplasmic reticulum residual peptide or a vacuolar translocation signal peptide added to a carboxyl terminus.   A recombinant vector comprising the DNA according to any one of claims 1 to 6.   A transformed plant cell or transformed plant transformed with the recombinant vector according to claim 7.   A seed obtained from the transformed plant according to claim 8.   A pig edema disease vaccine using the transformed plant cell or transformed plant according to claim 8 as a raw material. Including expressing the DNA according to any one of claims 1 to 6 in a plant cell,
A method for producing a protein having Stx2e immunogenicity. The modified Stx2e B subunit protein of the following (A) or (B).
(A) Modified Stx2e B subunit protein Asn-X-Cys (X is an arbitrary amino acid sequence) having an amino acid sequence obtained by modifying the following partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 (Indicates amino acid)
(B) an amino acid in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence obtained by modifying the partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 Modified Stx2e B subunit protein Asn-X1-Cys having a sequence and not including the following partial amino acid sequence (X1 represents an amino acid other than Pro or Asp) Two or three modified Stx2e B subunit proteins shown in (A) and / or (B) below are linked in tandem via peptides having the following characteristics (x) and (y), respectively: Hybrid protein.
(A) Modified Stx2e B subunit protein Asn-X-Cys (X is an arbitrary amino acid sequence) having an amino acid sequence obtained by modifying the following partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 (Indicates amino acid)
(B) an amino acid in which one or several amino acids are substituted, deleted, inserted, or added in the amino acid sequence obtained by modifying the partial amino acid sequence at positions 55 to 57 of the amino acid sequence represented by SEQ ID NO: 1 Modified Stx2e B subunit protein Asn-X1-Cys having a sequence and not including the following partial amino acid sequence (X1 represents an amino acid other than Pro or Asp)
(X) 12-30 amino acids (y) Pro content of 20-35%   A plant which retains the protein according to claim 12 or the hybrid protein according to claim 13 in a cell.