Bacterial Toxin Vaccine

Abstract

A bacterial toxin protein such as a Shiga toxin protein is efficiently produced using plant cells. The plant cells are transformed using a DNA construct containing DNA encoding a hybrid protein in which the bacterial toxin proteins such as the Shiga toxin proteins are tandemly linked through a peptide having the following characteristics (A) and (B) to produce the bacterial toxin protein in the plant cells: (A) a number of amino acids is 12 to 30; and (B) a content of proline is 20 to 35%.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. application Ser. No. 12/990,597, filed, Nov. 1, 2010, which is a 35 U.S.C. .sctn.371 National Stage patent application of International patent application PCT/JP2009/058345, filed on Apr. 28, 2009, which claims priority to Japanese patent application JP 2008-120573, filed on May 2, 2008.

TECHNICAL FIELD

[0002] The present invention relates to a hybrid protein used for vaccines for diseases caused by bacterial toxins such as Shiga toxins, cholera toxins, and Escherichia coli heat-labile toxins, and a DNA construct for producing the hybrid protein.

BACKGROUND ART

[0003] Shiga toxins (Stxs, verotoxins) are proteinous exotoxins produced by enterohemorrhagic Escherichia coli of pathogenic Escherichia coli species. The Shiga toxins cause hemorrhagic enteritis, hemolytic uremic syndrome, encephalopathy, and the like.

[0004] The Shiga toxins are broadly classified into Stx1 and Stx2, each of which is further classified into subclasses. An example of the Stx2 includes Stx2e which causes swine edema disease. The swine edema disease is known to frequently occur in baby pigs one to two weeks after weaning. A fatality due to infection with edema disease bacteria is 50 to 90%, which is extremely high.

[0005] Further, cholera toxins (CTs) are proteinous exotoxins produced by Vibrio cholerae. The CTs are known to cause severe diarrhea and emesis.

[0006] Still further, Escherichia coli heat-labile toxins (LTs) are proteinous endotoxins produced by enterotoxigenic Escherichia coli. The LTs are known to cause diarrhea and emesis.

[0007] The bacterial toxins, Stxs, LTs, and CTs are all known to include a B-subunit pentamer involved in adhesion to cells and an A-subunit monomer having a toxicity. The LTs and the CTs are also known to be similar structurally and functionally.

[0008] As a method of preventing the diseases caused by those bacterial toxins, methods of administering a vaccine by an injection or a nasal spray and administering the vaccine orally are known.

[0009] For example, a technology where an attenuated Stx2e protein is produced using recombinant Escherichia coli and administered to pigs by an injection is known (Non-patent Document 1). However, for example, an amount of the attenuated Stx2e protein produced by the recombinant Escherichia coli is not sufficient and the administration of the vaccine by an injection requires human labor. This has been a problem.

[0010] Further, the method of administering the vaccine orally draws increasing attention in terms of reducing the labor in a stockbreeding field. In such a context, a technology where the bacterial toxin protein is produced by plants using a transgenic technology has been being developed. For example, a transgenic plant containing DNA encoding an LT protein B subunit (LTB) and expressing the DNA has been described (Patent Documents 1 and 2). A transgenic plant expressing DNA encoding the LT protein or the CT protein has been also described (Patent Document 3). However, there has been a problem that the amount of the produced protein is not sufficient in those technologies. An example of producing the LTB in Lactuca sativa has been reported (Non-patent Document 2). In this study, a gene of the LT protein B subunit including modified codons is expressed in Lactuca sativa using both a cauliflower mosaic virus 35S RNA promoter (CaMV35S) which is a promoter expressed highly in a plant and Kozak sequence which is an enhancer. As a result, it has been reported that the LT protein B subunit is accumulated in an amount of about 2.0% by mass of a total soluble protein of Lactuca sativa. However, this extent of the accumulated protein is thought to be insufficient to efficiently prevent a bacterial disease by utilizing the transgenic plant. That is, it is necessary to efficiently produce and accumulate the target bacterial toxin protein in plant cells.

[0011] The inventors of the present invention found that the Stx2e protein could be produced efficiently in a plant such as Lactuca sativa and accumulated at a high concentration in a plant body by expressing the Stx2e protein where a secretory signal peptide derived from a plant had been added to its amino terminus, using a 5'-untranslated region (ADH 5'-UTR) of an alcohol dehydrogenase gene derived from a plant, and filed the patent (Patent Document 4). [0012] [Patent Document 1] JP 10-507916 A [0013] [Patent Document 2] JP 2000-166411 A [0014] [Patent Document 3] JP 2002-533068 A [0015] [Patent Document 4] WO 2009/004842 A1 [0016] [Non-patent Document 1] Makino et al., Microbial Pathogenesis, Volume 31, Number 1, July 2001, pp. 1-8(08) [0017] [Non-patent Document 2] Kim et al., Protein Expression and Purification, Volume 51, Number 1, January 2006, pp. 22-27(06)

SUMMARY OF INVENTION

[0018] It is an object of the present invention to more efficiently produce a Stx protein and other bacterial toxin proteins having a conformation similar thereto using plant cells.

[0019] Through production of a hybrid protein in which two or three bacterial toxin proteins such as Stx2e and CT are tandemly linked through a peptide having a particular sequence in plant cells, the inventors of the present invention have succeeded in accumulating the bacterial toxin protein at a high concentration in the plant cells and completed the present invention.

[0020] The present invention is as follows.

(1) a hybrid protein, in which two or three of Shiga toxin proteins, cholera toxin proteins, or Escherichia coli heat-labile toxin proteins are each tandemly linked through a peptide having the following characteristics (A) and (B):

[0021] (A) the number of amino acids is 12 to 30; and

[0022] (B) the content of proline is 20 to 35%;

(2) the hybrid protein according to Item (1), in which the peptide further has the following characteristic (C):

[0023] (C) proline is allocated every two or three amino acids;

(3) the hybrid protein according to Item (2), in which the peptide has an amino acid sequence represented by SEQ ID NO: 2, 82, or 84; (4) the hybrid protein according to Item (3), in which two of the Shiga toxin proteins, cholera toxin proteins, or Escherichia coli heat-labile toxin proteins are tandemly linked through the peptide having the amino acid sequence represented by SEQ ID NO: 2; (5) the hybrid protein according to any one of Items (1) to (4), in which the Shiga toxin proteins are Shiga toxin protein B subunits; (6) the hybrid protein according to any one of Items (1) to (5), in which the Shiga toxin proteins are Stx2e proteins; (7) the hybrid protein according to any one of Items (1) to (4), in which the cholera toxin proteins are cholera toxin protein B subunits; (8) the hybrid protein according to Item (4), including an amino acid sequence represented by SEQ ID NO: 10, 12, 14, or 16; (9) the hybrid protein according to Item (3), including an amino acid sequence represented by SEQ ID NO: 86, 88, 90, 92, 94, 96, 98, or 100; (10) the hybrid protein according to any one of Items (1) to (9), in which a secretory signal peptide derived from a plant is added to its amino terminus; (11) the hybrid protein according to Item (10), in which an endoplasmic reticulum retention signal peptide is added to its carboxyl terminus; (12) the hybrid protein according to any one of Items (1) to (9), in which a chloroplast transit signal peptide is added to its amino terminus; (13) a DNA construct, including DNA encoding the hybrid protein according to any one of Items (1) to (12); (14) the DNA construct according to Item (13), including DNA having a base sequence represented by SEQ ID NO: 9, 11, 13, or 15; (15) the DNA construct according to Item (13), including DNA having a base sequence represented by SEQ ID NO: 85, 87, 89, 91, 93, 95, 97, or 99; (16) the DNA construct according to any one of Items (13) to (15), in which DNA encoding a hybrid protein is operably-linked to a 5'-untranslated region of an alcohol dehydrogenase gene derived from a plant; (17) the DNA construct according to Item (16), in which the 5'-untranslated region of the alcohol dehydrogenase gene derived from the plant is derived from Nicotiana tabacum; (18) a DNA construct according to Item (17), including a base sequence represented by any one of SEQ ID NOS: 24 to 29; (19) a DNA construct according to Item (17), including a base sequence represented by any one of SEQ ID NOS: 101 to 111; (20) a recombinant vector, including the DNA construct according to any one of Items (13) to (19); (21) a transformant transformed with the recombinant vector according to Item (20); (22) a transformant according to Item (21), in which the transformant is a transformed plant cell or a transformed plant; (23) a seed, which is obtained from the transformant according to Item (21) or (22); and (24) a peptide, having an amino acid sequence represented by SEQ ID NO: 2, 82, or 84.

BRIEF DESCRIPTION OF DRAWINGS

[0024] In the accompanying drawings:

[0025] FIG. 1 is a view illustrating a design of Stx2eB expression vectors, in which an arrow denotes a translation initiation site, and an inverted triangle denotes a site to be cleaved after the translation (Figure discloses `HDEL` as SEQ ID NO: 20);

[0026] FIG. 2 is a photograph illustrating levels of accumulated Stx2eB obtained in a transient expression experiment using Lactuca sativa protoplasts;

[0027] FIG. 3 is a photograph illustrating levels of accumulated CTB obtained in a transient expression experiment using the Lactuca sativa protoplasts;

[0028] FIG. 4 is a view illustrating a design of DNA constructs encoding an Stx2eB-YFP fusion protein, in which an arrow denotes a translation initiation site, and an inverted triangle denotes a site to be cleaved after the translation (Figure discloses `HDEL` as SEQ ID NO: 20);

[0029] FIG. 5 is a photograph illustrating localization of the Stx2eB-YFP fusion protein obtained in a transient expression experiment using cultured tobacco cell protoplasts;

[0030] FIG. 6 is a photograph illustrating localization of the Stx2eB-YFP fusion protein obtained in a transient expression experiment using the cultured tobacco cell protoplasts;

[0031] FIG. 7 is a photograph illustrating localization of vacuole-type GFP and the Stx2eB-YFP fusion protein in co-expression of ARF1pWT and ARF1pDN obtained in a transient expression experiment using the cultured tobacco cell protoplasts;

[0032] FIG. 8 is a photograph illustrating the levels of accumulated Stx2eB obtained in a transformation experiment using cultured tobacco cells (BY2), in which a numeral in each lane denotes a clone number;

[0033] FIG. 9 is a photograph illustrating the levels of accumulated Stx2eB obtained in a transformation experiment using the cultured tobacco cells (BY2), in which the numeral in each lane denotes the clone number;

[0034] FIG. 10 is photographs illustrating the levels of accumulated Stx2eB obtained in a transformation experiment using the cultured Tobacco cells (BY2), in which the numeral in each lane denotes the clone number;

[0035] FIG. 11 is a graph illustrating the levels of accumulated Stx2eB obtained in a transformation experiment using the cultured tobacco cells (BY2), in which each numeral denotes the clone number;

[0036] FIG. 12 is a graph illustrating a relationship between mRNA levels of Stx2eB and the levels of accumulated Stx2eB;

[0037] FIG. 13 is a view illustrating the design of DNA constructs encoding Stx2eB, in which A, B, and C denote the designs of an endoplasmic reticulum type, a cytoplasm type, and a chloroplast type of the DNA constructs, respectively (Figure discloses `HDEL` as SEQ ID NO: 20);

[0038] FIG. 14 is a view illustrating the design of DNA constructs encoding CTB, in which A, B, and C denote the designs of an endoplasmic reticulum type, a cytoplasm type and a chloroplast type of the DNA constructs, respectively (Figure discloses `HDEL` as SEQ ID NO: 20);

[0039] FIG. 15 is a photograph illustrating the levels of accumulated Stx2eB obtained in a transient expression experiment using the Lactuca sativa protoplasts;

[0040] FIG. 16 is a photograph illustrating the levels of accumulated CTB obtained in a transient expression experiment using the Lactuca sativa protoplasts;

[0041] FIG. 17 is photographs illustrating the levels of accumulated Stx2eB obtained in a transformation experiment using the cultured tobacco cells (BY2), in which the numeral in each lane denotes the clone number;

[0042] FIG. 18 is photographs illustrating the levels of accumulated Stx2eB obtained in a transformation experiment using a tobacco plant body, in which the numeral in each lane denotes the clone number;

[0043] FIG. 19 is a photograph illustrating the levels of accumulated Stx2eB obtained in a transformation experiment using the tobacco plant body, in which the numeral in each lane denotes the clone number;

[0044] FIG. 20 is a view illustrating the design of DNA constructs encoding Stx2eB (Figure discloses `HDEL` as SEQ ID NO: 20);

[0045] FIG. 21 is a view illustrating the design of DNA constructs encoding CTB (Figure discloses `HDEL` as SEQ ID NO: 20);

[0046] FIG. 22 is photographs illustrating the levels of accumulated Stx2eB obtained in a transformation experiment using the cultured tobacco cells (BY2); and

[0047] FIG. 23 is a photograph illustrating the levels of accumulated CTB obtained in a transformation experiment using the cultured tobacco cells (BY2).

DESCRIPTION OF EMBODIMENTS

[0048] In a hybrid protein of the present invention, two or three of Shiga toxin (Stx) proteins, cholera toxin (CT) proteins, or Escherichia coli heat-labile toxin (LT) proteins are each tandemly linked through a peptide having the following characteristics (A) and (B):

[0049] (A) the number of amino acids is 12 to 30; and

[0050] (B) the content of proline is 20 to 35%.

[0051] Shiga toxins (Stxs) are classified into type 1 (Stx1) and type 2 (Stx2). The Stx1 is further classified into subclasses a to d, and the Stx2 is further classified into subclasses a to g, respectively. The Shiga toxin includes one A subunit which is a toxin main body and five B subunits involved in invasion into intestinal mucosa.

[0052] Of those, for example, Stx2e is known as a swine edema disease toxin, and its A subunit (Stx2eA) is represented by an amino acid sequence of SEQ ID NO: 4 and its B subunit (Stx2eB) is represented by an amino acid sequence of SEQ ID NO: 6.

[0053] In Stx2eA and Stx2eB, one or several amino acids may be substituted, deleted, inserted, or added in the amino acid sequences represented by SEQ ID NO: 4 or 6 as long as they can cause an immune response by administering to pigs. For example, the term "several" means the number of preferably 2 to 30, more preferably 2 to 20, and still more preferably 2 to 10, in Stx2eA, and means the number of preferably 2 to 10, more preferably 2 to 5, and still more preferably 2 to 3, in Stx2eB.

[0054] Further, Stx2eA and Stx2eB may be those having an identity of preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more to the amino acid sequences represented by SEQ ID NOS: 4 and 6, and being capable of causing the immune response by administering to the pig.

[0055] The cholera toxin (CT) includes one A subunit (CTA) which is the toxin main body and five B subunits (CTB) represented by SEQ ID NO: 8 and involved in the invasion into intestinal mucosa.

[0056] In CTB, one or several amino acids may be substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 8 as long as CTB can cause the immune response by administering to animals. The term "several" means preferably 2 to 10, more preferably 2 to 5, and still more preferably 2 to 3.

[0057] Further, CTB may be those having an identity of preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more to the amino acid sequences represented by SEQ ID NO: 8, and being capable of causing the immune response by administering to the animals.

[0058] The Escherichia coli heat-labile toxin (LT) protein includes one A subunit which is the toxin main body and five subunits involved in the invasion into intestinal mucosa.

[0059] The Shiga toxin, the cholera toxin, and the Escherichia coli heat-labile toxin are also collectively referred to as "bacterial toxins" herein.

[0060] The number of the amino acids in the peptide is preferably 12 to 25 and more preferably 12 to 22. The content of proline in the peptide is preferably 20 to 27% and more preferably 20 to 25%.

[0061] Further, proline is allocated preferably every two or three residues in the peptide. But, in this case, the amino acids other than proline may be consecutive within 5 residues and preferably 4 residues in the terminus of the peptide.

[0062] In addition, the total content of serine, glycine, arginine, lysine, threonine, glutamine, asparagine, histidine, and aspartic acid in the amino acids other than proline is preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more in the peptide. Further, the total content of serine, glycine, and asparagine in the amino acids other than proline is preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more in the peptide. Still further, the total content of serine and glycine in the amino acids other than proline is preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more in the peptide. This is because the peptide containing those amino acids abundantly is hard to form a secondary structure (.beta.-sheet structure and helix structure).

[0063] Meanwhile, the total content of alanine, methionine, and glutamic acid in the amino acids other than proline is preferably 30% or less, more preferably 20% or less, and still more preferably 10% or less in the peptide. This is because the peptide containing those amino acids abundantly easily forms the helix structure. The total content of tryptophan, leucine, isoleucine, tyrosine, phenylalanine, and valine in the amino acids other than proline is preferably 20% or less, more preferably 10% or less, and still more preferably 5% or less in the peptide. This is because the peptide containing those amino acids abundantly easily forms the .beta.-sheet structure and the helix structure.

[0064] The peptide is preferably selected from the peptide (PG12) having the amino acid sequence represented by SEQ ID NO: 2, the peptide (PG17) having the amino acid sequence represented by SEQ ID NO: 82, and the peptide (PG22) having the amino acid sequence represented by SEQ ID NO: 84.

[0065] In the hybrid protein of the present invention, two or three hybrid proteins of the A subunit and the B subunit may be tandemly linked through the above peptide, or two or three A subunits may be tandemly linked through the peptide, or two or three B subunits may be tandemly linked through the peptide. When the hybrid protein contains the A subunit, the A subunit is preferably attenuated. In the hybrid protein of the present invention, preferably two or three B subunits are tandemly linked through the peptide. In the hybrid protein of the present invention, preferably two B subunits are tandemly linked through PG12.

[0066] Further, in the hybrid protein of the present invention, the peptide is preferably added to its C terminus. PG12 is particularly preferably added to its C terminus in the hybrid protein of the present invention.

[0067] The hybrid protein of the present invention has the amino acid sequence represented by SEQ ID NO: 10, 12, 14, 16, 86, 88, 90, 92, 94, 96, 98, or 100, for example. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 10, two Stx2eBs are tandemly linked through PG12. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 12, two CTBs are tandemly linked through PG12. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 14, two Stx2eBs are tandemly linked through PG12 and further PG12 is linked to the C terminus thereof. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 16, two CTBs are tandemly linked through PG12 and further PG12 is linked to the C terminus thereof. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 86, three Stx2eBs are each tandemly linked through PG12. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 88, three Stx2eBs are each tandemly linked through PG12 and further PG12 is linked to the C terminus thereof. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 90, two Stx2eBs are tandemly linked through PG17 and further PG12 is linked to the C terminus thereof. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 92, two Stx2eBs are tandemly linked through PG22 and further PG12 is linked to the C terminus thereof. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 94, three CTBs are each tandemly linked through PG12. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 96, three CTBs are each tandemly linked through PG12 and further PG12 is linked to the C terminus thereof. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 98, two CTBs are tandemly linked through PG17 and further PG12 is linked to the C terminus thereof. In the hybrid protein having the amino acid sequence represented by SEQ ID NO: 100, two CTBs are tandemly linked through PG22 and further PG12 is linked to the C terminus thereof.

[0068] By using the peptide such as PG12, PG17, or PG22 as a linker for linking the bacterial toxin proteins, the level of the bacterial toxin protein accumulated in the plant cell is increased.

[0069] In the hybrid protein of the present invention, a secretory signal peptide derived from a plant or a chloroplast transit signal peptide is preferably added to its amino terminus. Here, the term "addition" is a concept including both the case where the secretory signal peptide is directly bound to the amino terminus of the two or three bacterial toxin proteins linked through the peptide and the case where the secretory signal peptide is bound thereto through another peptide.

[0070] The secretory signal peptide is derived from preferably a plant belonging to the family Solanaceae, Brassicaceae, or Asteraceae, further preferably a plant belonging to the genus Nicotiana, Arabidopsis, Lactuca, etc., and more preferably Nicotiana tabacum or Arabidopsis thaliana, Lactuca sativa, etc.

[0071] Moreover, it is preferably derived from a .beta.-D-glucan exohydrolase of Nicotiana tabacum or a 38-kDa peroxidase of Nicotiana tabacum (GenBank ACCESSION D42064).

[0072] An example of the secretory signal peptide includes a peptide that is derived from a .beta.-D-glucan exohydrolase of Nicotiana tabacum and has the amino acid sequence represented by SEQ ID NO: 18.

[0073] An example of the chloroplast transit signal peptide includes a chloroplast transit signal peptide (transit peptide, T.P., SEQ ID NO: 79) derived from Lactuca sativa Rbcs (Rubisco small subunit) (GenBank ACCESSION D14001). A base sequence of DNA which encodes the chloroplast transit signal peptide derived from Lactuca sativa Rbcs is represented by SEQ ID NO: 80. Herein, the hybrid protein including the chloroplast transit signal peptide added to its amino terminus is also referred to as a chloroplast-type (Chl) hybrid protein, and a DNA construct encoding the chloroplast-type (Chl) hybrid protein is also referred to as a chloroplast-type DNA construct. The chloroplast-type hybrid protein is efficiently accumulated particularly in a plant whose chloroplast is developed well such as Nicotiana tabacum.

[0074] The hybrid protein in which neither the secretory signal peptide nor the chloroplast transit signal protein is added to its amino terminus is referred to as a cytoplasm-type (Cyt) hybrid protein, and the DNA construct encoding the cytoplasm-type hybrid protein is referred to as a cytoplasmic-type DNA construct. In the cytoplasm-type hybrid protein, particularly preferably three bacterial toxin protein B subunits are tandemly linked through the peptide.

[0075] In the hybrid protein of the present invention, the signal peptide such as an endoplasmic reticulum retention signal peptide and a vacuolar transport signal peptide may be added to its carboxyl terminus. Here, the term "addition" is the concept including both the case where the signal peptide is directly bound to the carboxyl terminus of the hybrid protein and the case where the signal peptide is bound thereto through another peptide. Herein, the hybrid protein in which the secretory signal peptide is added to its amino terminus and the endoplasmic reticulum retention signal peptide is added to the carboxyl terminus is also referred to as an endoplasmic reticulum-type (ER) hybrid protein, and the DNA construct encoding the endoplasmic reticulum-type hybrid protein is also referred to as an endoplasmic reticulum-type DNA construct. The endoplasmic reticulum-type hybrid protein is efficiently accumulated particularly in a plant such as Lactuca sativa.

[0076] In the hybrid protein of the present invention, the endoplasmic reticulum retention signal peptide is preferably added to its carboxyl terminus. Examples of the endoplasmic reticulum retention signal peptide include an endoplasmic reticulum retention signal peptide including KDEL sequence (SEQ ID NO: 19), HDEL sequence (SEQ ID NO: 20), KDEF sequence (SEQ ID NO: 21), or HDEF sequence (SEQ ID NO: 22).

[0077] The vacuolar transport signal peptide is derived from preferably a plant belonging to the family Solanaceae, Brassicaceae, or Asteraceae, further preferably a plant belonging to the genus Nicotiana, Arabidopsis, Armoracia, etc., and more preferably Nicotiana tabacum, Arabidopsis thaliana, Armoracia rusticana, etc. In addition, the peptide is preferably derived from a chitinase. The amino acid sequence of a vacuolar transport signal peptide derived from a tobacco chitinase is represented by SEQ ID NO: 76. Meanwhile, the base sequence of DNA encoding a vacuolar transport signal peptide derived from a tobacco chitinase is represented by SEQ ID NO: 75, for example.

[0078] Moreover, the peptide is preferably derived from a horseradish peroxidase C1a isozyme. The amino acid sequence of a vacuolar transport signal peptide derived from a horseradish peroxidase C1a isozyme is represented by SEQ ID NO: 78. Meanwhile, the base sequence of DNA encoding a vacuolar transport signal peptide derived from a horseradish peroxidase C1a isozyme is represented by SEQ ID NO: 77, for example. Herein, the hybrid protein in which the secretory signal peptide is added to its amino terminus, and the vacuolar transport signal peptide is added to its carboxyl terminus is also referred to as a vacuole-type (Vac) hybrid protein, and a DNA construct encoding the vacuole-type hybrid protein is also referred to as a vacuole-type DNA construct.

[0079] The hybrid protein of the present invention may be synthesized chemically, or may be produced by genetic engineering. A method of producing by the genetic engineering is described later.

[0080] The DNA construct of the present invention is characterized by containing DNA encoding the hybrid protein of the present invention.

[0081] That is, the DNA construct of the present invention includes DNA in which DNAs encoding the two or three bacterial toxin proteins are tandemly linked through DNA encoding the peptide. The DNA encoding the peptide is represented by, for example, SEQ ID NO: 1 (PG12), SEQ ID NO: 81 (PG17), and SEQ ID NO: 83 (PG22). Examples of the DNA encoding the bacterial toxin protein include DNA (SEQ ID NO: 3) encoding Stx2eA, DNA (SEQ ID NO: 5) encoding Stx2eB, and DNA (SEQ ID NO: 7) encoding CTB. The DNA encoding the peptide and the DNA encoding the bacterial toxin protein are linked by matching their reading frames except stop codons.

[0082] The DNA encoding the bacterial toxin protein can be obtained by a common genetic engineering technique based on the base sequence of SEQ ID NO: 3, 5, or 7, for example. Specifically, a cDNA library is prepared from a bacterium which produces each bacterial toxin according to a conventional method, and a desired clone is selected using a probe prepared from the library based on the base sequence. Alternatively, the DNA can also be synthesized chemically based on the base sequence, or synthesized by PCR with genomic DNA as a template using a 5'- and 3'-terminal base sequence of the base sequence as primers, for example.

[0083] The DNA encoding the hybrid protein of the present invention is represented by SEQ ID NO: 9, 11, 13, 15, 85, 87, 89, 91, 93, 95, 97, or 99.

[0084] In the DNA encoding the hybrid protein, preferably a codon corresponding to an amino acid which composes the hybrid protein is appropriately modified so that the amount of the translated hybrid protein is increased depending on a host cell in which the hybrid protein is produced.

[0085] As the method of modifying the codon, a method of Kang et al. (2004) may serve as a reference, for example. And, the method of selecting the codon frequently used in the host cell, the method of selecting the codon in which the content of GC is high, or the method of selecting the codon frequently used in house keeping genes in the host cell is exemplified.

[0086] Further, the DNA encoding the hybrid protein may be DNA which hybridizes with DNA having the base sequence of SEQ ID NO: 9, 11, 13, 15, 85, 87, 89, 91, 93, 95, 97, or 99 under a stringent condition. The term "stringent condition" refers to the condition where a so-called specific hybrid is formed whereas no non-specific hybrid is formed. There is exemplified the condition where two DNAs with high identity, e.g., two DNAs having the identity of preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more are hybridized with each other whereas two DNAs with lower identity than that are not hybridized. Further, there is exemplified the condition of 2.times.SSC (330 mM NaCl, 30 mM citric acid) at 42.degree. C. and preferably 0.1.times.SSC (330 mM NaCl, 30 mM citric acid) at 60.degree. C.

[0087] In the DNA construct of the present invention, preferably the DNA encoding the hybrid protein is operably-linked to an enhancer. The term "operably" refers to the fact that the hybrid protein is produced in host cells when a vector obtained by inserting the DNA construct of the present invention into a vector including a suitable promoter is introduced into suitable host cells. In addition, the term "linked" refers to both a case where two DNAs are directly linked and a case where two DNAs are linked via another base sequence.

[0088] Examples of the enhancer include Kozak sequence and a 5'-untranslated region of an alcohol dehydrogenase gene derived from a plant. Particularly preferably, the DNA encoding the hybrid protein is operably-linked to the 5'-untranslated region of an alcohol dehydrogenase gene derived from a plant.

[0089] The 5'-untranslated region of an alcohol dehydrogenase gene refers to a region including a sequence between the base at the transcription initiation site of a gene encoding an alcohol dehydrogenase and the base before the translation initiation site (ATG, methionine). The region has a function to increase a translation level. The phrase "function to increase a translation level" refers to a function to increase an amount of a protein produced by translation when the information encoded in a structural gene is transcribed and then translated to produce a protein. The region may be derived from a plant, and it is preferably derived from a plant belonging to the family Solanaceae, Brassicaceae, or Asteraceae, further preferably derived from a plant belonging to the genus Nicotiana, Arabidopsis, Lactuca, etc., and more preferably derived from Nicotiana tabacum, Arabidopsis thaliana, Lactuca sativa, etc.

[0090] The 5'-untranslated region of an alcohol dehydrogenase gene is particularly preferably a region including the base sequence represented by SEQ ID NO: 23, which is the 5'-untranslated region of an alcohol dehydrogenase gene (NtADH 5'-UTR) derived from Nicotiana tabacum, for example.

[0091] The 5'-untranslated region of an alcohol dehydrogenase gene derived from a plant can be isolated from an alcohol dehydrogenase gene of a plant cultured cell where an alcohol dehydrogenase is highly expressed, for example (see JP 2003-79372 A). Meanwhile, in the case of a region having a determined base sequence, such as the 5'-untranslated region of an alcohol dehydrogenase gene derived from Nicotiana tabacum, the region can also be synthesized by chemical synthesis, PCR using a genomic DNA as a template and using the base sequences of the 5'- and 3'-termini of the region as primers, or the like. In addition, if a part of the region having a determined base sequence is used as a probe, the 5'-untranslated region of an alcohol dehydrogenase gene derived from another plant can be searched and isolated.

[0092] The 5'-untranslated region of an alcohol dehydrogenase gene represented by the base sequence of SEQ ID NO: 23 may have substitution, deletion, insertion, or addition of one or several bases as long as the region has a function to increase a translation level. The term "several" means the number of preferably 2 to 10, further preferably 2 to 5, and particularly preferably 2 to 3.

[0093] In addition, DNA having an identity of preferably 85% or more and particularly preferably 90% or more to the 5'-untranslated region of an alcohol dehydrogenase gene and having an ability to increase a translation level may be used.

[0094] Whether the region has an intended function to increase a translation level or not can be confirmed by, for example, a transient assay using a GUS (.beta.-glucuronidase) gene or a luciferase gene as a reporter gene in tobacco cultured cells, or an assay in transformed cells engineered to carry those genes in a chromosome.

[0095] The DNA construct of the present invention has the base sequence represented by any one of SEQ ID NOS: 24 to 29 and SEQ ID NOS: 101 to 111, for example.

[0096] The DNA construct having the base sequence represented by SEQ ID NO: 24 is the DNA construct in which DNA (SEQ ID NO: 9) encoding the hybrid protein in which two Stx2eB proteins are tandemly linked through PG12 is linked to the 5'-untranslated region (NtADH 5'-UTR, SEQ ID NO: 23) of an alcohol dehydrogenase gene derived from Nicotiana tabacum. Further, the DNA construct having the base sequence represented by SEQ ID NO: 25 is the DNA construct in which DNA (SEQ ID NO: 11) encoding the hybrid protein in which two CTB proteins are tandemly linked through PG12 is linked to NtADH 5'-UTR.

[0097] The DNA construct having the base sequence represented by SEQ ID NO: 26 is the DNA construct in which DNA encoding the hybrid protein in which two Stx2eB proteins are tandemly linked through PG12, the secretory signal peptide is added to its amino terminus, and the endoplasmic reticulum retention signal peptide is added to its carboxyl terminus is linked to NtADH 5'-UTR. Further, the DNA construct having the base sequence represented by SEQ ID NO: 27 is the DNA construct in which DNA encoding the hybrid protein in which two CTB proteins are tandemly linked through PG12, the secretory signal peptide is added to its amino terminus, and the endoplasmic reticulum retention signal peptide is added to its carboxyl terminus is linked to NtADH 5'-UTR.

[0098] The DNA construct having the base sequence represented by SEQ ID NO: 28 is the DNA construct in which DNA encoding the hybrid protein in which two Stx2eB proteins are tandemly linked through PG12, the secretory signal peptide is added to its amino terminus, PG12 is linked to its carboxyl terminus, and further the endoplasmic reticulum retention signal peptide is added to its carboxyl terminus is linked to NtADH 5'-UTR. Further, the DNA construct having the base sequence represented by SEQ ID NO: 29 is the DNA construct in which DNA encoding the hybrid protein in which two CTB proteins are tandemly linked through PG12, the secretory signal peptide is added to its amino terminus, PG12 is linked to its carboxyl terminus, and further the endoplasmic reticulum retention signal peptide is added to its carboxyl terminus is linked to NtADH 5'-UTR.

[0099] The DNA construct having the base sequence represented by SEQ ID NO: 101 is the DNA construct in which DNA encoding the hybrid protein in which two Stx2eB proteins are tandemly linked through PG12 and PG12 is linked to its carboxyl terminus is linked to NtADH 5'-UTR.

[0100] The DNA construct having the base sequence represented by SEQ ID NO: 102 is the DNA construct in which DNA encoding the hybrid protein in which two Stx2eB proteins are tandemly linked through PG17 to NtADH 5'-UTR, the secretory signal peptide is added to its amino terminus, PG12 is linked to its carboxyl terminus, and further the endoplasmic reticulum retention signal peptide is added to its carboxyl terminus is linked to NtADH 5'-UTR. Further, the DNA construct having the base sequence represented by SEQ ID NO: 103 is the DNA construct in which DNA encoding the hybrid protein in which two Stx2eB proteins are tandemly linked through PG22, the secretory signal peptide is added to its amino terminus, PG12 is linked to its carboxyl terminus, and further the endoplasmic reticulum retention signal peptide is added to its carboxyl terminus is linked to NtADH 5'-UTR.

[0101] The DNA construct having the base sequence represented by SEQ ID NO: 104 is the DNA construct in which DNA encoding the hybrid protein in which two Stx2eB proteins are tandemly linked through PG12, the chloroplast transit signal peptide is added to its amino terminus, and PG12 is linked to its carboxyl terminus is linked to NtADH 5'-UTR.

[0102] The DNA construct having the base sequence represented by SEQ ID NO: 105 is the DNA construct in which DNA encoding the hybrid protein in which three Stx2eB proteins are each tandemly linked through PG12 and PG12 is linked to its carboxyl terminus is linked to NtADH 5'-UTR.

[0103] The DNA construct having the base sequence represented by SEQ ID NO: 106 is the DNA construct in which DNA encoding the hybrid protein in which three Stx2eB proteins are each tandemly linked through PG12, the secretory signal peptide is added to its amino terminus, and PG12 is linked to its carboxyl terminus, and further the endoplasmic reticulum retention signal peptide is added to its carboxyl terminus is linked to NtADH 5'-UTR.

[0104] The DNA construct having the base sequence represented by SEQ ID NO: 107 is the DNA construct in which DNA encoding the hybrid protein in which three Stx2eB proteins are each tandemly linked through PG12, the chloroplast transit signal peptide is added to its amino terminus, and PG12 is linked to its carboxyl terminus is linked to NtADH 5'-UTR.

[0105] The DNA construct having the base sequence represented by SEQ ID NO: 108 is the DNA construct in which DNA encoding the hybrid protein in which two CTB proteins are tandemly linked through PG12 and PG12 is linked to its carboxyl terminus is linked to NtADH 5'-UTR.

[0106] The DNA construct having the base sequence represented by SEQ ID NO: 109 is the DNA construct in which DNA encoding the hybrid protein in which two CTB proteins are tandemly linked through PG17, the signal peptide is added to its amino terminus, PG12 is linked to its carboxyl terminus, and further the endoplasmic reticulum retention signal peptide is added to the its carboxyl terminus is linked to NtADH 5'-UTR. Further, the DNA construct having the base sequence represented by SEQ ID NO: 110 is the DNA construct in which DNA encoding the hybrid protein in which two CTB proteins are tandemly linked through PG22, the signal peptide is added to its amino terminus, PG12 is linked to its carboxyl terminus, and further the endoplasmic reticulum retention signal peptide is added to the its carboxyl terminus is linked to NtADH 5'-UTR.

[0107] The DNA construct having the base sequence represented by SEQ ID NO: 111 is the DNA construct in which DNA encoding the hybrid protein in which two CTB proteins are tandemly linked through PG12, the chloroplast transit signal peptide is added to its amino terminus, and PG12 is linked to its carboxyl terminus is linked to NtADH 5'-UTR.

[0108] The DNA construct of the present invention can be prepared by a general genetic engineering technique, which includes the following procedures: digesting DNAs including the 5'-untranslated region of an alcohol dehydrogenase gene derived from a plant, a DNA encoding a secretory signal peptide derived from a plant, a DNA encoding a chloroplast transit signal peptide, a DNA encoding a bacterial toxin protein, and a DNA encoding an endoplasmic reticulum retention signal peptide with suitable restriction enzymes; and ligating the resultant fragments with a suitable ligase.

[0109] The recombinant vector of the present invention is characterized by including the DNA construct of the present invention. The recombinant vector of the present invention may be a vector obtained by inserting a DNA encoding a hybrid protein of the present invention into a vector so that the DNA can be expressed in host cells to be introduced with the vector. The vector is not particularly limited as long as it can replicate in host cells, and examples thereof include a plasmid DNA and a viral DNA. In addition, the vector preferably includes a selective marker such as a drug resistance gene. The plasmid DNA can be prepared from Escherichia coli or Agrobacterium by the alkaline extraction method (Birnboim, H. C. & Doly, J. (1979) Nucleic acid Res 7: 1513) or a modified method thereof. Commercially available plasmids such as pBI221, pBI121, pBI101, and pIG121Hm may be used. The viral DNA may be, for example, pTB2 (Donson et al., 1991) (see Donson J., Kerney C M., Hilf M E., Dawson W O. Systemic expression of a bacterial gene by a tabacco mosaic virus-based vector. Proc. Natl. Acad. Sci. (1991) 88: 7204-7208).

[0110] Promoters to be used in vectors may be appropriately selected depending on host cells to be introduced with vectors. The promoters are preferably a cauliflower mosaic virus 35S promoter (Odell et al., 1985, Nature 313:810), a rice actin promoter (Zhang et al. 1991 Plant Cell 3:1155), a corn ubiquitin promoter (Cornejo et al., 1993, Plant Mol. Biol., 23:567), etc. for example. Meanwhile, terminators to be used in vectors may be appropriately selected depending on host cells to be introduced with vectors. The terminators are preferably a nopaline synthase gene transcription terminator, a cauliflower mosaic virus 35S terminator, etc.

[0111] The recombinant vector of the present invention may be prepared as follows.

[0112] First, a DNA construct of the present invention is digested with a suitable restriction enzyme, or a restriction enzyme site is added to the DNA construct by PCR. Subsequently, the DNA construct is inserted into the restriction enzyme site or multicloning site of a vector.

[0113] The transformant of the present invention is characterized by being transformed with the recombinant vector of the present invention. The host cells to be used for transformation may be eukaryotic cells or prokaryotic cells.

[0114] The eukaryotic cells are preferably plant cells, particularly preferably cells of plants belonging to the family Asteraceae, Solanaceae, Brassicaceae, and Chenopodiaceae. Moreover, the eukaryotic cells are preferably cells of plants belonging to the genus Lactuca, particularly preferably Lactuca sativa cells. In the case of using Lactuca sativa cells as host cells, a cauliflower mosaic virus 35S RNA promoter or the like may be used in the vector.

[0115] The prokaryotic cells may be cells of Escherichia coli, Agrobacterium tumefaciens, etc.

[0116] The transformant of the present invention can be prepared by a general genetic engineering technique by introducing a vector of the present invention into host cells. For example, the transformant can be prepared by the introduction method using Agrobacterium (Hood, et al., 1993, Transgenic, Res. 2:218, Hiei, et al., 1994 Plant J. 6:271), an electroporation method (Tada, et al., 1990, Theor. Appl. Genet, 80:475), a polyethylene glycol method (Lazzeri, et al., 1991, Theor. Appl. Genet. 81:437), a particle gun method (Sanford, et al., 1987, J. Part. Sci. tech. 5:27), a polycation method (Ohtsuki), etc.

[0117] After introduction of the vector of the present invention into host cells, a transformant of the present invention can be selected based on the phenotype of a selective marker. If the selected transformant is cultured, the bacterial toxin protein can be produced. The culture medium and conditions for culture may be suitably selected depending on the type of a transformant.

[0118] In addition, in the case of using a plant cell as a host cell, culture of selected plant cell in accordance with a conventional method can regenerate a plant and accumulate the bacterial toxin protein in the plant cells or outside the cell membranes of the plant cells. The method depends on the type of the plant cell, and examples thereof include the method of Visser et al. (Theor. Appl. Genet, 78:594(1989)) for potato and the method of Nagata and Takebe (Planta, 99:12(1971)) for Nicotiana tabacum.

[0119] In the case of Lactuca sativa, a shoot can be regenerated in MS medium containing 0.1 mg/l NAA (naphthalene acetic acid), 0.05 mg/l BA (benzyladenine), and 0.5 g/l polyvinylpyrrolidone, and culture of the regenerated shoot in a 1/2 MS medium containing 0.5 g/l polyvinylpyrrolidone may cause rooting.

[0120] The seed of the present invention can be obtained by collecting a seed from a plant regenerated as above. If the seed of the present invention are sown and cultivated by a suitable method, a plant capable of producing a bacterial toxin protein can be obtained and is included in the transformant of the present invention.

Examples

<1> Transient Expression Experiment

(1) Construction of Stx2eB Transient Expression Vector

[0121] A vector containing a DNA construct in which DNA (SEQ ID NO: 5) encoding an Stx2e protein B subunit (Stx2eB) was linked to a 5'-untranslated region (NtADH 5'-UTR) of a tobacco alcohol dehydrogenase gene was prepared as follows.

[0122] A design of the vector is shown in FIG. 1.

[0123] 1.times.Stx2eB (PG12) denotes a DNA construct containing DNA in which DNA encoding PG12 is linked to DNA encoding Stx2eB. 2.times.Stx2eB (PG12) denotes a DNA construct containing DNA in which two DNAs encoding Stx2eB are linked using DNA encoding PG12 as a spacer.

[0124] In addition, a DNA construct, 3.times.Stx2eB (PG12) in which three DNAs encoding Stx2eB are linked using DNA encoding PG12 as a spacer, and a DNA construct, 4.times.Stx2eB (PG12) in which four DNAs encoding Stx2eB are linked using DNA encoding PG12 as a spacer were prepared as well.

[0125] Specific techniques are shown below.

[0126] PCR using a Kozak-stx2eb-F primer (SEQ ID NO: 30) and an stx2eb-R primer (SEQ ID NO: 31) was performed to amplify a DNA fragment encoding a mature region (except for a secretory signal peptide to periplasm, Ala 19 to Asn 87) of Stx2eB. The resulting DNA fragment was cloned into an EcoRV gap in pBluescript II SK. The resulting plasmid was cleaved with HindIII, and treated with T4 DNA polymerase, followed by self-ligation to convert a HindIII site to a NheI site (plasmid 1).

[0127] Stx2eB was inserted as follows into the multicloning site (MCS) of a transient expression vector in plant cells, pBI221 (Clontech).

[0128] In order to introduce SalI, KpnI, and SmaI sites into the MCS, SalKpnSma-F (SEQ ID NO: 32) and SalKpnSma-R (SEQ ID NO: 33) were annealed and phosphorylated with T4 polynucleotide kinase (T4 PNK) (TaKaRa) and inserted into the SacI gap of pBI221 (plasmid 2). Stx2eB was cleaved out from plasmid 1 using XbaI and KpnI to insert into plasmid 2, and the resultant product was arranged between a cauliflower mosaic virus 35S RNA promoter (35S pro.) and a nopaline synthase gene transcription terminator (NOS-T) (plasmid 3).

[0129] The 5'-untranslated region (NtADH 5'-UTR, SEQ ID NO: 23) of a tobacco alcohol dehydrogenase gene was amplified by PCR with ADH-221 (Sato et al., 2004, (see below)) as a template using ADH XbaI-F primer (SEQ ID NO: 34) and ADH NsiI-R primer (SEQ ID NO: 35). A DNA region (SEQ ID NO: 17) encoding a secretory signal peptide (SEQ ID NO: 18) of .beta.-D glucan exohydrolase (GenBank ACCESSION AB017502) was amplified with a tobacco genomic DNA as a template using .beta.D NsiI-F primer (SEQ ID NO: 36) and .beta.D BamHI-R primer (SEQ ID NO: 37). The obtained respective DNA fragments of NtADH 5'-UTR and the secretory signal peptide were treated with NsiI (manufactured by Toyobo Co., Ltd.), ligated using Ligation High (manufactured by Toyobo Co., Ltd.), followed by being blunted, and cloned into the EcoRV gap in pBluescript II SK (manufactured by Stratagene) (plasmid 4).

[0130] Satoh et al., The 5'-untranslated region of the tobacco alcohol dehydrogenase gene functions as an effective translational enhancer in plant. J. Biosci. Bioeng. (2004) 98, 1-8

[0131] Plasmid 4 was treated with NsiI, and blunted with T4 DNA polymerase (manufactured by Toyobo Co., Ltd.), followed by performing self-ligation to be ligated so that the initiation codon (atg) of NtADH was matched to the initiation codon of the secretory signal peptide (plasmid 5).

[0132] A DNA obtained by ligating an NtADH 5'-UTR fragment and a secretory signal peptide was amplified using plasmid 5 as a template and using ADH XbaI-F primer (SEQ ID NO: 34) and .beta.D BamHI-R primer (SEQ ID NO: 35). The resultant DNA fragment was treated with XbaI and BamHI and inserted into the XbaI-BamHI gap of plasmid 3 (plasmid 6).

[0133] In order to add an endoplasmic reticulum retention signal (SEQ ID NO: 38), an HDEL-F primer (SEQ ID NO: 39; `HDEL` disclosed as SEQ ID NO: 20) and an HDEL-R primer (SEQ ID NO: 40; `HDEL` disclosed as SEQ ID NO: 20) were annealed and phosphorylated with T4 PNK, and the resultant product was inserted into the BglII gap of plasmid 6, which had been dephosphorylated with alkaline phosphatase (AP) (TakaRa) (plasmid 7).

[0134] An HA tag was added as a peptide tag for detecting Stx2eB. In order to add the HA tag, an HA-F primer (SEQ ID NO: 41) and an HA-R primer (SEQ ID NO: 42) were annealed and phosphorylated with T4 PNK. The resultant phosphorylated HA fragment was inserted into the BglII gap of plasmid 7 (plasmid 8).

[0135] A PG12 spacer (SEQ ID NO: 2) was inserted between Stx2eB and the HA tag. A PG12-F primer (SEQ ID NO: 43) and a PG12-R primer (SEQ ID NO: 44) were annealed and phosphorylated with T4 PNK. The resulting phosphorylated DNA fragment was inserted into the BglII gap of plasmid 8 (1.times.Stx2eB (PG12)).

[0136] 2.times.Stx2eB (PG12) was obtained by cleaving a 2eB-PG12 fragment out from 1.times.Stx2eB (PG12) with BamHI and BglII and then inserting the fragment into the BamHI gap of the 1.times.Stx2eB (PG12). 3.times.Stx2eB (PG12) was obtained by cleaving an Stx2eB-PG12 fragment out from 1.times.Stx2eB (PG12) with BamHI and BglII and then inserting the fragment into the BamHI gap of 2.times.Stx2eB (PG12). Further, 4.times.Stx2eB (PG12) was obtained by cleaving a 2.times.(Stx2eB-PG12) fragment out from 2.times.Stx2eB (PG12) with BamHI and BglII and then inserting the fragment into the BamHI gap of the 2.times.Stx2eB (PG12).

[0137] (2) Construction of CTB Transient Expression Vector

[0138] A vector containing a DNA construct (2.times.CTB (PG12)) in which DNA encoding a CT protein B subunit (CTB) had been linked to the 5'-untranslated region of a tobacco alcohol dehydrogenase gene was prepared as follows.

[0139] A DNA fragment (SEQ ID NO: 7) encoding the mature region (except for the secretory signal to the periplasm, Thr 22 to Asn 124) (SEQ ID NO: 8) of CTB was prepared. First, the following ten primers were prepared.

[0140] CTB1: SEQ ID NO: 45

[0141] CTB2: SEQ ID NO: 46

[0142] CTB3: SEQ ID NO: 47

[0143] CTB4: SEQ ID NO: 48

[0144] CTB5: SEQ ID NO: 49

[0145] CTB6: SEQ ID NO: 50

[0146] CTB7: SEQ ID NO: 51

[0147] CTB8: SEQ ID NO: 52

[0148] CTB9: SEQ ID NO: 53

[0149] CTB10: SEQ ID NO: 54

[0150] PCR using the primers synthesized above was performed under the condition described in Kang et al. (2004). That is, PCR was performed in combination of CTB1 and CTB2, CTB3 and CTB4, CTB5 and CTB6, CTB7 and CTB8, and CTB9 and CTB10, and DNA fragments of 72 bp (1+2), 74 bp (3+4), 67 bp (5+6), 82 bp (7+8) and 68 bp (9+10) were synthesized, respectively. Subsequently, the second PCR was performed in combination of CTB1+2 and CTB3+4, CTB3+4 and CTB5+6, CTB5+6 and CTB7+8, and CTB7+8 and CTB9+10, and DNA fragments of 135 bp (1+2+3+4), 132 bp (3+4+5+6), 138 bp (5+6+7+8), and 141 bp (7+8+9+10) were synthesized, respectively. Then, the third PCR was performed in combination of CTB1+2+3+4 and CTB3+4+5+6, and CTB5+6+7+8 and CTB7+8+9+10, and DNA fragments of 194 bp (1+2+3+4+5+6) and 198 bp (5+6+7+8+9+10) were synthesized, respectively. Finally, PCR was performed in combination of CTB1+2+3+4+5+6 and CTB5+6+7+8+9+10, and a DNA fragment of 315 bp in which a BamHI site and a BglII site were added to a CTB coding region was synthesized.

[0151] The DNA fragment prepared above was treated with BamHI and BglII, and inserted into a BamHI-BglII gap in plasmid 8 (plasmid 9).

[0152] The PG12 spacer (SEQ ID NO: 2) was inserted between CTB and the HA tag. A PG12-F primer (SEQ ID NO: 43) and a PG12-R primer (SEQ ID NO: 44) were annealed and phosphorylated with T4 PNK. The resulting phosphorylated DNA fragment was inserted into the BglII gap of plasmid 9 (1.times.CTB (PG12)).

[0153] A CTB-PG12 fragment was cut out from 1.times.CTB (PG12) using BamHI and BglII, and inserted into the BamHI gap of 1.times.CTB (PG12) (2.times.CTB (PG12)).

[0154] (3) Transient Expression Test Using Lactuca sativa Protoplast

[0155] A leaf of potted Lactuca sativa (green wave) (about 1 g) was cut into 0.5-cm square pieces using a surgical knife, to thereby prepare leaf discs. The leaf discs were immersed in 500 mM mannitol, and shaken for 1 hour. The leaf discs were immersed in 50 ml of a protoplastization enzyme solution (1.0% cellulose RS (Yakult Honsha Co., Ltd.), 0.25% macerozyme R-10 (Yakult Honsha Co., Ltd.), 400 mM mannitol, 8 mM CaCl.sub.2, and 5 mM Mes-KOH, pH 5.6), and the whole was shaken at room temperature for 2 hours. The protoplast suspension was passed through meshes of 100 .mu.m and 40 .mu.m to remove the leaf discs. The protoplast suspension was centrifuged at 60 g for 5 minutes to precipitate the protoplast. The protoplast was resuspended in an aqueous solution containing 167 mM mannitol and 133 mM CaCl.sub.2, and the suspension was centrifuged at 40 g for 5 minutes. The protoplast was resuspended in an aqueous solution containing 333 mM mannitol and 66.7 mM CaCl.sub.2, and the suspension was centrifuged at 40 g for 5 minutes. The protoplast was suspended in W5 solution (154 mM NaCl, 125 mM CaCl.sub.2, 5 mM KCl, 2 mM Mes-KOH, pH 5.6), and the suspension was allowed to stand on ice for 1 hour. The protoplast suspension was centrifuged at 40 g for 5 minutes, and the protoplast was suspended in an MaMg solution (400 mM mannitol, 15 mM MgCl.sub.2, and 4 mM Mes-KOH, pH 5.6) to have a protoplast concentration of 2.times.10.sup.6 cells/ml.

[0156] Each of the Stx2eB transient expression vector and the CTB transient expression vector prepared above was mixed with 120 .mu.L of a protoplast suspension, subsequently 140 .mu.L of PEG solution (400 mM mannitol, 100 mM Ca(NO.sub.3).sub.2 and 40% PEG) was added thereto, and the resulting mixture was blended gently and incubated for 7 minutes. Then, 1 mL of W5 solution was added to the protoplast suspension over about 20 minutes. A solution (1 mL) obtained by mixing 400 mM mannitol and the W5 solution at a ratio of 4:1 was added to the protoplast precipitated by centrifugation. LS medium (1 mL) containing 1% sucrose, 400 mM mannitol, and 0.3 mM carbenicillin was added to the protoplast precipitated by centrifugation, and the mixture was then cultured in a dark place at 25.degree. C. for 24 hours.

[0157] (4) Western Analysis

[0158] To the protoplast collected by centrifugation were added 30 .mu.l of SDS-sample buffer (4% (w/v) SDS, 20% (w/v) glycerol, 0.05% (w/v) bromophenol blue, 300 mM .beta.-mercaptoethanol, 125 mM Tris-HCl, pH 6.8), followed by thermal denaturation at 95.degree. C. for 2 minutes, to thereby prepare samples. Proteins were separated using a 15% acrylamide gel and blotted on a PVDF membrane (Hybond-P; Amersham) using an electro transfer system. An anti-HA antibody (No. 11 867 423 001, Roche) was used to detect Stx2eB and CTB.

[0159] (a) Effects of Linking Number of Stx2eB

[0160] A result is shown in FIG. 2. When 1.times.Stx2eB (PG12) was expressed, a signal was detected at a position of about 8.5 kDa. When 2.times.Stx2eB (PG12) was expressed, a signal was detected at a position of about 17 kDa at the same level as when 1.times.Stx2eB (PG12) was expressed. When 3.times.Stx2eB (PG12) was expressed, a signal was detected at a position of about 26 kDa, the signal being smaller than that when 1.times.Stx2eB (PG12) was expressed. These corresponded to the molecular weights estimated from the design of the DNA constructs. When 4.times.Stx2eB (PG12) was expressed, the specific signal was below a detection limit.

[0161] From the above result, it was demonstrated that when 2.times.Stx2eB (PG12) and 3.times.Stx2eB (PG12) were expressed, hybrid proteins in which multiple Stx2eB proteins were linked could be produced.

[0162] Since each of the above DNA constructs contains one molecule of the HA tag (see FIG. 1), it is conceivable that a protein amount corresponding to about 2 times an Stx2eB protein amount when 1.times.Stx2eB (PG12) is expressed is accumulated when 2.times.Stx2eB (PG12) is expressed. That is, it has been found that when two DNAs encoding the Stx2eB protein are linked through DNA encoding PG12, the Stx2eB protein can be produced with very high efficiency.

[0163] Meanwhile, it has been also found that when three DNAs encoding the Stx2eB protein or four DNAs encoding the Stx2eB protein are linked through DNA encoding PG12, the amount of the produced Stx2eB protein is equal to or less than the amount when one Stx2eB protein is linked to PG12.

[0164] It has been found that the level of accumulated proteins tends to be higher in the Stx2eB protein (1.times.Stx2eB (PG12)) having PG12 added at its carboxyl terminus prepared in this experiment, compared with the Stx2eB protein having no PG12. This speculates that it is a favorable form that PG12 is added to the carboxyl terminus in the hybrid protein of the present invention.

[0165] (b) Effects of Linking Number of CTB

[0166] The results are shown in FIG. 3.

[0167] When 1.times.CTB (PG12) was expressed, a signal was detected at the position of about 20 kDa. When 2.times.CTB (PG12) was expressed, a larger signal than that when 1.times.CTB (PG12) was expressed was detected at the positions of about 33 kDa and about 35 kDa.

[0168] From the above results, it was demonstrated that the hybrid protein in which two CTB proteins were linked could be produced when 2.times.CTB (PG12) was expressed. These corresponded to the molecular weights estimated from the design of the DNA constructs.

[0169] Since each of the above DNA constructs contains one molecule of the HA tag, it is conceivable that the protein amount corresponding to the CTB protein amount which is larger than two times the protein amount when 1.times.CTB (PG12) is expressed is accumulated when 2.times.CTB (PG12) is expressed. That is, it has been found that when two DNAs encoding the CTB protein are linked through DNA encoding PG12, the CTB protein can be produced with very high efficiency.

[0170] (5) Analysis of Localization of Stx2eB

[0171] In order to analyze the localization of Stx2eB in the cell, a transient expression vector for the hybrid protein of Stx2eB and a yellow fluorescent protein YFP was prepared. The design for the vector is shown in FIG. 4. 1.times.Stx2eB (PG12)-YFP denotes a DNA construct in which DNA encoding the Stx2eB protein is linked to DNA encoding YFP. 2.times.Stx2eB (PG12)-YFP denotes a DNA construct in which DNA encoding the hybrid protein in which two Stx2eB proteins are linked through PG12 is linked to DNA encoding YFP. 2.times.Stx2eB (RS)-YFP using RS (Arg Ser) in place of PG12 as a spacer was also prepared.

[0172] A specific technique is shown below.

[0173] First, a DNA fragment of YFP was amplified by PCR with pEYFP (Clontech) as a template using a YFP-F primer (SEQ ID NO: 55) and a YEP-R primer (SEQ ID NO: 56). The resulting DNA fragment was treated with BamHI and BglII, and inserted into the BamHI-BglII gap of plasmid 8 (ER-YFP).

[0174] An Stx2eB fragment was cleaved out from plasmid 1 with BamHI and BglII and then inserted into the BamHI gap of the 1.times.Stx2eB (PG12) (2.times.Stx2eB (RS)).

[0175] An Stx2eB-PG12 fragment, a 2.times.(Stx2eB-RS) fragment, and a 2.times.(Stx2eB-PG12) fragment were cleaved out from 1.times.Stx2eB (PG12), 2.times.Stx2eB (RS), and 2.times.Stx2eB (PG12) with BamHI-BglII, respectively, and each fragment was inserted into the BamHI gap of ER-YFP (1.times.Stx2eB (PG12)-YFP, 2.times.Stx2eB (RS)-YFP, and 2.times.Stx2eB (PG12)-YFP).

[0176] Meanwhile, an expression vector for a red fluorescent protein (mRFP, Campbell R. E. et al., 2002, (see below)) localized in the endoplasmic reticulum was prepared as a vector for visualizing the endoplasmic reticulum. PCR was performed using an mRFP-F primer (SEQ ID NO: 57) and an mRFP-R primer (SEQ ID NO: 58). The resulting DNA fragment was treated with BamHI and BglII, and inserted into the BamHI-BglII gap of plasmid 8 (ER-mRFP).

Campbell R. E. et al., A monomeric red fluorescent protein (2002), Proc. Nat. Acad. Sci., 99: 7877-7882.

[0177] The Stx2eB expression vector and the mRFP expression vector were introduced into the protoplasts of cultured tobacco cells (BY2) in the same way as the above methods, and observed using a confocal microscope observation system (LSM510, Zeiss).

[0178] The results are shown in FIGS. 5 and 6.

[0179] FIG. 5 shows the localization of the hybrid protein of Stx2eB-YFP. When 2.times.Stx2eB (PG12)-YFB was expressed, it was observed that the hybrid protein of Stx2eB-YFP was localized granularly at about 100 granules/cell. When 1.times.Stx2eB (PG12)-YFP and 2.times.Stx2eB (RS)-YFP were expressed, no granule was observed.

[0180] In FIG. 6, an image A in a leftmost column shows the localization of mRFP in a certain protoplast. The localization of mRFP reflects the location of the endoplasmic reticulum. An image B in a middle column shows the localization of the hybrid protein of Stx2eB-YFP in the identical protoplast. An image in a rightmost column is a composite image of the image A and the image B. It is found from this composite image that the hybrid protein of Stx2eB-YFP is localized granularly in the endoplasmic reticula.

[0181] (6) Effect of Vesicular Transport Function

[0182] It was examined in which process after the protein translation the accumulation and aggregation of 2.times.Stx2eB (PG12) occur.

[0183] 2.times.Stx2eB (PG12)-YFP was co-expressed with an Arabidopsis vesicular transport regulation protein ARF1 or a dominant negative mutant thereof ARF1 (Q71L) (ARF1DN). As a reporter for inhibition of protein transport to Golgi apparatus by the co-expression with ARF1DN, vacuole-GFP was co-expressed with each ARF1. An expression vector for each ARF1 was constructed as follows. An expression vector for the vacuole-GFP can be prepared with reference to the following document.

[0184] Di Sansebastiano et. al., Specific accumulation of GFP in a non-acidic vacuolar compartment via a C-terminal propeptide-mediated sorting pathway. Plant J. (1998) 15, 449-457

[0185] As the vesicular transport regulation protein, expression vectors for Arabidopsis ARF1 (GenBank ACCESSION No. M95166) and for the dominant negative mutant thereof ARF1 (Q71L) (Misaki Takeuchi et al., 2002, (see below)) were constructed. PCR using cDNA prepared from Arabidopsis embryo plant as a template was performed using an ARF1-F primer (SEQ ID NO: 59) and an ARF1-R primer (SEQ ID NO: 60). The resulting DNA fragment was subcloned into the EcoRV gap in pBluescript (Stratagene). Another PCR was performed using an ARFQL-F primer (SEQ ID NO: 61) and an ARFQL-R primer (SEQ ID NO: 62) to substitute a glutamine residue at position 71 with a leucine residue. Each resulting ARF1 fragment was subcloned into a transient expression vector pBI221,

[0186] Each of the vectors prepared was introduced into the protoplast of the cultured tobacco cell in the same manner as that described above, and the respective proteins were co-expressed to examine the localization of 2.times.Stx2eB (PG12).

[0187] The result is shown in FIG. 7.

[0188] Both when 2.times.Stx2eB (PG12)-YFP was co-expressed with ARF1 and co-expressed with ARF1 (Q71L), granules were formed as observed in an expression of 2.times.Stx2eB (PG12)-YFP alone. On the other hand, co-expression of ARF1 (Q71L) inhibited the exit of vacuole-GFP from ER. It was speculated from these that the granule formation is not dependent on vesicular transport process from the ER to Golgi apparatus.

<2> Transformation Experiments Using Cultured Tobacco Cells

(1) Construction of Vectors for Transformation

[0189] 1.times.Stx2eB (PG12), 2.times.Stx2eB (PG12), 3.times.Stx2eB (PG12), and 4.times.Stx2eB (PG12) were prepared in the same way as above.

[0190] Further, by using RS (Arg, Ser), PG7 (SEQ ID NO: 63), or SG12 (SEQ ID NO: 64) as a spacer instead of PG12, DNA constructs, 2.times.Stx2eB (RS), 2.times.Stx2eB (PG7), and 2.times.Stx2eB (SG12) were prepared by the following methods.

[0191] A PG7 spacer (SEQ ID NO: 63) was inserted between Stx2eB and the HA tag in the plasmid 8. A PG7-F primer (SEQ ID NO: 65) and a PG7-R primer (SEQ ID NO: 66) were annealed and phosphorylated with T4 PNK. The obtained phosphorylated DNA fragment was inserted into the BglII gap of plasmid 8 (plasmid 10).

[0192] An SG12 spacer (SEQ ID NO: 64) was inserted between Stx2eB and the HA tag. An SG12-F primer (SEQ ID NO: 67) and an SG12-R primer (SEQ ID NO: 68) were annealed and phosphorylated with T4 PNK. The obtained phosphorylated DNA fragment was inserted into the BglII gap of plasmid 8 (plasmid 11).

[0193] An Stx2eB fragment was cleaved out from plasmid 1 with BamHI and BglII and then inserted into the BamHI gap of 1.times.Stx2eB (PG12) (2.times.Stx2eB (RS)). An Stx2eB-PG7 fragment was cleaved out from plasmid 10 with BamHI and BglII and then inserted into the BamHI gap of 1.times.Stx2eB (PG12) (2.times.Stx2eB (PG7)). An Stx2eB-SG12 fragment was cleaved out from plasmid 11 with BamHI and BglII and then inserted into the BamHI gap of 1.times.Stx2eB (PG12) (2.times.Stx2eB (SG12)).

[0194] In order to produce Stx2eB using a stable transformant of the plant, each of the DNA constructs of the above Stx2eB was subcloned into a vector for transformation (for the design of the vectors, see FIG. 1). Each of 1.times.Stx2eB (PG12), 2.times.Stx2eB (PG12), 3.times.Stx2eB (PG12), 4.times.Stx2eB (PG12), 2.times.Stx2eB (RS), 2.times.Stx2eB (PG7), and 2.times.Stx2eB (SG12) was inserted into pBI121 (Clontech) using XbaI and SacI, and allocated between a cauliflower mosaic virus 35S RNA promoter (35S pro.) and a nopaline synthetase gene transcription terminator (NOS-T).

[0195] (2) Transformation of Cultured Tobacco Cells

[0196] The produced vector for transformation was introduced into Agrobacterium tumefacience EHA105 using an electroporation method. An Agrobacterium medium (100 .mu.L) cultured in 5 mL of LB medium containing 100 mg/L of kanamycin at 28.degree. C. for 2 nights was mixed with 5 to 10 mL of a suspension of cultured tobacco cells (Nicotiana tabacum, cv BY2) on the fourth day of the culture in a petri-dish, and the mixture was co-cultured by being left to stand in a dark place at 25.degree. C. for 2 nights. In order to remove Agrobacterium, the medium in the petri-dish was transferred into a 15-mL centrifuging tube, which was then centrifuged (1,000 rpm, 5 minutes, 4.degree. C.), and a supernatant was removed. A modified LS medium was charged, and the tube was centrifuged (1,000 rpm, 5 minutes, 4.degree. C.) to wash the cells. This washing operation was repeated four times to remove Agrobacterium. The resultant BY2 cells were then placed in a modified LS agar medium containing kanamycin (100 mg/L), and cultured by being left to stand in the dark place at 25.degree. C. After about 2 to 3 weeks, cells which has formed a callus were transferred to a new plate, and a growing clone was selected.

[0197] (3) Semi-Quantification of Stx2eB Protein Using Western Analysis

[0198] Cultured tobacco cells cultured in a plate medium were collected in a centrifuging tube, and 1 .mu.L of SDS sample buffer per mg of cell weight was added. The cells were denatured at 95.degree. C. for 2 minutes to make a sample for electrophoresis. Proteins were separated using 15% acrylamide gel, and then blotted onto a PVDF membrane (Hybond-P; Amersham) using an electrotransfer apparatus. An Stx2eB protein was detected using an anti-HA antibody (No. 11 867 423 001, Roche). Serial dilution of Stx2eB having the HA tag at known concentrations was prepared, and loaded on the gel. A calibration curve was prepared based on their signal intensities, and the amount of Stx2eB proteins in each sample was calculated.

[0199] The results are shown below.

[0200] (a) Effects of Linking Number of Stx2eB

[0201] The results are shown in FIGS. 8 and 9.

[0202] When 1.times.Stx2eB (PG12) was expressed, signals were detected at the positions of about 10 kDa and about 17 kDa. They are presumed to be Stx2eB in which the signal peptide was cleaved and Stx2eB in which the signal peptide is not cleaved, respectively. When 2.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 19 kDa at the similar level to that when 1.times.Stx2eB (PG12) was expressed. When 3.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 26 kDa, the signal being smaller than that when 1.times.Stx2eB (PG12) was expressed. These corresponded to the molecular weights estimated from the design of the DNA constructs. When 4.times.Stx2eB (PG12) was expressed, a specific signal was below the detection limit (data not shown).

[0203] It has been found from the above that 2.times.Stx2eB (PG12) accumulates the larger amount of Stx2eB than 1.times.Stx2eB (PG12) and 3.times.Stx2eB (PG12).

[0204] (b) Effects of Spacer

[0205] The results are shown in FIGS. 10 and 11.

[0206] Based on the intensity of the signal corresponding to Stx2eB, the level of accumulated Stx2eB was the highest when PG12 was used as a spacer. The levels were secondly high when PG7 and SG12 were used, which were in the similar degree. The level was lowest when RS was used. This indicates that the length and the amino acid sequence of the spacer between two Stx2eBs affect the level of the accumulated 2.times.Stx2eB protein.

[0207] (4) Quantification of mRNA by Real-Time PCR

[0208] It was examined whether the level of the accumulated protein was influenced by a transcription level or not.

[0209] RNA was prepared using RNeasy Mini Kit (Qiagen) from each of the transformed BY2 cells obtained above. The resulting RNA was treated with DNase, and then reversely transcribed using Transcriptor Reverse Transcriptase (Roche). Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). A primer set (SEQ ID NO: 69 and SEQ ID NO: 70) which amplified the region containing NtADH 5'-UTR and the signal peptide which were common in the respective constructs was used for the quantification of Stx2eB mRNA. The amount of an expressed BY2 ubiquitin gene was quantified using a UBQ-F primer (SEQ ID NO: 71) and a UBQ-R primer (SEQ ID NO: 72) to compensate the mRNA level of an Stx2eB gene. Note that the mRNA level of 1.times.Stx2eB (PG12) was calculated by multiplying a quantified value by 1/2.

[0210] The results are shown in FIG. 12.

[0211] The accumulation levels of Stx2eB protein per mRNA tend to be higher in cells expressing 2.times.Stx2eB (PG12) than those in cells expressing 2.times.Stx2eB (RS) or 1.times.Stx2eB (PG12). This indicates that the difference of the spacer does not influence on the transcription level but influences on a translation level or stability of the protein after the translation. Considering together the result that the 2.times.Stx2eB protein was localized granularly, it is conceivable that the spacer influences on the stability of the protein after the translation.

<3> Transient Expression Experiments

[0212] (1) Construction of Stx2eB Transient Expression Vectors

[0213] Transient expression vectors for 1.times.Stx2eB (PG12), 2.times.Stx2eB (PG12), 3.times.Stx2eB (PG12), and 4.times.Stx2eB (PG12) were constructed by the method in above <1> (1) (FIG. 13-A). These vectors are referred to as ER-1.times.Stx2eB (PG12), ER-2.times.Stx2eB (PG12), ER-3.times.Stx2eB (PG12), and ER-4.times.Stx2eB (PG12), respectively. Note that "ER" means the endoplasmic reticulum type.

[0214] Further, transient expression vectors containing the cytoplasm (Cyt) type of DNA construct (FIG. 13-B) and expression vectors containing the chloroplast (Chl) type of DNA construct (FIG. 13-C) were constructed by the following method. These DNA constructs were designed to contain DNA encoding the endoplasmic reticulum retention signal peptide, for the purpose of expressing the hybrid protein having as close a structure as possible to that of the endoplasmic reticulum type of hybrid protein. But, since these DNA constructs do not contain DNA encoding the secretory signal peptide, the endoplasmic reticulum retention signal peptide does not exert its function (retention of the protein in the endoplasmic reticulum) in the produced hybrid protein.

[0215] An NtADH 5'-UTR fragment was amplified by PCR using an ADH XbaI-F primer (SEQ ID NO: 34) and an ADH BamHI-R primer (SEQ ID NO: 112), and the resulting DNA fragment was treated with XbaI and BamHI. The XbaI-BamHI fragment of NtADH 5'-UTR was inserted into the XbaI-BamHI gap of each of ER-1.times.Stx2eB (PG12), ER-2.times.Stx2eB (PG12), ER-3.times.Stx2eB (PG12), and ER-4.times.Stx2eB (PG12) to prepare Cyt-1.times.Stx2eB (PG12), Cyt-2.times.Stx2eB (PG12), Cyt-3.times.Stx2eB (PG12), and Cyt-4.times.Stx2eB (PG12) which were cytoplasm type Stx2eB vectors.

[0216] The NtADH 5'-UTR fragment was amplified by PCR using an ADH XbaI-F primer (SEQ ID NO: 34) and an ADH NsiI-R primer (SEQ ID NO: 35). A DNA fragment (SEQ ID NO: 80) encoding the transit signal peptide, the chloroplast being derived from Lactuca sativa Rbcs (Rubisco small subunit) (GenBank ACCESSION D14001) (transit peptide, T.P.), was amplified by PCR with cDNA of a Lactuca sativa leaf as a template using a TP NsiI-F primer (SEQ ID NO: 113) and a TP BamHI-R primer (SEQ ID NO: 114). Each resulting DNA fragment of NtADH 5'-UTR and each DNA fragment of the secretory signal peptide was treated with NsiI (manufactured by Toyobo Co., Ltd.), ligated using Ligation High (Toyobo Co., Ltd.) followed by being blunted, and cloned into the EcoRV gap of pBluescript II SK (manufactured by Stratagene) (plasmid 12). Plasmid 12 was treated with NsiI, blunted with T4 DNA polymerase (Toyobo Co., Ltd.), and then self-ligated to be fused so that the initiation codon of NtADH and the initiation codon of Rbcs were matched (plasmid 13). An NtADH 5'-UTR-T.P. fusion fragment was cut out from plasmid 13 using XbaI and BamHI, and inserted into the XbaI-BamHI gap of each of ER-1.times.Stx2eB (PG12), ER-2.times.Stx2eB (PG12), ER-3.times.Stx2eB (PG12), and ER-4.times.Stx2eB (PG12) to prepare Chl-1.times.Stx2eB (PG12), Chl-2.times.Stx2eB (PG12), Chl-3.times.Stx2eB (PG12), and Chl-4.times.Stx2eB (PG12), which were chloroplast type Stx2eB vectors.

[0217] (2) Production of CTB Transient Expression Vectors

[0218] Transient expression vectors for 1.times.CTB (PG12) and 2.times.CTB (PG12) were constructed by the method in above <1> (2) (FIG. 14-A). Hereinafter, these vectors are referred to as ER-1.times.CTB (PG12) and ER-2.times.CTB (PG12).

[0219] Transient expression vectors containing the cytoplasm (Cyt) type of DNA construct (FIG. 14-B) and expression vectors containing the chloroplast (Chl) type of DNA construct (FIG. 14-C) were constructed by the following methods.

[0220] A CTB-PG12 fragment was cut out from ER-1.times.CTB (PG12) using BamHI and BglII, and inserted into the BamHI-BglII gap in Cyt-1.times.Stx2eB (PG12) and the BamHI-BglII gap in Chl-1.times.Stx2eB (PG12) produced in above <3> (1) to prepare cytoplasm type of 1.times.CTB (PG12) and chloroplast type of 1.times.CTB (PG12) (Cyt-1.times.CTB (PG12), Chl-1.times.CTB (PG12)).

[0221] Subsequently, a 2.times.(CTB-PG12) fragment was cut out from ER-2.times.CTB (PG12) using BamHI and BglII, and inserted into the BamHI-BglII gap of Cyt-1.times.Stx2eB (PG12) and the BamHI-BglII gap of Chl-1.times.Stx2eB (PG12) to prepare cytoplasm type of 2.times.CTB (PG12) and chloroplast type of 2.times.CTB (PG12) (Cyt-2.times.CTB (PG12), Chl-2.times.CTB (PG12)).

[0222] (3) Transient Expression Experiments and Western Analysis

[0223] Transient expression experiments were carried out using Lactuca sativa protoplasts in the same way as in above <1> (3). Subsequently, Stx2eB and CTB were detected in the same way as in <1> (4).

[0224] (a) Effects of Linking Number of Stx2eB

[0225] The results are shown in FIG. 15.

[0226] When ER-1.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 10 kDa. When ER-2.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 19 kDa, the signal being larger than that when ER-1.times.Stx2eB (PG12) was expressed. When ER-3.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 27 kDa, the signal being larger than that when ER-1.times.Stx2eB (PG12) was expressed. These corresponded to the molecular weights estimated from the design of the DNA constructs. When ER-4.times.Stx2eB (PG12) was expressed, a specific signal was below the detection limit.

[0227] When Cyt-1.times.Stx2eB (PG12) was expressed, a specific signal was below the detection limit. When Cyt-2.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 20 kDa. When Cyt-3.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 30 kDa. These corresponded to the molecular weights estimated from the design of the DNA constructs. Further, when Cyt-4.times.Stx2eB (PG12) was expressed, a specific signal was below the detection limit.

[0228] When Chl-1.times.Stx2eB (PG12) was expressed, a signal was detected faintly at the position of about 14 kDa. When Chl-2.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 22 kDa, the signal being at the similar level to that when ER-3.times.Stx2eB (PG12) was expressed. When Chl-3.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 30 kDa, the signal being at the similar level to that when Chl-2.times.Stx2eB (PG12) was expressed. These corresponded to the molecular weights estimated from the design of the DNA constructs. When Chl-4.times.Stx2eB (PG12) was expressed, a signal was detected faintly at the position of about 34 kDa.

[0229] Since each of the above DNA constructs contains one molecule of the HA tag (see FIG. 13), when DNA encoding two Stx2eBs is expressed and when DNA encoding three Stx2eBs is expressed, the amounts of the accumulated proteins are thought to correspond to about two times and about three times, respectively, the amount when DNA containing one Stx2eB is expressed.

[0230] Therefore, it has been found that when any of the endoplasmic reticulum type (ER), cytoplasm type (Cyt), and chloroplast type (Chl) of DNA constructs is expressed, the Stx2eB protein can be more efficiently accumulated when the protein in which two or three Stx2eBs are tandemly linked through the spacer is expressed than when one Stx2eB protein is expressed.

[0231] (b) Effects of Linking Number of CTB

[0232] The results are shown in FIG. 16.

[0233] When ER-1.times.CTB (PG12) was expressed, a signal was detected at the position of about 17 kDa. When ER-2.times.CTB (PG12) was expressed, a larger signal than that when ER-1.times.Stx2eB (PG12) was expressed was detected at the positions of about 28 kDa and about 30 kDa. These corresponded to the molecular weights estimated from the design of the DNA constructs.

[0234] When Cyt-1.times.CTB (PG12) was expressed, a signal was faintly detected at the position of about 14 kDa. When Cyt-2.times.CTB (PG12) was expressed, a signal was detected at the position of about 26 kDa, the signal being at the similar level to that when ER-2.times.CTB (PG12) was expressed. These corresponded to the molecular weights estimated from the design of the DNA constructs.

[0235] When Chl-1.times.CTB (PG12) was expressed, a signal was detected at the position of about 14 kDa. When Chl-2.times.CTB (PG12) was expressed, a signal was detected at the position of about 26 kDa, the signal being at the similar level to that when ER-2.times.CTB (PG12) was expressed. These corresponded to the molecular weights estimated from the design of the DNA constructs.

[0236] From the above, it has been found that when any of the endoplasmic reticulum type (ER), cytoplasm type (Cyt), and chloroplast type (Chl) of DNA constructs is expressed, CTB proteins can be more efficiently accumulated when the protein in which two CTBs are tandemly linked through the spacer is expressed than when one CTB protein is expressed.

<4> Transformation Experiments Using Cultured Tobacco Cells

(1) Construction of Vectors for Transformation

[0237] Transformation experiments were performed using ER-2.times.Stx2eB (PG12), Cyt-1.times.Stx2eB (PG12), Cyt-2.times.Stx2eB (PG12), and Cyt-3.times.Stx2eB (PG12) prepared above.

[0238] Vectors for transformation were prepared in the same way as the method in above <2> (1).

(2) Transformation and Western Analysis of Cultured Tobacco Cells

[0239] Transformation experiments and the western analysis were carried out in the same ways as the methods in above <2> (2) and (3).

[0240] The results are shown in FIG. 17.

[0241] When ER-2.times.Stx2eB (PG12) was expressed, a signal was detected at the positions of about 19, 21, and 23 kDa. When Cyt-1.times.Stx2eB (PG12) was expressed, a specific signal was below the detection limit. When Cyt-2.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 19 kDa. When Cyt-3.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 27 kDa, the signal being larger than that when Cyt-2.times.Stx2eB (PG12) was expressed.

[0242] From the above, it has been found that also in the transformant of the cultured tobacco cell, Stx2eB proteins can be more efficiently accumulated when the protein in which two or three Stx2eBs are tandemly linked through the spacer is expressed than when one Stx2eB protein is expressed. It has been also found that when the cytoplasm type of DNA construct is expressed in the transformant of the cultured tobacco cell, in particular when the protein in which three Stx2eBs are tandemly linked through the spacer is expressed, the Stx2eB proteins can be efficiently accumulated.

[0243] It has been also found that in the transformant of the cultured tobacco cell, the Stx2eB proteins can be more efficiently accumulated when the endoplasmic reticulum type of DNA construct is expressed than when the cytoplasm type of DNA construct is expressed.

<5> Transformation Experiments Using Tobacco Plant Body

[0244] (1) Construction of Vectors for Transformation

[0245] Transformation experiments were performed using ER-2.times.Stx2eB (PG12), Chl-1.times.Stx2eB (PG12), Chl-2.times.Stx2eB (PG12), and Chl-3.times.Stx2eB (PG12) prepared above.

[0246] Vectors for transformation were prepared in the same way as the method in above <2> (1).

[0247] (2) Transformation of Tobacco Plant Body

[0248] Tobacco plant bodies were transformed by the following method using the vectors prepared above.

[0249] Seeds of the tobacco plant body (Nicotiana tabacum L. cv. Petit habana SR1) were sterilized and seeded on an MS medium. A leaf portion of the sterilized Nicotiana tabacum was cut into pieces each having a size of about 1.times.1 cm without including leaf veins, and placed to face up the backside of the leaf in a petri-dish containing sterile water. An Agrobacterium suspension cultured for two nights in the LB medium containing 100 mg/L of kanamycin and obtained in above <2> (2) was poured in the petri-dish, and the leaf piece was immersed therein for 3 to 5 minutes. The leaf piece was picked up, and an extra bacterial medium on the leaf piece was wiped with sterile kim-towel. The leaf piece was placed on a callus formation medium and cultured at 25.degree. C. After 2 to 3 days, when Agrobacterium became visible on the medium, the leaf piece was transferred into a 50-mL tube, washed five times with sterile water, placed on a callus formation medium (containing 100 mg/L of kanamycin and 250 mg/L of carbenicillin), and cultured at 25.degree. C. for 1 to 2 weeks. When the leaf piece curled up compared with the original and showed a concavoconvex surface, the leaf piece was transferred into a shoot formation medium (containing 100 mg/L of kanamycin and 250 mg/L of carbenicillin). After additional 4 to 6 weeks, a shoot having a developed stem and leaf portion was cut off, transferred to a root formation medium (containing 100 mg/L of kanamycin and 250 mg/L of carbenicillin), and cultured at 25.degree. C. until rhizogenesis was observed. A plant body grown to a certain size was grown as a pot plant.

[0250] (3) Western Analysis

[0251] The leaf of the genetically engineered tobacco plant body produced above was sampled, and an SDS sample buffer was added thereto in the proportion of 1 .mu.L of SDS to 1 mg of leaf. The sample was thermally denatured at 95.degree. C. for 2 minutes to serve as the sample for electrophoresis. Proteins were separated using 15% acrylamide gel, and then blotted onto a PVDF membrane (Hybond-P; Amersham) using an electrotransfer apparatus. The Stx2eB protein was detected using an anti-HA antibody (No. 11 867 423 001, Roche).

[0252] The results are shown in FIGS. 18 and 19.

[0253] Clones in which Stx2eB was accumulated with high efficiency were obtained in the plant body transformed with one of ER-2.times.Stx2eB (PG12), Chl-1.times.Stx2eB (PG12), Chl-2.times.Stx2eB (PG12), and Chl-3.times.Stx2eB (PG12). Also in the chloroplast type of DNA constructs, it has been found that the clone accumulating Stx2eB efficiently is obtained with a higher probability when the protein in which two or three Stx2eBs are tandemly linked through the spacer is expressed than when one Stx2eB protein is expressed.

[0254] When ER-2.times.Stx2eB (PG12) was expressed, signals were detected at the positions of about 15 kDa, about 19 kDa, and about 22 kDa. When Chl-1.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 12 kDa. When Chl-2.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 19 kDa. When Chl-3.times.Stx2eB (PG12) was expressed, a signal was detected at the position of about 27 kDa. These corresponded to the molecular weights estimated from the design of the DNA constructs.

<6> Transformation Experiments Using Cultured Tobacco Cells

(1) Construction of Vectors for Stx2eB Transformation

[0255] ER-2.times.Stx2eB (PG12) was prepared by the method in above <1> (1). ER-2.times.Stx2eB (PG17) and ER-2.times.Stx2eB (PG22) were prepared by the following method. The design of the DNA constructs is shown in FIG. 20.

[0256] A PG7-F primer (SEQ ID NO: 65) and a PG7-R primer (SEQ ID NO: 66) were annealed and phosphorylated with T4 PNK. The resulting phosphorylated DNA fragment was inserted into the BglII gap of ER-1.times.Stx2eB (PG12) obtained in <1> (1) (plasmid 14).

[0257] A PG12-F primer (SEQ ID NO: 43) and a PG12-R primer (SEQ ID NO: 44) were annealed and phosphorylated with T4 PNK. The resulting phosphorylated DNA fragment was inserted into the BglII gap of ER-1.times.Stx2eB (PG12) (plasmid 15).

[0258] An Stx2eB-PG17 fragment was cut out from plasmid 14 using BamHI and BglII, and inserted into the BamHI gap of ER-1.times.Stx2eB (PG12) (2.times.Stx2eB (PG17)). An Stx2eB-PG22 fragment was cut out from plasmid 15 using BamHI and BglII, and inserted into the BamHI gap of ER-1.times.Stx2eB (PG12) (ER-2.times.Stx2eB (PG22)).

[0259] In order to produce Stx2eB using the stable transformant of the plant, each of the above DNA constructs for Stx2eB was subcloned into a vector for transformation. That is, each of ER-2.times.Stx2eB (PG12), ER-2.times.Stx2eB (PG17), and ER-2.times.Stx2eB (PG22) was inserted into pBI121 (Clontech) using XbaI and SacI, and allocated between the cauliflower mosaic virus 35S RNA promoter (35S pro.) and the nopaline synthetase gene transcription terminator (NOS-T).

[0260] (2) Construction of Vectors for CTB Transformation

[0261] ER-2.times.CTB (PG12) was prepared by the method in above <1> (2). ER-2.times.CTB (PG17) and ER-2.times.CTB (PG22) were prepared by the following method. The design of the DNA constructs is shown in FIG. 21.

[0262] A PG7-F primer (SEQ ID NO: 65) and a PG7-R primer (SEQ ID NO: 66) were annealed and phosphorylated with T4 PNK. The resulting phosphorylated DNA fragment was inserted into the BglII gap of ER-1.times.CTB (PG12) obtained in <1> (2) (plasmid 16). A PG12-F primer (SEQ ID NO: 43) and a PG12-R primer (SEQ ID NO: 44) were annealed and phosphorylated with T4 PNK. The resulting phosphorylated DNA fragment was inserted into the BglII gap of ER-1.times.CTB (PG12) (plasmid 17).

[0263] A CTB-PG17 fragment was cut out from plasmid 16 using BamHI and BglII, and inserted into the BamHI gap of ER-1.times.CTB (PG12) (ER-2.times.CTB (PG17)). A CTB-PG22 fragment was cut out from plasmid 17 using BamHI and BglII, and inserted into the BamHI gap of ER-1.times.CTB (PG12) (ER-2.times.CTB (PG22)).

[0264] In order to produce CTB using the stable transformant of the plant, each of the above DNA constructs for CTB was subcloned into a vector for transformation. That is, each of ER-2.times.CTB (PG12), ER-2.times.CTB (PG17), and ER-2.times.CTB (PG22) was inserted into pBI121 (Clontech) using XbaI and SacI, and allocated between the cauliflower mosaic virus 35S RNA promoter (35S pro.) and the nopaline synthetase gene transcription terminator (NOS-T).

[0265] (3) Transformation Experiments and Western Analysis

[0266] Transformation experiments and western analysis were carried out by the methods in above <2> (2) and (3).

[0267] (a) Effects of Length of Spacer on Tandem Linking of Stx2eB

[0268] The results are shown in FIG. 22.

[0269] When one of ER-2.times.Stx2eB (PG17) and ER-2.times.Stx2eB (PG22) was expressed, a signal was detected at the similar level to that when ER-2.times.Stx2eB (PG12) was expressed. This indicates that any of PG17 and PG22 exhibits the same effect as PG12.

[0270] When ER-2.times.Stx2eB (PG12) was expressed, signals were detected at the positions of about 19 kDa and about 22 kDa. When ER-2.times.Stx2eB (PG17) was expressed, signals were detected at the positions of about 19 kDa and about 22 kDa. When ER-2.times.Stx2eB (PG22) was expressed, signals were detected at the positions of about 20 kDa and about 23 kDa. These corresponded to the molecular weights estimated from the design of the DNA constructs.

[0271] (b) Effects of Length of Spacer on Tandem Linking of CTB

[0272] The results are shown in FIG. 23.

[0273] When one of ER-2.times.CTB (PG17) and ER-2.times.CTB (PG22) was expressed, a signal was detected at the similar level to that when ER-2.times.CTB (PG12) was expressed. This indicates that any of PG17 and PG22 exhibits the same effect as PG12.

[0274] When ER-2.times.CTB (PG12) was expressed, signals were detected at the positions of about 32 kDa, about 34 kDa, and about 36 kDa. When ER-2.times.CTB (PG17) was expressed, signals were detected at the positions of about 32 kDa, about 34 kDa, and about 36 kDa. When ER-2.times.CTB (PG22) was expressed, signals were detected at the positions of about 32 kDa, about 34 kDa, and about 36 kDa. These corresponded to the molecular weights estimated from the design of the DNA constructs.

INDUSTRIAL APPLICABILITY

[0275] The hybrid protein of the present invention is highly stable and accumulated at a high level in plant cells. Besides, by producing the hybrid protein of the present invention in the plant using the DNA construct of the present invention, it is possible to efficiently produce oral vaccines for Shiga toxin, cholera toxin, and Escherichia coli heat-labile toxin.

[0276] The present invention enables to express a bacterial antigen in the plant at the level which is enough to induce immunity. The present invention enables to give the immunity against the bacterial antigen to the animal at low cost by giving a transgenic plant as food to the animal. For example, the present invention is useful for developing swine edema disease vaccine and cholera vaccine.

Sequence CWU 1

1

114136DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 1agatcccctg gttctggtcc tggttctcct agatcc 36212PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 2Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg Ser 1 5 10 3959DNAEscherichia coli 3atgaagtgta tattgttaaa gtggatactg tgtctgttac tgggtttttc ttcggtatcc 60tattcccagg agtttacgat agacttttcg actcaacaaa gttatgtatc ttcgttaaat 120agtatacgga cagcgatatc gacccctctt gaacatatat ctcagggagc tacatcggta 180tccgttatta atcatacacc accaggaagt tatatttccg taggtatacg agggcttgat 240gtttatcagg agcgttttga ccatcttcgt ctgattattg aacgaaataa tttatatgtg 300gctggatttg ttaatacgac aacaaatact ttctacagat tttcagattt gcacatatat 360cattgcccgg tgtgacaact atttccatga caacggacag cagttatacc actctgcaac 420gtgtcgcagc gctggaacgt tccggaatgc aaatcagtcg tcactcactg gtttcatcat 480atctggcgtt aatggagttc agtggtaata caatgaccag agatgcatca agagcagttc 540tgcgttttgt cactgtcaca gcagaagcct tacggttcag gcaaatacag agagaatttc 600gtctggcact gtctgaaact gctcctgttt atacgatgac gccggaagac gtggacctca 660ctctgaactg ggggagaatc agcaatgtgc ttccggagta tcggggagag gctggtgtca 720gagtggggag aatatccttt aataatatat cagcgatact tggtactgtg gccgttatac 780tgaattgcca tcatcagggc gcacgttctg ttcgcgccgt gaatgaagag agtcaaccag 840aatgtcagat aactggcgac aggcccgtta taaaaataaa caatacatta tgggaaagta 900atacagcagc agcgtttctg aacagaaagt cacagccttt atatacaact ggtgaatga 9594304PRTEscherichia coli 4Met Lys Cys Ile Leu Leu Lys Trp Ile Leu Cys Leu Leu Leu Gly Phe 1 5 10 15 Ser Ser Val Ser Tyr Ser Gln Glu Phe Thr Ile Asp Phe Ser Thr Gln 20 25 30 Gln Ser Tyr Val Ser Ser Leu Asn Ser Ala Ile Ser Thr Pro Leu Glu 35 40 45 His Ile Ser Gln Gly Ala Thr Ser Val Ser Val Ile Asn His Thr Pro 50 55 60 Pro Gly Ser Tyr Ile Ser Val Gly Ile Arg Gly Leu Asp Val Tyr Gln 65 70 75 80 Glu Arg Phe Asp His Leu Arg Leu Ile Ile Glu Arg Asn Asn Leu Tyr 85 90 95 Phe Val Asn Thr Thr Thr Asn Thr Phe Tyr Arg Phe Ser Asp Phe Ala 100 105 110 His Ile Ser Leu Pro Gly Val Thr Thr Ile Ser Met Thr Thr Asp Ser 115 120 125 Ser Tyr Thr Thr Leu Gln Arg Val Ala Ala Leu Glu Arg Ser Gly Met 130 135 140 Gln Ile Ser Arg His Ser Leu Tyr Leu Ala Leu Met Glu Phe Ser Gly 145 150 155 160 Asn Thr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg Phe Val Thr 165 170 175 Val Thr Ala Glu Ala Leu Arg Phe Arg Gln Ile Gln Arg Glu Phe Arg 180 185 190 Leu Ala Leu Ser Glu Thr Ala Pro Val Tyr Thr Met Thr Pro Asp Leu 195 200 205 Thr Leu Asn Trp Gly Arg Ile Ser Asn Val Leu Pro Glu Tyr Arg Gly 210 215 220 Glu Ala Gly Val Arg Val Gly Arg Ile Ser Phe Asn Asn Ile Ser Ala 225 230 235 240 Ile Leu Gly Thr Val Ala Val Ile Leu Asn Cys His His Gln Gly Ala 245 250 255 Arg Ser Val Arg Ala Glu Ser Gln Pro Glu Cys Gln Ile Thr Gly Asp 260 265 270 Arg Pro Val Ile Lys Ile Asn Asn Thr Leu Trp Glu Ser Asn Thr Ala 275 280 285 Ala Ala Phe Leu Asn Arg Lys Ser Gln Pro Leu Tyr Thr Thr Gly Glu 290 295 300 5210DNAEscherichia coli 5gcggcggatt gtgctaaagg taaaattgag ttttccaagt ataatgagga taataccttt 60actgtgaagg tgtcaggaag agaatactgg acgaacagat ggaatttgca gccattgtta 120caaagtgctc agctgacagg gatgactgta acaatcatat ctaatacctg cagttcaggc 180tcaggctttg cccaggtgaa gtttaactga 210669PRTEscherichia coli 6Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu 1 5 10 15 Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn 20 25 30 Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met 35 40 45 Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala 50 55 60 Gln Val Lys Phe Asn 65 7312DNAVibrio cholerae 7accccccaga acatcaccga cctctgcgcc gagagccaca acacccaaat ctacaccctc 60aacgacaaga ttttcagcta caccgagagc ctcgccggca agagggagat ggccatcatc 120accttcaaga acggcgccat cttccaggtc gaggtccccg gcagccagca catcgacagc 180cagaagaagg ccatcgagag gatgaaggac accctcagga tcgcctacct caccgaggcc 240aaggtcgaga agctctgcgt ctggaacaac aagacccccc acgccatcgc cgccatcagc 300atggccaact ga 3128103PRTVibrio cholerae 8Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His Asn Thr Gln 1 5 10 15 Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala 65 70 75 80 Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95 Ala Ala Ile Ser Met Ala Asn 100 9453DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 9gcggcggatt gtgctaaagg taaaattgag ttttccaagt ataatgagga taataccttt 60actgtgaagg tgtcaggaag agaatactgg acgaacagat ggaatttgca gccattgtta 120caaagtgctc agctgacagg gatgactgta acaatcatat ctaatacctg cagttcaggc 180tcaggctttg cccaggtgaa gtttaacaga tcccctggtt ctggtcctgg ttctcctaga 240tccgcggcgg attgtgctaa aggtaaaatt gagttttcca agtataatga ggataatacc 300tttactgtga aggtgtcagg aagagaatac tggacgaaca gatggaattt gcagccattg 360ttacaaagtg ctcagctgac agggatgact gtaacaatca tatctaatac ctgcagttca 420ggctcaggct ttgcccaggt gaagtttaac tga 45310150PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 10Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu 1 5 10 15 Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn 20 25 30 Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met 35 40 45 Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala 50 55 60 Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg 65 70 75 80 Ser Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn 85 90 95 Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr 100 105 110 Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly 115 120 125 Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe 130 135 140 Ala Gln Val Lys Phe Asn 145 150 11657DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 11accccccaga acatcaccga cctctgcgcc gagagccaca acacccaaat ctacaccctc 60aacgacaaga ttttcagcta caccgagagc ctcgccggca agagggagat ggccatcatc 120accttcaaga acggcgccat cttccaggtc gaggtccccg gcagccagca catcgacagc 180cagaagaagg ccatcgagag gatgaaggac accctcagga tcgcctacct caccgaggcc 240aaggtcgaga agctctgcgt ctggaacaac aagacccccc acgccatcgc cgccatcagc 300atggccaaca gatcccctgg ttctggtcct ggttctccta gatccacccc ccagaacatc 360accgacctct gcgccgagag ccacaacacc caaatctaca ccctcaacga caagattttc 420agctacaccg agagcctcgc cggcaagagg gagatggcca tcatcacctt caagaacggc 480gccatcttcc aggtcgaggt ccccggcagc cagcacatcg acagccagaa gaaggccatc 540gagaggatga aggacaccct caggatcgcc tacctcaccg aggccaaggt cgagaagctc 600tgcgtctgga acaacaagac cccccacgcc atcgccgcca tcagcatggc caactga 65712218PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 12Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His Asn Thr Gln 1 5 10 15 Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala 65 70 75 80 Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95 Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser 100 105 110 Pro Arg Ser Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His 115 120 125 Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu 130 135 140 Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly 145 150 155 160 Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln 165 170 175 Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu 180 185 190 Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro 195 200 205 His Ala Ile Ala Ala Ile Ser Met Ala Asn 210 215 13489DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 13gcggcggatt gtgctaaagg taaaattgag ttttccaagt ataatgagga taataccttt 60actgtgaagg tgtcaggaag agaatactgg acgaacagat ggaatttgca gccattgtta 120caaagtgctc agctgacagg gatgactgta acaatcatat ctaatacctg cagttcaggc 180tcaggctttg cccaggtgaa gtttaacaga tcccctggtt ctggtcctgg ttctcctaga 240tccgcggcgg attgtgctaa aggtaaaatt gagttttcca agtataatga ggataatacc 300tttactgtga aggtgtcagg aagagaatac tggacgaaca gatggaattt gcagccattg 360ttacaaagtg ctcagctgac agggatgact gtaacaatca tatctaatac ctgcagttca 420ggctcaggct ttgcccaggt gaagtttaac agatcccctg gttctggtcc tggttctcct 480agatcttga 48914162PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 14Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu 1 5 10 15 Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn 20 25 30 Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met 35 40 45 Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala 50 55 60 Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg 65 70 75 80 Ser Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn 85 90 95 Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr 100 105 110 Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly 115 120 125 Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe 130 135 140 Ala Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro 145 150 155 160 Arg Ser 15693DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 15accccccaga acatcaccga cctctgcgcc gagagccaca acacccaaat ctacaccctc 60aacgacaaga ttttcagcta caccgagagc ctcgccggca agagggagat ggccatcatc 120accttcaaga acggcgccat cttccaggtc gaggtccccg gcagccagca catcgacagc 180cagaagaagg ccatcgagag gatgaaggac accctcagga tcgcctacct caccgaggcc 240aaggtcgaga agctctgcgt ctggaacaac aagacccccc acgccatcgc cgccatcagc 300atggccaaca gatcccctgg ttctggtcct ggttctccta gatccacccc ccagaacatc 360accgacctct gcgccgagag ccacaacacc caaatctaca ccctcaacga caagattttc 420agctacaccg agagcctcgc cggcaagagg gagatggcca tcatcacctt caagaacggc 480gccatcttcc aggtcgaggt ccccggcagc cagcacatcg acagccagaa gaaggccatc 540gagaggatga aggacaccct caggatcgcc tacctcaccg aggccaaggt cgagaagctc 600tgcgtctgga acaacaagac cccccacgcc atcgccgcca tcagcatggc caacagatcc 660cctggttctg gtcctggttc tcctagatct tga 69316230PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 16Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His Asn Thr Gln 1 5 10 15 Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala 65 70 75 80 Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95 Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser 100 105 110 Pro Arg Ser Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His 115 120 125 Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu 130 135 140 Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly 145 150 155 160 Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln 165 170 175 Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu 180 185 190 Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro 195 200 205 His Ala Ile Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly 210 215 220 Pro Gly Ser Pro Arg Ser 225 230 1772DNANicotiana tabacum 17atggggagaa tgtcaatacc catgatgggt tttgtggtgt tatgtctatg ggcagtggta 60gcagaaggat cc 721824PRTNicotiana tabacum 18Met Gly Arg Met Ser Ile Pro Met Met Gly Phe Val Val Leu Cys Leu 1 5 10 15 Trp Ala Val Val Ala Glu Gly Ser 20 194PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 19Lys Asp Glu Leu 1 204PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 20His Asp Glu Leu 1 214PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 21Lys Asp Glu Phe 1 224PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 22His Asp Glu Phe 1 2391DNANicotiana tabacum 23tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata a 9124547DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 24tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggcggcg gattgtgcta aaggtaaaat 120tgagttttcc aagtataatg aggataatac ctttactgtg aaggtgtcag gaagagaata 180ctggacgaac agatggaatt tgcagccatt gttacaaagt gctcagctga cagggatgac 240tgtaacaatc atatctaata cctgcagttc aggctcaggc tttgcccagg tgaagtttaa 300cagatcccct ggttctggtc ctggttctcc tagatccgcg gcggattgtg ctaaaggtaa 360aattgagttt tccaagtata atgaggataa tacctttact gtgaaggtgt caggaagaga 420atactggacg aacagatgga atttgcagcc attgttacaa agtgctcagc tgacagggat 480gactgtaaca atcatatcta atacctgcag ttcaggctca ggctttgccc aggtgaagtt 540taactga 54725751DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 25tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatgaccccc cagaacatca ccgacctctg 120cgccgagagc cacaacaccc aaatctacac cctcaacgac aagattttca gctacaccga 180gagcctcgcc ggcaagaggg agatggccat catcaccttc aagaacggcg ccatcttcca 240ggtcgaggtc cccggcagcc agcacatcga cagccagaag aaggccatcg agaggatgaa 300ggacaccctc aggatcgcct

acctcaccga ggccaaggtc gagaagctct gcgtctggaa 360caacaagacc ccccacgcca tcgccgccat cagcatggcc aacagatccc ctggttctgg 420tcctggttct cctagatcca ccccccagaa catcaccgac ctctgcgccg agagccacaa 480cacccaaatc tacaccctca acgacaagat tttcagctac accgagagcc tcgccggcaa 540gagggagatg gccatcatca ccttcaagaa cggcgccatc ttccaggtcg aggtccccgg 600cagccagcac atcgacagcc agaagaaggc catcgagagg atgaaggaca ccctcaggat 660cgcctacctc accgaggcca aggtcgagaa gctctgcgtc tggaacaaca agacccccca 720cgccatcgcc gccatcagca tggccaactg a 75126637DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 26tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccgcggcgg attgtgctaa 180aggtaaaatt gagttttcca agtataatga ggataatacc tttactgtga aggtgtcagg 240aagagaatac tggacgaaca gatggaattt gcagccattg ttacaaagtg ctcagctgac 300agggatgact gtaacaatca tatctaatac ctgcagttca ggctcaggct ttgcccaggt 360gaagtttaac agatcccctg gttctggtcc tggttctcct agatccgcgg cggattgtgc 420taaaggtaaa attgagtttt ccaagtataa tgaggataat acctttactg tgaaggtgtc 480aggaagagaa tactggacga acagatggaa tttgcagcca ttgttacaaa gtgctcagct 540gacagggatg actgtaacaa tcatatctaa tacctgcagt tcaggctcag gctttgccca 600ggtgaagttt aacagatctg aacatgatga attgtga 63727841DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 27tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccacccccc agaacatcac 180cgacctctgc gccgagagcc acaacaccca aatctacacc ctcaacgaca agattttcag 240ctacaccgag agcctcgccg gcaagaggga gatggccatc atcaccttca agaacggcgc 300catcttccag gtcgaggtcc ccggcagcca gcacatcgac agccagaaga aggccatcga 360gaggatgaag gacaccctca ggatcgccta cctcaccgag gccaaggtcg agaagctctg 420cgtctggaac aacaagaccc cccacgccat cgccgccatc agcatggcca acagatcccc 480tggttctggt cctggttctc ctagatccac cccccagaac atcaccgacc tctgcgccga 540gagccacaac acccaaatct acaccctcaa cgacaagatt ttcagctaca ccgagagcct 600cgccggcaag agggagatgg ccatcatcac cttcaagaac ggcgccatct tccaggtcga 660ggtccccggc agccagcaca tcgacagcca gaagaaggcc atcgagagga tgaaggacac 720cctcaggatc gcctacctca ccgaggccaa ggtcgagaag ctctgcgtct ggaacaacaa 780gaccccccac gccatcgccg ccatcagcat ggccaacaga tctgaacatg atgaattgtg 840a 84128667DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 28tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccgcggcgg attgtgctaa 180aggtaaaatt gagttttcca agtataatga ggataatacc tttactgtga aggtgtcagg 240aagagaatac tggacgaaca gatggaattt gcagccattg ttacaaagtg ctcagctgac 300agggatgact gtaacaatca tatctaatac ctgcagttca ggctcaggct ttgcccaggt 360gaagtttaac agatcccctg gttctggtcc tggttctcct agatccgcgg cggattgtgc 420taaaggtaaa attgagtttt ccaagtataa tgaggataat acctttactg tgaaggtgtc 480aggaagagaa tactggacga acagatggaa tttgcagcca ttgttacaaa gtgctcagct 540gacagggatg actgtaacaa tcatatctaa tacctgcagt tcaggctcag gctttgccca 600ggtgaagttt aacagatccc ctggttctgg tcctggttct cctagatctg aacatgatga 660attgtga 66729871DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 29tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccacccccc agaacatcac 180cgacctctgc gccgagagcc acaacaccca aatctacacc ctcaacgaca agattttcag 240ctacaccgag agcctcgccg gcaagaggga gatggccatc atcaccttca agaacggcgc 300catcttccag gtcgaggtcc ccggcagcca gcacatcgac agccagaaga aggccatcga 360gaggatgaag gacaccctca ggatcgccta cctcaccgag gccaaggtcg agaagctctg 420cgtctggaac aacaagaccc cccacgccat cgccgccatc agcatggcca acagatcccc 480tggttctggt cctggttctc ctagatccac cccccagaac atcaccgacc tctgcgccga 540gagccacaac acccaaatct acaccctcaa cgacaagatt ttcagctaca ccgagagcct 600cgccggcaag agggagatgg ccatcatcac cttcaagaac ggcgccatct tccaggtcga 660ggtccccggc agccagcaca tcgacagcca gaagaaggcc atcgagagga tgaaggacac 720cctcaggatc gcctacctca ccgaggccaa ggtcgagaag ctctgcgtct ggaacaacaa 780gaccccccac gccatcgccg ccatcagcat ggccaacaga tcccctggtt ctggtcctgg 840ttctcctaga tctgaacatg atgaattgtg a 8713038DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30tatctagagc caccatggga tccgcggcgg attgtgct 383130DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31ttcaagatct gttaaacttc acctgggcaa 303223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 32gtcgacggta cccccgggga gct 233323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33ccccgggggt accgtcgaca gct 233441DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 34aatctagagt ctatttaact cagtattcag aaacaacaaa a 413530DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 35aaatgcatta tttttcttga tttccttcac 303630DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36aaatgcatgg ggagaatgtc aatacccatg 303730DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37tataggatcc cattattttt cttgatttcc 30387PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 38Gly Ser Glu His Asp Glu Leu 1 5 3921DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39gatctgaaca tgatgaattg t 214021DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 40gatcacaatt catcatgttc a 214133DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 41gatcttatcc ttatgattat cctgattatg ctg 334233DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 42gatccagcat aatcaggata atcataagga taa 334330DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 43gatcccctgg ttctggtcct ggttctccta 304430DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 44gatctaggag aaccaggacc agaaccaggg 304540DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 45ttggatccac cccccagaac atcaccgacc tctgcgccga 404639DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 46cgttgagggt gtagatttgg gtgttgtggc tctcggcgc 394740DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 47ccctcaacga caagattttc agctacaccg agagcctcgc 404840DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 48cttgaaggtg atgatggcca tctccctctt gccggcgagg 404932DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 49caccttcaag aacggcgcca tcttccaggt cg 325045DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 50cttctggctg tcgatgtgct ggctgccggg gacctcgacc tggaa 455145DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 51agccagaaga aggccatcga gaggatgaag gacaccctca ggatc 455243DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 52gcagagcttc tcgaccttgg cctcggtgag gtaggcgatc ctg 435330DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 53aagctctgcg tctggaacaa caagaccccc 305445DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 54aaagatctgt tggccatgct gatggcggcg atggcgtggg gggtc 455531DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 55tttggatcca gcaagggcga ggagctgttc a 315633DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 56tttagatctc ttgtacagct cgtccatgcc gag 335730DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 57aaaggatccg cctcctccga ggacgtcatc 305830DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 58aaaagatctg gcgccggtgg agtggcggcc 305930DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 59gatcaaaatg gggttgtcat tcggaaagtt 306030DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 60attccatcta tgccttgctt gcgatgttgt 306124DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 61ctagacaaga tccgtccatt gtgg 246224DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 62acccccaaca tcccacacgg tgaa 24637PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 63Arg Ser Pro Gly Ser Arg Ser 1 5 6412PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 64Arg Ser Gly Ser Gly Ser Gly Ser Gly Ser Arg Ser 1 5 10 6515DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 65gatcccctgg ttcca 156615DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 66gatctggaac caggg 156730DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 67gatccggttc tggttctggt tctggttcca 306830DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 68gatctggaac cagaaccaga accagaaccg 306925DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 69gtgatcagtg aaggaaatca agaaa 257023DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 70cataacacca caaaacccat cat 237122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 71ccaagccaaa gaagatcaag ca 227224DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 72ccctgaatca tcgaccttgt agaa 2473700DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 73tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccgcggcgg attgtgctaa 180aggtaaaatt gagttttcca agtataatga ggataatacc tttactgtga aggtgtcagg 240aagagaatac tggacgaaca gatggaattt gcagccattg ttacaaagtg ctcagctgac 300agggatgact gtaacaatca tatctaatac ctgcagttca ggctcaggct ttgcccaggt 360gaagtttaac agatcccctg gttctggtcc tggttctcct agatccgcgg cggattgtgc 420taaaggtaaa attgagtttt ccaagtataa tgaggataat acctttactg tgaaggtgtc 480aggaagagaa tactggacga acagatggaa tttgcagcca ttgttacaaa gtgctcagct 540gacagggatg actgtaacaa tcatatctaa tacctgcagt tcaggctcag gctttgccca 600ggtgaagttt aacagatccc ctggttctgg tcctggttct cctagatctt atccttatga 660ttatcctgat tatgctggat ctgaacatga tgaattgtga 70074904DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 74tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccacccccc agaacatcac 180cgacctctgc gccgagagcc acaacaccca aatctacacc ctcaacgaca agattttcag 240ctacaccgag agcctcgccg gcaagaggga gatggccatc atcaccttca agaacggcgc 300catcttccag gtcgaggtcc ccggcagcca gcacatcgac agccagaaga aggccatcga 360gaggatgaag gacaccctca ggatcgccta cctcaccgag gccaaggtcg agaagctctg 420cgtctggaac aacaagaccc cccacgccat cgccgccatc agcatggcca acagatcccc 480tggttctggt cctggttctc ctagatccac cccccagaac atcaccgacc tctgcgccga 540gagccacaac acccaaatct acaccctcaa cgacaagatt ttcagctaca ccgagagcct 600cgccggcaag agggagatgg ccatcatcac cttcaagaac ggcgccatct tccaggtcga 660ggtccccggc agccagcaca tcgacagcca gaagaaggcc atcgagagga tgaaggacac 720cctcaggatc gcctacctca ccgaggccaa ggtcgagaag ctctgcgtct ggaacaacaa 780gaccccccac gccatcgccg ccatcagcat ggccaacaga tcccctggtt ctggtcctgg 840ttctcctaga tcttatcctt atgattatcc tgattatgct ggatctgaac atgatgaatt 900gtga 9047521DNANicotiana tabacum 75gatttgttgg ttgatactat g 21767PRTNicotiana tabacum 76Asp Leu Leu Val Asp Thr Met 1 5 7745DNAArmoracia rusticana 77ctactccatg atatggtgga ggtcgttgac tttgttagct ctatg 457815PRTArmoracia rusticana 78Leu Leu His Asp Met Val Glu Val Val Asp Phe Val Ser Ser Met 1 5 10 15 7975PRTLactuca sativa 79Met Ala Ser Ile Ser Ser Ser Ala Ile Ala Thr Val Asn Arg Thr Thr 1 5 10 15 Ser Thr Gln Ala Ser Leu Ala Ala Pro Phe Thr Gly Leu Lys Ser Asn 20 25 30 Val Ala Phe Pro Val Thr Lys Lys Ala Asn Asn Asp Phe Ser Ser Leu 35 40 45 Pro Ser Asn Gly Gly Arg Val Gln Cys Met Lys Val Trp Pro Pro Ile 50 55 60 Gly Leu Lys Lys Tyr Glu Thr Leu Ser Tyr Leu 65 70 75 80225DNALactuca sativa 80atggcctcca tctcctcctc agccatcgcc accgtcaacc ggaccacctc cacccaagct 60agcttggcag ctccattcac cggcctcaag tctaacgtag ctttcccagt taccaagaag 120gctaacaatg acttttcatc cctacccagc aacggtggaa gagtacaatg catgaaggtg 180tggccaccaa ttgggttgaa gaagtacgag actctttcat accta 2258151DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 81agatcccctg gttctggtcc tggttctcct agatcccctg gttccagatc t 518217PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 82Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg Ser Pro Gly Ser Arg 1 5 10 15 Ser 8366DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 83agatcccctg gttctggtcc tggttctcct agatcccctg gttctggtcc tggttctcct 60agatct 668422PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 84Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg Ser Pro Gly Ser Gly 1 5 10 15 Pro Gly Ser Pro Arg Ser 20 85696DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 85gcggcggatt gtgctaaagg taaaattgag ttttccaagt ataatgagga taataccttt 60actgtgaagg tgtcaggaag agaatactgg acgaacagat ggaatttgca gccattgtta 120caaagtgctc agctgacagg gatgactgta acaatcatat ctaatacctg cagttcaggc 180tcaggctttg cccaggtgaa gtttaacaga tcccctggtt ctggtcctgg ttctcctaga 240tccgcggcgg attgtgctaa aggtaaaatt gagttttcca agtataatga ggataatacc 300tttactgtga aggtgtcagg aagagaatac tggacgaaca gatggaattt gcagccattg 360ttacaaagtg ctcagctgac agggatgact gtaacaatca tatctaatac ctgcagttca 420ggctcaggct ttgcccaggt gaagtttaac agatcccctg gttctggtcc tggttctcct 480agatccgcgg cggattgtgc taaaggtaaa attgagtttt ccaagtataa tgaggataat 540acctttactg tgaaggtgtc aggaagagaa tactggacga acagatggaa tttgcagcca 600ttgttacaaa gtgctcagct gacagggatg actgtaacaa tcatatctaa tacctgcagt 660tcaggctcag gctttgccca ggtgaagttt aactga 69686231PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 86Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu 1 5 10 15 Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn 20 25 30 Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met 35 40 45 Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala 50 55 60 Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg 65 70 75 80 Ser Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn 85 90 95 Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr 100 105 110 Asn Arg Trp Asn Leu Gln Pro Leu

Leu Gln Ser Ala Gln Leu Thr Gly 115 120 125 Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe 130 135 140 Ala Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro 145 150 155 160 Arg Ser Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr 165 170 175 Asn Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp 180 185 190 Thr Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr 195 200 205 Gly Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly 210 215 220 Phe Ala Gln Val Lys Phe Asn 225 230 87732DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 87gcggcggatt gtgctaaagg taaaattgag ttttccaagt ataatgagga taataccttt 60actgtgaagg tgtcaggaag agaatactgg acgaacagat ggaatttgca gccattgtta 120caaagtgctc agctgacagg gatgactgta acaatcatat ctaatacctg cagttcaggc 180tcaggctttg cccaggtgaa gtttaacaga tcccctggtt ctggtcctgg ttctcctaga 240tccgcggcgg attgtgctaa aggtaaaatt gagttttcca agtataatga ggataatacc 300tttactgtga aggtgtcagg aagagaatac tggacgaaca gatggaattt gcagccattg 360ttacaaagtg ctcagctgac agggatgact gtaacaatca tatctaatac ctgcagttca 420ggctcaggct ttgcccaggt gaagtttaac agatcccctg gttctggtcc tggttctcct 480agatccgcgg cggattgtgc taaaggtaaa attgagtttt ccaagtataa tgaggataat 540acctttactg tgaaggtgtc aggaagagaa tactggacga acagatggaa tttgcagcca 600ttgttacaaa gtgctcagct gacagggatg actgtaacaa tcatatctaa tacctgcagt 660tcaggctcag gctttgccca ggtgaagttt aacagatccc ctggttctgg tcctggttct 720cctagatcct ga 73288243PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 88Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu 1 5 10 15 Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn 20 25 30 Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met 35 40 45 Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala 50 55 60 Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg 65 70 75 80 Ser Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn 85 90 95 Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr 100 105 110 Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly 115 120 125 Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe 130 135 140 Ala Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro 145 150 155 160 Arg Ser Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr 165 170 175 Asn Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp 180 185 190 Thr Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr 195 200 205 Gly Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly 210 215 220 Phe Ala Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser 225 230 235 240 Pro Arg Ser 89504DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 89gcggcggatt gtgctaaagg taaaattgag ttttccaagt ataatgagga taataccttt 60actgtgaagg tgtcaggaag agaatactgg acgaacagat ggaatttgca gccattgtta 120caaagtgctc agctgacagg gatgactgta acaatcatat ctaatacctg cagttcaggc 180tcaggctttg cccaggtgaa gtttaacaga tcccctggtt ctggtcctgg ttctcctaga 240tcccctggtt ccagatctgc ggcggattgt gctaaaggta aaattgagtt ttccaagtat 300aatgaggata atacctttac tgtgaaggtg tcaggaagag aatactggac gaacagatgg 360aatttgcagc cattgttaca aagtgctcag ctgacaggga tgactgtaac aatcatatct 420aatacctgca gttcaggctc aggctttgcc caggtgaagt ttaacagatc ccctggttct 480ggtcctggtt ctcctagatc ttga 50490167PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 90Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu 1 5 10 15 Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn 20 25 30 Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met 35 40 45 Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala 50 55 60 Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg 65 70 75 80 Ser Pro Gly Ser Arg Ser Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu 85 90 95 Phe Ser Lys Tyr Asn Glu Asp Asn Thr Phe Thr Val Lys Val Ser Gly 100 105 110 Arg Glu Tyr Trp Thr Asn Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser 115 120 125 Ala Gln Leu Thr Gly Met Thr Val Thr Ile Ile Ser Asn Thr Cys Ser 130 135 140 Ser Gly Ser Gly Phe Ala Gln Val Lys Phe Asn Arg Ser Pro Gly Ser 145 150 155 160 Gly Pro Gly Ser Pro Arg Ser 165 91519DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 91gcggcggatt gtgctaaagg taaaattgag ttttccaagt ataatgagga taataccttt 60actgtgaagg tgtcaggaag agaatactgg acgaacagat ggaatttgca gccattgtta 120caaagtgctc agctgacagg gatgactgta acaatcatat ctaatacctg cagttcaggc 180tcaggctttg cccaggtgaa gtttaacaga tcccctggtt ctggtcctgg ttctcctaga 240tcccctggtt ctggtcctgg ttctcctaga tctgcggcgg attgtgctaa aggtaaaatt 300gagttttcca agtataatga ggataatacc tttactgtga aggtgtcagg aagagaatac 360tggacgaaca gatggaattt gcagccattg ttacaaagtg ctcagctgac agggatgact 420gtaacaatca tatctaatac ctgcagttca ggctcaggct ttgcccaggt gaagtttaac 480agatcccctg gttctggtcc tggttctcct agatcttga 51992172PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 92Ala Ala Asp Cys Ala Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu 1 5 10 15 Asp Asn Thr Phe Thr Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn 20 25 30 Arg Trp Asn Leu Gln Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met 35 40 45 Thr Val Thr Ile Ile Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala 50 55 60 Gln Val Lys Phe Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg 65 70 75 80 Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg Ser Ala Ala Asp Cys Ala 85 90 95 Lys Gly Lys Ile Glu Phe Ser Lys Tyr Asn Glu Asp Asn Thr Phe Thr 100 105 110 Val Lys Val Ser Gly Arg Glu Tyr Trp Thr Asn Arg Trp Asn Leu Gln 115 120 125 Pro Leu Leu Gln Ser Ala Gln Leu Thr Gly Met Thr Val Thr Ile Ile 130 135 140 Ser Asn Thr Cys Ser Ser Gly Ser Gly Phe Ala Gln Val Lys Phe Asn 145 150 155 160 Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg Ser 165 170 931002DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 93accccccaga acatcaccga cctctgcgcc gagagccaca acacccaaat ctacaccctc 60aacgacaaga ttttcagcta caccgagagc ctcgccggca agagggagat ggccatcatc 120accttcaaga acggcgccat cttccaggtc gaggtccccg gcagccagca catcgacagc 180cagaagaagg ccatcgagag gatgaaggac accctcagga tcgcctacct caccgaggcc 240aaggtcgaga agctctgcgt ctggaacaac aagacccccc acgccatcgc cgccatcagc 300atggccaaca gatcccctgg ttctggtcct ggttctccta gatccacccc ccagaacatc 360accgacctct gcgccgagag ccacaacacc caaatctaca ccctcaacga caagattttc 420agctacaccg agagcctcgc cggcaagagg gagatggcca tcatcacctt caagaacggc 480gccatcttcc aggtcgaggt ccccggcagc cagcacatcg acagccagaa gaaggccatc 540gagaggatga aggacaccct caggatcgcc tacctcaccg aggccaaggt cgagaagctc 600tgcgtctgga acaacaagac cccccacgcc atcgccgcca tcagcatggc caacagatcc 660cctggttctg gtcctggttc tcctagatcc accccccaga acatcaccga cctctgcgcc 720gagagccaca acacccaaat ctacaccctc aacgacaaga ttttcagcta caccgagagc 780ctcgccggca agagggagat ggccatcatc accttcaaga acggcgccat cttccaggtc 840gaggtccccg gcagccagca catcgacagc cagaagaagg ccatcgagag gatgaaggac 900accctcagga tcgcctacct caccgaggcc aaggtcgaga agctctgcgt ctggaacaac 960aagacccccc acgccatcgc cgccatcagc atggccaact ga 100294333PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 94Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His Asn Thr Gln 1 5 10 15 Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala 65 70 75 80 Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95 Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser 100 105 110 Pro Arg Ser Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His 115 120 125 Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu 130 135 140 Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly 145 150 155 160 Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln 165 170 175 Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu 180 185 190 Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro 195 200 205 His Ala Ile Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly 210 215 220 Pro Gly Ser Pro Arg Ser Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala 225 230 235 240 Glu Ser His Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser 245 250 255 Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe 260 265 270 Lys Asn Gly Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile 275 280 285 Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile 290 295 300 Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn 305 310 315 320 Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn 325 330 951038DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 95accccccaga acatcaccga cctctgcgcc gagagccaca acacccaaat ctacaccctc 60aacgacaaga ttttcagcta caccgagagc ctcgccggca agagggagat ggccatcatc 120accttcaaga acggcgccat cttccaggtc gaggtccccg gcagccagca catcgacagc 180cagaagaagg ccatcgagag gatgaaggac accctcagga tcgcctacct caccgaggcc 240aaggtcgaga agctctgcgt ctggaacaac aagacccccc acgccatcgc cgccatcagc 300atggccaaca gatcccctgg ttctggtcct ggttctccta gatccacccc ccagaacatc 360accgacctct gcgccgagag ccacaacacc caaatctaca ccctcaacga caagattttc 420agctacaccg agagcctcgc cggcaagagg gagatggcca tcatcacctt caagaacggc 480gccatcttcc aggtcgaggt ccccggcagc cagcacatcg acagccagaa gaaggccatc 540gagaggatga aggacaccct caggatcgcc tacctcaccg aggccaaggt cgagaagctc 600tgcgtctgga acaacaagac cccccacgcc atcgccgcca tcagcatggc caacagatcc 660cctggttctg gtcctggttc tcctagatct accccccaga acatcaccga cctctgcgcc 720gagagccaca acacccaaat ctacaccctc aacgacaaga ttttcagcta caccgagagc 780ctcgccggca agagggagat ggccatcatc accttcaaga acggcgccat cttccaggtc 840gaggtccccg gcagccagca catcgacagc cagaagaagg ccatcgagag gatgaaggac 900accctcagga tcgcctacct caccgaggcc aaggtcgaga agctctgcgt ctggaacaac 960aagacccccc acgccatcgc cgccatcagc atggccaaca gatcccctgg ttctggtcct 1020ggttctccta gatcttga 103896345PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 96Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His Asn Thr Gln 1 5 10 15 Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala 65 70 75 80 Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95 Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser 100 105 110 Pro Arg Ser Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His 115 120 125 Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu 130 135 140 Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly 145 150 155 160 Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln 165 170 175 Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu 180 185 190 Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro 195 200 205 His Ala Ile Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly 210 215 220 Pro Gly Ser Pro Arg Ser Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala 225 230 235 240 Glu Ser His Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser 245 250 255 Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe 260 265 270 Lys Asn Gly Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile 275 280 285 Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile 290 295 300 Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn 305 310 315 320 Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn Arg Ser Pro 325 330 335 Gly Ser Gly Pro Gly Ser Pro Arg Ser 340 345 97708DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 97accccccaga acatcaccga cctctgcgcc gagagccaca acacccaaat ctacaccctc 60aacgacaaga ttttcagcta caccgagagc ctcgccggca agagggagat ggccatcatc 120accttcaaga acggcgccat cttccaggtc gaggtccccg gcagccagca catcgacagc 180cagaagaagg ccatcgagag gatgaaggac accctcagga tcgcctacct caccgaggcc 240aaggtcgaga agctctgcgt ctggaacaac aagacccccc acgccatcgc cgccatcagc 300atggccaaca gatcccctgg ttctggtcct ggttctccta gatcccctgg ttccagatct 360accccccaga acatcaccga cctctgcgcc gagagccaca acacccaaat ctacaccctc 420aacgacaaga ttttcagcta caccgagagc ctcgccggca agagggagat ggccatcatc 480accttcaaga acggcgccat cttccaggtc gaggtccccg gcagccagca catcgacagc 540cagaagaagg ccatcgagag gatgaaggac accctcagga tcgcctacct caccgaggcc 600aaggtcgaga agctctgcgt ctggaacaac aagacccccc acgccatcgc cgccatcagc 660atggccaaca gatcccctgg ttctggtcct ggttctccta gatcttga 70898235PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 98Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His Asn Thr Gln 1 5 10 15 Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55

60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala 65 70 75 80 Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95 Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser 100 105 110 Pro Arg Ser Pro Gly Ser Arg Ser Thr Pro Gln Asn Ile Thr Asp Leu 115 120 125 Cys Ala Glu Ser His Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile 130 135 140 Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile 145 150 155 160 Thr Phe Lys Asn Gly Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln 165 170 175 His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu 180 185 190 Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp 195 200 205 Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn Arg 210 215 220 Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg Ser 225 230 235 99723DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 99accccccaga acatcaccga cctctgcgcc gagagccaca acacccaaat ctacaccctc 60aacgacaaga ttttcagcta caccgagagc ctcgccggca agagggagat ggccatcatc 120accttcaaga acggcgccat cttccaggtc gaggtccccg gcagccagca catcgacagc 180cagaagaagg ccatcgagag gatgaaggac accctcagga tcgcctacct caccgaggcc 240aaggtcgaga agctctgcgt ctggaacaac aagacccccc acgccatcgc cgccatcagc 300atggccaaca gatcccctgg ttctggtcct ggttctccta gatcccctgg ttctggtcct 360ggttctccta gatctacccc ccagaacatc accgacctct gcgccgagag ccacaacacc 420caaatctaca ccctcaacga caagattttc agctacaccg agagcctcgc cggcaagagg 480gagatggcca tcatcacctt caagaacggc gccatcttcc aggtcgaggt ccccggcagc 540cagcacatcg acagccagaa gaaggccatc gagaggatga aggacaccct caggatcgcc 600tacctcaccg aggccaaggt cgagaagctc tgcgtctgga acaacaagac cccccacgcc 660atcgccgcca tcagcatggc caacagatcc cctggttctg gtcctggttc tcctagatct 720tga 723100240PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 100Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Ser His Asn Thr Gln 1 5 10 15 Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala 65 70 75 80 Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95 Ala Ala Ile Ser Met Ala Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser 100 105 110 Pro Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg Ser Thr Pro Gln 115 120 125 Asn Ile Thr Asp Leu Cys Ala Glu Ser His Asn Thr Gln Ile Tyr Thr 130 135 140 Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg 145 150 155 160 Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Ile Phe Gln Val Glu 165 170 175 Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg 180 185 190 Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu 195 200 205 Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile 210 215 220 Ser Met Ala Asn Arg Ser Pro Gly Ser Gly Pro Gly Ser Pro Arg Ser 225 230 235 240 101583DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 101tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggcggcg gattgtgcta aaggtaaaat 120tgagttttcc aagtataatg aggataatac ctttactgtg aaggtgtcag gaagagaata 180ctggacgaac agatggaatt tgcagccatt gttacaaagt gctcagctga cagggatgac 240tgtaacaatc atatctaata cctgcagttc aggctcaggc tttgcccagg tgaagtttaa 300cagatcccct ggttctggtc ctggttctcc tagatccgcg gcggattgtg ctaaaggtaa 360aattgagttt tccaagtata atgaggataa tacctttact gtgaaggtgt caggaagaga 420atactggacg aacagatgga atttgcagcc attgttacaa agtgctcagc tgacagggat 480gactgtaaca atcatatcta atacctgcag ttcaggctca ggctttgccc aggtgaagtt 540taacagatcc cctggttctg gtcctggttc tcctagatcc tga 583102682DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 102tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccgcggcgg attgtgctaa 180aggtaaaatt gagttttcca agtataatga ggataatacc tttactgtga aggtgtcagg 240aagagaatac tggacgaaca gatggaattt gcagccattg ttacaaagtg ctcagctgac 300agggatgact gtaacaatca tatctaatac ctgcagttca ggctcaggct ttgcccaggt 360gaagtttaac agatcccctg gttctggtcc tggttctcct agatcccctg gttccagatc 420tgcggcggat tgtgctaaag gtaaaattga gttttccaag tataatgagg ataatacctt 480tactgtgaag gtgtcaggaa gagaatactg gacgaacaga tggaatttgc agccattgtt 540acaaagtgct cagctgacag ggatgactgt aacaatcata tctaatacct gcagttcagg 600ctcaggcttt gcccaggtga agtttaacag atcccctggt tctggtcctg gttctcctag 660atctgaacat gatgaattgt ga 682103697DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 103tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccgcggcgg attgtgctaa 180aggtaaaatt gagttttcca agtataatga ggataatacc tttactgtga aggtgtcagg 240aagagaatac tggacgaaca gatggaattt gcagccattg ttacaaagtg ctcagctgac 300agggatgact gtaacaatca tatctaatac ctgcagttca ggctcaggct ttgcccaggt 360gaagtttaac agatcccctg gttctggtcc tggttctcct agatcccctg gttctggtcc 420tggttctcct agatctgcgg cggattgtgc taaaggtaaa attgagtttt ccaagtataa 480tgaggataat acctttactg tgaaggtgtc aggaagagaa tactggacga acagatggaa 540tttgcagcca ttgttacaaa gtgctcagct gacagggatg actgtaacaa tcatatctaa 600tacctgcagt tcaggctcag gctttgccca ggtgaagttt aacagatccc ctggttctgg 660tcctggttct cctagatctg aacatgatga attgtga 697104805DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 104tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggcctcc atctcctcct cagccatcgc 120caccgtcaac cggaccacct ccacccaagc tagcttggca gctccattca ccggcctcaa 180gtctaacgta gctttcccag ttaccaagaa ggctaacaat gacttttcat ccctacccag 240caacggtgga agagtacaat gcatgaaggt gtggccacca attgggttga agaagtacga 300gactctttca tacctagcgg cggattgtgc taaaggtaaa attgagtttt ccaagtataa 360tgaggataat acctttactg tgaaggtgtc aggaagagaa tactggacga acagatggaa 420tttgcagcca ttgttacaaa gtgctcagct gacagggatg actgtaacaa tcatatctaa 480tacctgcagt tcaggctcag gctttgccca ggtgaagttt aacagatccc ctggttctgg 540tcctggttct cctagatccg cggcggattg tgctaaaggt aaaattgagt tttccaagta 600taatgaggat aataccttta ctgtgaaggt gtcaggaaga gaatactgga cgaacagatg 660gaatttgcag ccattgttac aaagtgctca gctgacaggg atgactgtaa caatcatatc 720taatacctgc agttcaggct caggctttgc ccaggtgaag tttaacagat cccctggttc 780tggtcctggt tctcctagat cctga 805105826DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 105tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggcggcg gattgtgcta aaggtaaaat 120tgagttttcc aagtataatg aggataatac ctttactgtg aaggtgtcag gaagagaata 180ctggacgaac agatggaatt tgcagccatt gttacaaagt gctcagctga cagggatgac 240tgtaacaatc atatctaata cctgcagttc aggctcaggc tttgcccagg tgaagtttaa 300cagatcccct ggttctggtc ctggttctcc tagatccgcg gcggattgtg ctaaaggtaa 360aattgagttt tccaagtata atgaggataa tacctttact gtgaaggtgt caggaagaga 420atactggacg aacagatgga atttgcagcc attgttacaa agtgctcagc tgacagggat 480gactgtaaca atcatatcta atacctgcag ttcaggctca ggctttgccc aggtgaagtt 540taacagatcc cctggttctg gtcctggttc tcctagatcc gcggcggatt gtgctaaagg 600taaaattgag ttttccaagt ataatgagga taataccttt actgtgaagg tgtcaggaag 660agaatactgg acgaacagat ggaatttgca gccattgtta caaagtgctc agctgacagg 720gatgactgta acaatcatat ctaatacctg cagttcaggc tcaggctttg cccaggtgaa 780gtttaacaga tcccctggtt ctggtcctgg ttctcctaga tcctga 826106910DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 106tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccgcggcgg attgtgctaa 180aggtaaaatt gagttttcca agtataatga ggataatacc tttactgtga aggtgtcagg 240aagagaatac tggacgaaca gatggaattt gcagccattg ttacaaagtg ctcagctgac 300agggatgact gtaacaatca tatctaatac ctgcagttca ggctcaggct ttgcccaggt 360gaagtttaac agatcccctg gttctggtcc tggttctcct agatccgcgg cggattgtgc 420taaaggtaaa attgagtttt ccaagtataa tgaggataat acctttactg tgaaggtgtc 480aggaagagaa tactggacga acagatggaa tttgcagcca ttgttacaaa gtgctcagct 540gacagggatg actgtaacaa tcatatctaa tacctgcagt tcaggctcag gctttgccca 600ggtgaagttt aacagatccc ctggttctgg tcctggttct cctagatccg cggcggattg 660tgctaaaggt aaaattgagt tttccaagta taatgaggat aataccttta ctgtgaaggt 720gtcaggaaga gaatactgga cgaacagatg gaatttgcag ccattgttac aaagtgctca 780gctgacaggg atgactgtaa caatcatatc taatacctgc agttcaggct caggctttgc 840ccaggtgaag tttaacagat cccctggttc tggtcctggt tctcctagat ccgaacatga 900tgaattgtga 9101071048DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 107tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggcctcc atctcctcct cagccatcgc 120caccgtcaac cggaccacct ccacccaagc tagcttggca gctccattca ccggcctcaa 180gtctaacgta gctttcccag ttaccaagaa ggctaacaat gacttttcat ccctacccag 240caacggtgga agagtacaat gcatgaaggt gtggccacca attgggttga agaagtacga 300gactctttca tacctagcgg cggattgtgc taaaggtaaa attgagtttt ccaagtataa 360tgaggataat acctttactg tgaaggtgtc aggaagagaa tactggacga acagatggaa 420tttgcagcca ttgttacaaa gtgctcagct gacagggatg actgtaacaa tcatatctaa 480tacctgcagt tcaggctcag gctttgccca ggtgaagttt aacagatccc ctggttctgg 540tcctggttct cctagatccg cggcggattg tgctaaaggt aaaattgagt tttccaagta 600taatgaggat aataccttta ctgtgaaggt gtcaggaaga gaatactgga cgaacagatg 660gaatttgcag ccattgttac aaagtgctca gctgacaggg atgactgtaa caatcatatc 720taatacctgc agttcaggct caggctttgc ccaggtgaag tttaacagat cccctggttc 780tggtcctggt tctcctagat ccgcggcgga ttgtgctaaa ggtaaaattg agttttccaa 840gtataatgag gataatacct ttactgtgaa ggtgtcagga agagaatact ggacgaacag 900atggaatttg cagccattgt tacaaagtgc tcagctgaca gggatgactg taacaatcat 960atctaatacc tgcagttcag gctcaggctt tgcccaggtg aagtttaaca gatcccctgg 1020ttctggtcct ggttctccta gatcctga 1048108787DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 108tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatgaccccc cagaacatca ccgacctctg 120cgccgagagc cacaacaccc aaatctacac cctcaacgac aagattttca gctacaccga 180gagcctcgcc ggcaagaggg agatggccat catcaccttc aagaacggcg ccatcttcca 240ggtcgaggtc cccggcagcc agcacatcga cagccagaag aaggccatcg agaggatgaa 300ggacaccctc aggatcgcct acctcaccga ggccaaggtc gagaagctct gcgtctggaa 360caacaagacc ccccacgcca tcgccgccat cagcatggcc aacagatccc ctggttctgg 420tcctggttct cctagatcca ccccccagaa catcaccgac ctctgcgccg agagccacaa 480cacccaaatc tacaccctca acgacaagat tttcagctac accgagagcc tcgccggcaa 540gagggagatg gccatcatca ccttcaagaa cggcgccatc ttccaggtcg aggtccccgg 600cagccagcac atcgacagcc agaagaaggc catcgagagg atgaaggaca ccctcaggat 660cgcctacctc accgaggcca aggtcgagaa gctctgcgtc tggaacaaca agacccccca 720cgccatcgcc gccatcagca tggccaacag atcccctggt tctggtcctg gttctcctag 780atcctga 787109886DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 109tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccacccccc agaacatcac 180cgacctctgc gccgagagcc acaacaccca aatctacacc ctcaacgaca agattttcag 240ctacaccgag agcctcgccg gcaagaggga gatggccatc atcaccttca agaacggcgc 300catcttccag gtcgaggtcc ccggcagcca gcacatcgac agccagaaga aggccatcga 360gaggatgaag gacaccctca ggatcgccta cctcaccgag gccaaggtcg agaagctctg 420cgtctggaac aacaagaccc cccacgccat cgccgccatc agcatggcca acagatcccc 480tggttctggt cctggttctc ctagatcccc tggttccaga tctacccccc agaacatcac 540cgacctctgc gccgagagcc acaacaccca aatctacacc ctcaacgaca agattttcag 600ctacaccgag agcctcgccg gcaagaggga gatggccatc atcaccttca agaacggcgc 660catcttccag gtcgaggtcc ccggcagcca gcacatcgac agccagaaga aggccatcga 720gaggatgaag gacaccctca ggatcgccta cctcaccgag gccaaggtcg agaagctctg 780cgtctggaac aacaagaccc cccacgccat cgccgccatc agcatggcca acagatcccc 840tggttctggt cctggttctc ctagatctga acatgatgaa ttgtga 886110901DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 110tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggggaga atgtcaatac ccatgatggg 120ttttgtggtg ttatgtctat gggcagtggt agcagaagga tccacccccc agaacatcac 180cgacctctgc gccgagagcc acaacaccca aatctacacc ctcaacgaca agattttcag 240ctacaccgag agcctcgccg gcaagaggga gatggccatc atcaccttca agaacggcgc 300catcttccag gtcgaggtcc ccggcagcca gcacatcgac agccagaaga aggccatcga 360gaggatgaag gacaccctca ggatcgccta cctcaccgag gccaaggtcg agaagctctg 420cgtctggaac aacaagaccc cccacgccat cgccgccatc agcatggcca acagatcccc 480tggttctggt cctggttctc ctagatcccc tggttctggt cctggttctc ctagatctac 540cccccagaac atcaccgacc tctgcgccga gagccacaac acccaaatct acaccctcaa 600cgacaagatt ttcagctaca ccgagagcct cgccggcaag agggagatgg ccatcatcac 660cttcaagaac ggcgccatct tccaggtcga ggtccccggc agccagcaca tcgacagcca 720gaagaaggcc atcgagagga tgaaggacac cctcaggatc gcctacctca ccgaggccaa 780ggtcgagaag ctctgcgtct ggaacaacaa gaccccccac gccatcgccg ccatcagcat 840ggccaacaga tcccctggtt ctggtcctgg ttctcctaga tctgaacatg atgaattgtg 900a 9011111009DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotide 111tatttaactc agtattcaga aacaacaaaa gttcttctct acataaaatt ttcctatttt 60agtgatcagt gaaggaaatc aagaaaaata aatggcctcc atctcctcct cagccatcgc 120caccgtcaac cggaccacct ccacccaagc tagcttggca gctccattca ccggcctcaa 180gtctaacgta gctttcccag ttaccaagaa ggctaacaat gacttttcat ccctacccag 240caacggtgga agagtacaat gcatgaaggt gtggccacca attgggttga agaagtacga 300gactctttca tacctaaccc cccagaacat caccgacctc tgcgccgaga gccacaacac 360ccaaatctac accctcaacg acaagatttt cagctacacc gagagcctcg ccggcaagag 420ggagatggcc atcatcacct tcaagaacgg cgccatcttc caggtcgagg tccccggcag 480ccagcacatc gacagccaga agaaggccat cgagaggatg aaggacaccc tcaggatcgc 540ctacctcacc gaggccaagg tcgagaagct ctgcgtctgg aacaacaaga ccccccacgc 600catcgccgcc atcagcatgg ccaacagatc ccctggttct ggtcctggtt ctcctagatc 660caccccccag aacatcaccg acctctgcgc cgagagccac aacacccaaa tctacaccct 720caacgacaag attttcagct acaccgagag cctcgccggc aagagggaga tggccatcat 780caccttcaag aacggcgcca tcttccaggt cgaggtcccc ggcagccagc acatcgacag 840ccagaagaag gccatcgaga ggatgaagga caccctcagg atcgcctacc tcaccgaggc 900caaggtcgag aagctctgcg tctggaacaa caagaccccc cacgccatcg ccgccatcag 960catggccaac agatcccctg gttctggtcc tggttctcct agatcctga 100911230DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 112tataggatcc cattattttt cttgatttcc 3011330DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 113aaatgcatgg cctccatctc ctcctcagcc 3011430DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 114tttggatcct aggtatgaaa gagtctcgta 30

Claims

1-24. (canceled)

25. A hybrid protein comprising two or three toxin proteins selected from the group consisting of Shiga toxin protein (Stx), cholera toxin protein (CT) and Escherichia coli heat-labile toxin protein (LT), wherein said toxin proteins are tandemly linked through a peptide having the following characteristics (A) to (D): (A) a number of amino acids is 12 to 30; and (B) a content of proline is 20 to 35%, (C) proline is allocated with an interval of two or three amino acids, (D) the total content of serine and glycine in the amino acids other than proline is 70% or more.

26. The hybrid protein according to claim 25, wherein said peptide further has the following characteristics (D): (D) the total content of serine and glycine in the amino acids other than proline is 80% or more.

27. The hybrid protein according to claim 25, wherein Shiga toxin protein (Stx) or cholera toxin protein (CT) is tandemly linked to Escherichia coli heat-labile toxin protein (LT) through said peptide.

28. The hybrid protein according to claim 25, wherein said peptide further has the following characteristics (E): (E) the total content of alanine, methionine and glutamic acid in the amino acids other than proline is 10% or less.

29. The hybrid protein according to claim 25, wherein said peptide further has the following characteristics (F): (F) the total content of tryptophan, leucine, isoleucine, tyrosine, phenylalanine and valine in the amino acids other than proline is 5% or less.

30. The hybrid protein according to claim 25, wherein said peptide further has the following characteristics (G): (G) the total content of serine, glycine and asparagine in the amino acids other than proline is 90% or more.

31. The hybrid protein according to claim 25, wherein the Shiga toxin proteins are Stx2e protein B subunits, and the cholera toxin proteins are cholera toxin protein B subunits.

32. A DNA construct comprising DNA encoding the hybrid protein according in claim 25.

33. The DNA construct according to claim 32, wherein DNA encoding the hybrid protein is operably-linked to a 5'-untranslated region of an alcohol dehydrogenase gene derived from a plant.

34. The DNA construct according to claim 33, wherein said plant is Nicotiana tabacum.

35. A recombinant vector comprising the DNA construct according to claim 32.

36. A transformant transformed with the recombinant vector according to claim 35.

37. The transformant according to claim 36, wherein the transformant is a transformed plant cell or a transformed plant.

38. A seed, which is obtained from the transformant according to claim 36.

39. A hybrid protein comprising: (a) two or three toxin proteins selected from the group consisting of Shiga toxin proteins, cholera toxin proteins, Escherichia coli heat-labile toxin proteins and combinations thereof, and (b) a peptide having an amino acid sequence which has at least 60% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84 and which is tandemly linked to the C-terminus of each of the toxin proteins, wherein said hybrid protein causes an immune response when administered to an animal.

40. The hybrid protein according to claim 39, wherein said peptide has an amino acid sequence which has at least 70% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84.

41. The hybrid protein according to claim 39, wherein said peptide has an amino acid sequence which has at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84.

42. A peptide having an amino acid sequence which has at least 70% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84.

43. The peptide according to claim 42, wherein said peptide has at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84.

44. The peptide according to claim 42, wherein said peptide has at least 90% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84.

45. A hybrid protein comprising one toxin protein selected from the group consisting of Shiga toxin protein (Stx), cholera toxin protein (CT) and Escherichia coli heat-labile toxin protein (LT), wherein said toxin protein is linked with a peptide having the following characteristics (A) to (D): (A) a number of amino acids is 12 to 30; and (B) a content of proline is 20 to 35%. (C) proline is allocated with an interval of two or three amino acids. (D) the total content of serine and glycine in the amino acids other than proline is 70% or more.

46. A hybrid protein comprising: (a) one toxin protein selected from the group consisting of Shiga toxin protein (Stx), cholera toxin protein (CT) and Escherichia coli heat-labile toxin protein (LT) and combinations thereof, and (b) a peptide having the amino acid sequence represented by SEQ ID NO:2, 82, 84 or an amino acid sequence which has at least 80% identity to the amino acid sequence represented by SEQ ID NO:2, 82, 84 and which is linked with the toxin protein, wherein said hybrid protein cause an immune response when administered to an animal.

47. A hybrid protein comprising: (a) two Shiga toxin proteins, and (b) a peptide having an amino acid sequence which has at least 70% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84 and which links said two Shiga toxin proteins, wherein said hybrid protein causes an immune response when administered to an animal.

48. The hybrid protein according to claim 47, wherein said peptide has an amino acid sequence which has at least 80% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84.

49. The hybrid protein according to claim 47, wherein said peptide has an amino acid sequence which has at least 90% identity to the amino acid sequence represented by SEQ ID NO: 2, 82, or 84.