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%.

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.