Bacterial toxin vaccine
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. 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 the amino acid sequence represented by SEQ ID NO: 2, 82, or 84 or an amino acid sequence which has at least 90% 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.
2. The hybrid protein of claim 1 , wherein two of the Shiga toxin proteins, cholera toxin proteins, or Escherichia coli heat-labile toxin proteins are tandemly linked through a peptide having the amino acid sequence represented by SEQ ID NO: 2.
3. The hybrid protein of claim 1 or claim 2 , wherein each of the toxin proteins is a Shiga toxin protein B subunit.
4. The hybrid protein of claim 1 , wherein each of the toxin proteins are Stx2e proteins.
5. The hybrid protein of claim 1 , wherein each of the toxin proteins are cholera toxin protein B subunits.
6. The hybrid protein of claim 1 , wherein each of the toxin proteins are Escherichia coli heat-labile toxin protein B subunits.
7. The hybrid protein of claim 2 , which comprises the amino acid sequence represented by SEQ ID NO: 10, 12, 14, or 16.
8. The hybrid protein of claim 1 , which comprises the amino acid sequence represented by SEQ ID NO: 86, 88, 90, 92, 94, 96, 98, or 100.
9. The hybrid protein of claim 1 , which further comprises a secretory signal peptide derived from a plant at the amino terminus of the hybrid protein.
10. The hybrid protein of claim 9 , which further comprises an endoplasmic reticulum retention signal peptide at the carboxyl terminus of the hybrid protein.
11. The hybrid protein of claim 1 , which further comprises a chloroplast transit signal peptide at the amino terminus of the hybrid protein.
12. A DNA construct comprising a nucleotide sequence encoding the hybrid protein of claim 1 .
13. The DNA construct of claim 12 , which comprises a nucleotide sequence having the nucleotide sequence represented by SEQ ID NO: 9, 11, 13, or 15.
14. The DNA construct of claim 12 , which comprises a nucleotide sequence having the nucleotide sequence represented by SEQ ID NO: 85, 87, 89, 91, 93, 95, 97, or 99.
15. The DNA construct of claim 12 , wherein the nucleotide sequence encoding a hybrid protein is operably-linked to a 5′-untranslated region of an alcohol dehydrogenase gene derived from a plant.
16. The DNA construct of claim 15 , wherein the 5′-untranslated region of an alcohol dehydrogenase gene derived from a plant is derived from Nicotiana tabacum.
17. The DNA construct of claim 16 , which comprises the nucleotide sequence represented by any one of SEQ ID NOS: 24 to 29.
18. The DNA construct of claim 16 , which comprises the nucleotide sequence represented by any one of SEQ ID NOS: 101 to 111.
19. A recombinant vector comprising the DNA construct of claim 12 .
20. A transformant transformed with the recombinant vector of claim 19 .
21. The transformant of claim 20 , which is a transformed plant cell or a transformed plant.
22. A peptide having the amino acid sequence represented by one of SEQ ID NO: 2, 82, or 84.
23. The hybrid protein of claim 1 , wherein the peptide has the sequence represented by SEQ ID NO: 2.
24. The hybrid protein of claim 1 , wherein the peptide has the sequence represented by SEQ ID NO: 82.
25. The hybrid protein of claim 1 , wherein the peptide has the sequence represented by SEQ ID NO: 84.
26. The hybrid protein of claim 1 , which causes an immune response when administered to pigs.