Antibodies, recombinant antibodies, recombinant antibody fragments and fusions mediated plant disease resistance against fungi

• US 7,531,522 B2
• Filed: 04/14/2003
• Issued: 05/12/2009
• Est. Priority Date: 04/22/2002
• Status: Expired due to Fees

• Current Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Original Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Inventors: Fischer, Rainer, Schillberg, Stefan, Liao, Yu-Cai, Dorfmuller, Simone, Peschen, Dieter
• Primary Examiner(s): Navarro; Mark
• Assistant Examiner(s): Hines; JaNa
• Application Number:
  US10/512,184
• Publication Number:
  US 20050244901A1
• Time in Patent Office: 2,220 Days
• Field of Search: None
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: C07K 16/14 against material from fungi, algea or lichens
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2317/13 isolated from plants
• CPC Class Codes: C07K 2317/23 from birds
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/41 containing a Myc-tag
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/8258 for the production of oral vaccines (antigens) or immunoglobulins
• CPC Class Codes: C12N 15/8282 for fungal resistance

Abstract

A method for the production of fungus resistant transgenic plants, plant cells or plant tissue comprising the introduction of an Ab, rAb, rAb fragment or fusion or vector of the invention or the vectors of the composition of the invention into the genome of a plant, plant cell or plant cell tissue and a transgenic plant cell comprising stably integrated into the genome a polynucleotide or vector of the invention or the vectors of the composition of the invention.

Claims

1. A polynucleotide comprising a nucleic acid sequence coding for a fusion protein, the fusion protein comprising

a) an anti-fungal protein or peptide (AFP) and an antibody fragment, specifically recognising an epitope of an Ascomyceta, and

b) a cellular targeting signal,

wherein the antibody fragment is a scFv encoded by Seq. ID. No. 1.

2. The polynucleotide of claim 1, further comprising a regulatory sequence for expression in a plant, plant organ, plant tissue, or plant cell.

3. A vector comprising the polynucleotide of claim 1.

4. An expression cassette comprising the polynucleotide of claim 1.

5. The polynucleotide according to claim 2, wherein the regulatory sequence is selected from the group consisting of constitutive, chimeric, tissue specific, inducible synthetic, and cryptic promoters.

6. The polynucleotide according to claim 1, wherein the AFP, and the antibody fragment are arranged in any order relative to each other.

7. A composition comprising a purified preparation of a polynucleotide comprising a nucleic acid sequence coding for a fusion protein, the fusion protein comprising

a) an anti-fungal protein or peptide (AFP) and an antibody fragment, specifically recognising an epitope of an Ascomyceta, and

b) a cellular targeting signal

wherein the antibody fragment is a scFv encoded by Seq. ID. No. 1.

8. A method for the production of Ascomyceta-resistant transgenic plants, plant cells, or plant tissues, the method comprising the introduction of a fusion protein into a genome of a plant, plant cell, or plant tissue, the fusion protein comprising

a) an anti-fungal protein or peptide (AFP) and an antibody fragment, specifically recognising an epitope of an Ascomyceta, and

b) a cellular targeting signal

wherein the antibody fragment is a scFv encoded by Seq. ID. No. 1.

9. A kit comprising a fusion protein, a polynucleotide coding for the fusion protein, or a combination thereof, with the fusion protein comprising

a) an anti-fungal protein or peptide (AFP) and an antibody fragment, specifically recognising an epitope of an Ascomyceta, and

b) a cellular targeting signal

wherein the antibody fragment is a scFv encoded by Seq. ID. No. 1.

10. The kit according to claim 9, wherein the antibody fragment comprises an enzyme for ELISA or immunoblot detection.

11. The kit according to claim 10, wherein the enzyme is an alkaline phosphatase or a horse radish peroxidase.

12. A polynucleotide comprising a nucleic acid sequence coding for a fusion protein, the fusion protein comprising

a) an anti-fungal protein or peptide (AFP) and an antibody fragment, specifically recognising an epitope of an Ascomyceta, and

b) a cellular targeting signal,

wherein the AFP is encoded by Seq. ID. No. 12.

13. The polynucleotide of claim 12, further comprising a regulatory sequence for expression in a plant, plant organ, plant tissue, or plant cell.

14. The polynucleotide according to claim 13, wherein the regulatory sequence is selected from the group consisting of constitutive, chimeric, tissue specific, inducible synthetic, and cryptic promoters.

15. A vector comprising the polynucleotide of claim 12.

16. An expression cassette comprising the polynucleotide of claim 12.

17. The polynucleotide of claim 12, wherein a scFv encoding sequence is linked to an AFP encoding sequence via Seq. ID. No. 19, and

the scFv encoding sequence has the 5′

position with the AFP encoding portion in the 3′

position, or

the AFP encoding portion has the 5′

position and the and scFv encoding sequence has the 3′

position.

18. The polynucleotide according to claim 12, wherein the AFP, and the antibody fragment are arranged in any order relative to each other.

19. A composition comprising a purified preparation of a polynucleotide comprising a nucleic acid sequence coding for a fusion protein, the fusion protein comprising

a) an anti-fungal protein or peptide (AFP) and an antibody fragment, specifically recognising an epitope of an Ascomyceta, and

b) a cellular targeting signal

wherein the AFP is encoded by Seq. ID. No. 12.

20. A method for the production of Ascomyceta-resistant transgenic plants, plant cells, or plant tissues, the method comprising the introduction of a fusion protein into a genome of a plant, plant cell, or plant tissue, the fusion protein comprising

a) an anti-fungal protein or peptide (AFP) and an antibody fragment, specifically recognising an epitope of an Ascomyceta, and

b) a cellular targeting signal

wherein the AFP is encoded by Seq. ID. No. 12.

21. A kit comprising a fusion protein, a polynucleotide coding for the fusion protein, or a combination thereof, with the fusion protein comprising

a) an anti-fungal protein or peptide (AFP) and an antibody fragment, specifically recognising an epitope of an Ascomyceta, and

b) a cellular targeting signal

wherein the AFP is encoded by Seq. ID. No. 12.

22. The kit according to claim 21, wherein the antibody fragment comprises an enzyme for ELISA or immunoblot detection.

23. The kit according to claim 22, wherein the enzyme is an alkaline phosphatase or a horse radish peroxidase.