DNA encoding hyaluronan synthase from pasturella multocida and methods of use

• US 20030113845A1
• Filed: 08/12/2002
• Published: 06/19/2003
• Est. Priority Date: 04/02/1998
• Status: Active Grant

• Current Assignee: Paul Deangelis
• Original Assignee: Paul Deangelis
• Inventors: DeAngelis, Paul
• Granted Patent: US 6,987,023 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: A61K 39/102 Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
• CPC Class Codes: A61K 47/36 Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• CPC Class Codes: A61L 24/08 Polysaccharides A61L24/043 takes precedence
• CPC Class Codes: A61L 29/085 Macromolecular materials
• CPC Class Codes: C08L 5/08 Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates

Abstract

The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active Pasturella multocida hyaluronate synthase (PmHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product. Hyaluronate is also known as hyaluronic acid or hyaluronan. The present invention also relates to the use of the PmHAS in constructing “knock-out” mutant strains of P. multocida for use in vaccinations. The present invention further relates to the use of the PmHAS in diagnostic tests in the field determinations of livestock P. multocida infection.

Claims

1. A purified nucleic acid segment comprising a coding region encoding enzymatically active hyaluronate synthase from Pasturella, wherein the purified nucleic acid segment encodes the Pasturella hyaluronate synthase of SEQ ID NO:

1.

2. The purified nucleic acid segment of claim 1, wherein the purified nucleic acid segment comprises a nucleotide sequence as set forth in SEQ ID NO:

2.

21. A procaryotic or eucaryotic host cell transformed or transfected with an isolated nucleic acid segment according to claim 1 in a manner allowing the host cell to express hyaluronic acid.

24. A procaryotic or eucaryotic host cell transformed or transfected with a nucleic acid segment according to claim 1, 19, 20, or 22 in a manner allowing the host cell to express hyaluronic acid.

3. A recombinant vector selected from the group consisting of a plasmid, cosmid, phage, or virus vector and wherein the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasturella, wherein the purified nucleic acid segment encodes the Pasturella hyaluronan synthase of SEQ ID NO:

1.

4. The recombinant vector of claim 3, wherein the purified nucleic acid segment comprises a nucleotide sequence as set forth in SEQ ID NO:

2.

5. The recombinant vector of claim 3, wherein the plasmid further comprises an expression vector.

6. The recombinant vector of claim 5, wherein the expression vector comprises a promoter operatively linked to the coding region.

7. A recombinant host cell, wherein the recombinant host cell is a prokaryotic cell transformed with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasturella, wherein the purified nucleic acid segment encodes the Pasturella hyaluronan synthase of SEQ ID NO:

1.

8. The recombinant host cell of claim 7, wherein the purified nucleic acid segment comprises a nucleotide sequence as set forth in SEQ ID NO:

2.

9. The recombinant host cell of claim 8, wherein the host cell produces hyaluronic acid.

10. A recombinant host cell, wherein the recombinant host cell is a eukaryotic cell transfected with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasturella, wherein the purified nucleic acid segment encodes the Pasturella hyaluronan synthase of SEQ ID NO:

1.

11. The recombinant host cell of claim 10, wherein the purified nucleic acid segment comprises a nucleotide sequence as set forth in SEQ ID NO:

2.

12. The recombinant host cell of claim 11, wherein the host cell produces hyaluronic acid.

13. A recombinant host cell, wherein the recombinant host cell is transformed to introduce a recombinant vector into the recombinant host cell, wherein the recombinant vector comprises a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasturella, wherein the purified nucleic acid segment encodes the Pasturella hyaluronan synthase of SEQ ID NO:

1.

14. The recombinant host cell of claim 13, wherein the purified nucleic acid segment comprises a nucleotide sequence as set forth in SEQ ID NO:

2.

15. The recombinant host cell of claim 14, wherein the host cell produces hyaluronic acid.

16. A recombinant host cell, wherein the recombinant host cell is transduced with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase from Pasturella, wherein the purified nucleic acid segment encodes the Pasturella hyaluronan synthase of SEQ ID NO:

1.

17. The recombinant host cell of claim 16, wherein the purified nucleic acid segment comprises a nucleotide sequence as set forth in SEQ ID NO:

2.

18. The recombinant host cell of claim 17, wherein the host cell produces hyaluronic acid.

19. A purified and isolated nucleic acid sequence encoding enzymatically active hyaluronan synthase, the nucleic acid sequence selected from the group consisting of:

(a) the nucleic acid sequence as set forth in SEQ ID NO;

2; and

(b) complementary nucleic acid sequences to the nucleic acid sequence as set forth in SEQ ID NO;

2 and which encode an enzymatically active hyaluronan synthase.

20. A purified and isolated nucleic acid segment consisting essentially of a nucleic acid segment encoding enzymatically active hyaluronan synthase from Pasturella, wherein the purified nucleic acid segment encodes the Pasturella hyaluronan synthase of SEQ ID NO:

1.

22. An isolated nucleic acid segment consisting essentially of a nucleic acid segment encoding hyaluronan synthase from Pasturella having a nucleic acid segment sufficiently duplicative of the nucleic acid segment as set forth in SEQ ID NO:

2 to allow possession of the biological property of coding for Pasturella hyaluronan synthase.

23. A cDNA sequence according to claim 22.