Polynucleotides of Haemophilus parasuis and its use
Inventors
Bensaid, Albert • Pina Pedrero, Sonia • Rivas Adan, Raquel • Oliveira, Simone • ESPUNA MASO ENRIC, null • Espuña Maso, Enric • Herrero Molina, Carmen
Assignees
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Publication Number
US-8350018-B2
Publication Date
2013-01-08
Expiration Date
Abstract
The present invention relates to Haemophilus parasuis polynucleotides produced by recombinant technology. It also relates to polypeptides that are expressed by said polynucleotides and also to a vaccine against H. parasuis that comprises said polypeptides. In another aspect, the invention also relates to the use of polynucleotides to determine if a strain of H. parasuis is virulent or avirulent.
Core Innovation
The invention relates to Haemophilus parasuis virulence-associated polynucleotides and the translated polypeptides. The polynucleotides are provided as isolated polynucleotides with nucleotide sequences identified by SEQ ID NOs and the translated polypeptides are provided as amino acid sequence identifiers. The polynucleotides and polypeptides are grouped into three structural groups based on conserved 5′ and 3′ regions.
The invention further describes PCR primer sets using sequence identifiers pADH-F and pADH-R1, pADH-R2, and pADH-R3. Virulent strains are reported to show genomic amplification products for one or more of the invention polynucleotides, whereas avirulent strains show none. This enables classification of virulent versus avirulent strains that is described as independent of antigenic variability.
The invention also outlines recombinant expression of the polypeptides. Expression vectors and host cells are used to express the virulence-associated polypeptides, and the resulting polypeptides are described in relation to vaccine and immunogenic compositions for swine.
Claims Coverage
The independent claims cover 7 inventive features across isolated polynucleotides, expression vectors, host cells, processes for preparing polypeptides, and a PCR-based kit to determine whether Haemophilus parasuis strains are virulent or avirulent. Across the independent claims, the core inventive features are defined by sequence-identity or complete complementarity and by specified PCR primer components for virulence determination.
Isolated polynucleotide encoding or complementary to a defined polypeptide identity set
An isolated polynucleotide consisting of a nucleotide sequence that encodes, or consisting of a nucleotide sequence which is entirely complementary to the entirety of a polynucleotide sequence that encodes, a polypeptide which has at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
Expression vector encoding or complementary to a defined polypeptide identity set
An expression vector which comprises a nucleotide sequence that encodes, or which comprises a nucleotide sequence which is entirely complementary to the entirety of a polynucleotide sequence that encodes, a polypeptide which has at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
Expression vector comprising the polynucleotide sequence of SEQ ID NO:1
An expression vector comprising the polynucleotide sequence of SEQ ID NO:1.
Host cell transformed with an expression vector comprising the polynucleotide sequence of SEQ ID NO:1
A host cell transformed with an expression vector, wherein said expression vector comprises the polynucleotide sequence of SEQ ID NO:1.
Process for preparing a polypeptide by culturing transformed host cells and expressing the polynucleotide
A process for the preparation of a polypeptide, comprising the following steps: a) culturing a host cell transformed with an expression vector comprising a polynucleotide that encodes a polypeptide which has at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; and b) expressing said polynucleotide to produce said polypeptide.
Process for preparing a polypeptide by culturing transformed host cells and expressing a SEQ ID NO:1 polynucleotide
A process for preparing a polypeptide, comprising: culturing a host cell transformed with an expression vector, wherein said expression vector contains a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1; and expressing said polynucleotide to produce said polypeptide.
PCR kit for determining virulent versus avirulent Haemophilus parasuis strains using SEQ ID NO:1 and defined primers
A kit to determine if a strain of Haemophilus parasuis is virulent or avirulent, comprising an amplification product produced by PCR amplification of a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1 using a forward primer comprising the nucleotide sequence of SEQ ID NO: 27 and a reverse primer comprising the nucleotide sequence of SEQ ID NO: 28; oligonucleotide primers comprising SEQ ID NO: 27, 28, 29 and 30; and PCR reagents necessary to perform PCR amplification.
Overall, the claim set is grounded on virulence-associated Haemophilus parasuis polynucleotides and translated polypeptides defined by sequence identity (at least 80%) or by specified polynucleotide sequence identifiers, including SEQ ID NO:1. Coverage extends from isolated polynucleotides to expression vectors and transformed host cells, to processes for producing polypeptides, and to a PCR-based kit for determining virulent versus avirulent strains using specified primer and reagent components.
Stated Advantages
Enables classification of virulent versus avirulent strains independent of antigenic variability.
Documented Applications
A kit and PCR-based approach to determine whether a strain of Haemophilus parasuis is virulent or avirulent.
Inclusion of recombinant polypeptides in vaccine and immunogenic compositions for swine.
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