Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1998-07-21
2002-12-17
Navarro, Mark (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S300000, C530S317000, C530S325000, C530S326000, C530S350000, C930S200000, C930S270000
Reexamination Certificate
active
06495661
ABSTRACT:
BACKGROUND OF THE INVENTION
Fowl cholera is a severe respiratory disease in domestic poultry and wild birds. This disease occurs throughout the year and causes great losses in the poultry industry around the world (Rhoades et al., 1989). Fowl cholera has two clinical forms, acute disease and chronic disease. The acute disease is a septicemia with high morbidity and mortality. Clinical signs of the acute disease include fever, anorexia, ruffled feathers, mucous discharge from the mouth, diarrhea, increased respiratory rate and cyanosis. Death may be the first evidence of the acute disease. The chronic form of fowl cholera is characterized by localized infections including swelling in wattles, sinuses, periorbital subcutaneous tissues, leg or wing joints, sternal bursae and foot pads, exudative conjunctivitis, pharyngitis, emaciation and lethargy (Rhoades et al., 1991; Rhoades et al., 1989).
Pasteurella multocida
is the causative agent of fowl cholera. Almost all types of birds including chickens, turkeys, ducks and geese are susceptible to
P. multocida
infection. In addition to causing fowl cholera in birds,
P. multocida
also causes disease in mammals, including cattle, swine, horses, sheep, goats, rabbits and humans. Differences in the capsule antigen are employed to classify
P. multocida
into serogroups, i.e., serogroups A, B, D, E and F (Carter et al., 1955; Rimler et al., 1987). Differences in lipopolysaccharide antigen are used to subtype the bacteria into 16 somatic serotypes (Brogden et al., 1978; Heddleston et al., 1992). Most of the avian isolates are serotype A: 1, A:3, and A:4 (Hofacre et al., 1986).
Current control of fowl cholera mainly relies on vaccination. Two kinds of vaccines are used in poultry, the inactivated vaccines (bacterins) and attenuated live vaccines. However, a bacterin induces only homologous, i.e., serotype-specific, protection. As there are currently 16 somatic serotypes, the efficacy of bacterins is very limited. While live vaccines such as those which employ the CU, PM-1 and M-9 strains, can induce heterologous immunity, they can induce disease (Carpenter et al., 1988; Hofacre et al., 1989a; Hofacre et al., 1989b; Hofacre et al., 1986; Schlink et al., 1987a; Schlink et al., 1987b).
The outer membrane of Gram-negative bacteria contains a number of components: phospholipid layer, outer membrane proteins (OMP), and lipopolysaccharide (LPS). The outer membrane contains a number of proteins including major outer membrane porins and other proteins. There are reports that
P. multocida
outer membrane protein was able to induce protective immunity in poultry and other animals (Lu et al., 1991a; Lu et al., 1991b; Lu et al., 1988a; Lu et al., 1988b; Ruffolo et al., 1996; Zhao et al., 1995). Moreover, it has been reported that outer membrane proteins expressed by in vivo grown bacteria induced cross-protection in poultry (heterologous protection) (Rimler et al., 1994; Wang et al., 1994a; Wang et al., 1994b).
Thus, what is needed is the identification and isolation of the gene encoding the major outer membrane protein of
P. multocida
. Moreover, there is a need for improved immunogenic compositions useful to prevent or inhibit fowl cholera.
SUMMARY OF THE INVENTION
The invention provides an isolated and purified nucleic acid molecule comprising a preselected nucleic acid sequence which encodes an avian
Pasteurella multocida
outer membrane protein or polypeptide (OmpH), a biologically active subunit or a biologically active variant thereof. Preferably, the nucleic acid molecule of theinvention encodes a porin. Porins are major outer membrane proteins which have pore-forming function. They serve as molecular sieves allowing polar solutes to pass through but excluding non-polar molecules of comparable sizes. As described hereinbelow, the genes (ompH) encoding OmpHs from all 16
P. multocida
serotypes were cloned and sequenced. The OmpHs from different serotypes showed high homology in amino acid sequence (72.3% overall identity). Computer-aided secondary structure predictions revealed 16 anti-parallel beta-strands connected by 8 external loops and 8 short periplasmic turns. Preferred isolated nucleic acid molecules of the invention include those having a nucleic acid sequence comprising SEQ ID NO:1 or SEQ ID NO:3, molecules encode a polypeptide having an amino acid sequence such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 or SEQ ID NO:43. The nucleic acid molecules of the invention, subunits or variants thereof, are useful to prepare probes, primers or expression cassettes which, in turn, are useful to detect, amplify and express other ompH genes and related genes.
Therefore, the invention also provides an expression cassette comprising: a preselected DNA sequence which is operably linked to a promoter functional in a host cell, which DNA sequence encodes a
Pasteurella multocida
outer membrane polypeptide, a biologically active subunit or a biologically active variant thereof. The host cell may be prokaryotic or eukaryotic in origin. Preferably, the preselected DNA sequence comprises SEQ ID NO:1 or SEQ ID NO:3, or the complement thereto. Also preferably, the preselected DNA segment encodes a polypeptide having an amino acid sequence such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or a fragment thereof. These cassettes may be employed to prepare recombinant polypeptides. For example, a nucleic acid molecule of the invention, a variant or a fragment thereof, e.g., an expression cassette of the invention, may be introduced and expressed in a cultured host cell, preferably a host cell that is stably transformed with the nucleic acid molecule, so as to yield recombinant
Pasteurella multocida
outer membrane polypeptide, a biologically active subunit, variant or derivative thereof. Preferably, the recombinant polypeptide is recovered from the host cell. The recombinant polypeptide may be recovered from cell lysates in a soluble fraction, i.e., the recombinant OmpH is not associated with membranes, in the insoluble fraction, or from supernatants, i.e., the recombinant polypeptide is secreted into the extracellular environment of the host cell. As described herein, the genes encoding mature OmpH polypeptides of
Pasteurella multocida
strains X-73 and P-1059 were expressed as fusion proteins in
Escherichia coli
. Thus, another embodiment of the invention is a fusion polypeptide comprising at least an immunogenic or antigenic portion of the
Pasteurella multocida
outer membrane polypeptide.
Yet another embodiment of the invention is isolated and purified native
Pasteurella mullocida
outer membrane protein, which native polypeptide is obtained from a particular isolate or strain of
Pasteurella multocida
. As shown hereinbelow, the major outer membrane proteins of
Pasteurella multocida
strains X-73 and P-1059 were purified by selective extraction with detergents and size exclusion chromatography. The planar lipid bilayer assay showed that the isolated and purified native OmpH of X-73 and P-1059 had pore-forming activity. Thus, a preferred embodiment of the invention is isolated and purified
Pasteurella multocida
strain X-73 outer membrane protein, a biologically active subunit, variant or derivative thereof. Another preferred embodiment is isolated and purified
Pasteurella multocida
strain P-1059 outer membrane porin protein, a biologically active subunit, variant or derivative thereof.
The invention also provides an isolated and purified peptide of the
Pasteurella multocida
outer membrane polypeptide, a biologically active variant or derivative thereof. Preferably, a peptide of the invention corresponds to extracellular domains of
Pasteurella multocida
outer membrane polypeptide. More preferably, a peptide of the invention
Glisson John Robert
Luo Yugang
Hines Ja-Na A.
Navarro Mark
Schwegman Lundberg Woessner & Kluth P.A.
University of Georgia Research Foundation Inc.
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