Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-07-13
2003-04-08
Smith, Lynette R. F. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023400, C514S04400A, C435S069900, C435S069100
Reexamination Certificate
active
06545140
ABSTRACT:
BACKGROUND OF THE INVENTION
One of the defense mechanisms against infection by both animals and plants is the production of peptides that have antimicrobial and antiviral activity. Various classes of these peptides have been isolated from tissues of plants and animals. These classes include the tachyplesins (Nakamura et al., 1988), the “protegrins” (Kokryakov et al., 1993) and the defensins, which include beta-defensins and classical defensins. The defensins are characterized by six invariant cysteines and three intramolecular cystine disulfide bonds (Lehrer et al., 1991; Lehrer et al., 1993). Classical defensins are short (29-35 amino acid residues) cationic peptides containing three pair of disulfide-linked cysteines. Although beta-defensins are nearly identical in their three-dimensional structure to classical defensins, beta-defensins are slightly larger (38-42 amino acid residues) and differ in the spacing of the conserved cysteine residues and connectivity of the cysteine linkages relative to classical defensins (Zimmerman et al., 1995).
Heterophils are the major granulated leukocyte of birds and are similar to the mammalian neutrophil. Avian heterophils lack myeloperoxidase and alkaline phosphatase activities but their ultrastructure, cytochemistry, and functions are otherwise similar to mammalian neutrophils (Pennial and Spitznagel, 1975; Montali, 1988). Both heterophils and neutrophils possess cationic antimicrobial peptides, which are important mediators of innate disease resistance in tissues exposed to microbial pathogens. Cationic antimicrobial peptides likely exert their antimicrobial activity by interacting with cell membranes or viral envelopes initially via electrostatic forces then by membrane insertion in which they form voltage gated ion channels resulting in increased permeability (Boman, 1991; Kagan et al., 1990).
Numerous mammalian neutrophil classical defensins have been isolated and sequenced (Selsted and Harwig, 1987; Belcourt et al., 1992; Wilde et al., 1989; U.S. Pat. No. 5,202,420). Three chicken heterophil beta-defensin mature peptide amino acid sequences (Gal 1/CHP 1, Gal 1&agr;/CHP 2, and Gal 2) (Harwig et al., 1994; Evans et al., 1994; U.S. Pat. No. 5,202,420) and three partial turkey heterophil beta-defensins (THP 1, THP 2, and THP 3) have been reported (Evans et al., 1994). These avian beta-defensins are bacteriocidal in vitro for both avian and human bacterial pathogens (Evans et al., 1995). Thirteen mature bovine neutrophil beta-defensins amino acid sequences have also been reported (Selsted et al., 1993; U.S. Pat. No. 5,459,235).
Defensins are synthesized as 93-95 residue prepro defensins with a hydrophobic 19 amino acid signal sequence which is necessary for insertion into the endoplasmic reticulum prior to transport to granules. It has been proposed that neutrophil storage granule peptides (classical defensins) have a negatively charged propiece (of about 40-45 amino acids) to neutralize or balance the positive charge of the mature peptide (Michaelson et al., 1992). This propiece also is necessary for proper peptide folding and in targeting of the mature peptide into storage granules (Liu and Ganz, 1995). Thus far, all characterized classical defensins from mammalian granulocytes and from intestinal Paneth cells have a negatively charged propiece (Jones and Bevins, 1993; Yount et al., 1995).
In contrast, storage granule-free epithelial cell beta-defensin propeptides of the respiratory and oral cavity, i.e., bovine tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP), have an abridged propiece that has no neutralizing negative charge (Diamond et al., 1991; Schonwetter et al., 1995). These antimicrobial peptides are synthesized de novo upon stimulation and are not stored in granules (Russell et al., 1996; Schonwetter et al., 1995). The propieces for beta-defensins found in bovine neutrophils have not been characterized (Selsted et al., 1993).
Thus, what is needed is the identification and isolation of a variety of genes encoding avian peptides having specific antimicrobial activities.
SUMMARY OF THE INVENTION
The invention provides an isolated and purified nucleic acid molecule comprising a nucleic acid sequence which encodes an avian beta-defensin, a biologically active fragment or a biologically active variant thereof. As described hereinbelow, the complete cDNA for two chicken (Gal 1/CHP 1 and Gal 2) and two turkey (THP 1 and THP 2) beta-defensins were obtained. Surprisingly, the four deduced beta-defensin pro regions lack the long, negatively charged propiece reported for classical defensin pro regions, a region which is thought to stabilize and inactivate the positively charged mature peptide and target the propeptide to the storage granule.
Preferred isolated nucleic acid molecules of the invention include those having a nucleic acid sequence comprising a full-length avian beta-defensin gene, i.e., encoding the prepro form of the peptide (i.e., one having the signal sequence and the propiece), e.g., a nucleic acid sequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or a variant thereof, or comprising a nucleic acid sequence encoding the mature form of an avian beta-defensin, such as a nucleic acid sequence comprising SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or a variant thereof. Also preferably, the nucleic acid molecules of the invention encode a peptide having an amino acid corresponding to the prepro form of an avian beta-defensin, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or a variant thereof, or an amino acid sequence corresponding to the mature form of an avian beta-defensin, such as SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or a variant thereof. It is preferred that the nucleic acid molecules of the invention encode a peptide having antimicrobial (e.g., antibacterial, antiviral, antiprotozoal or antifungal) activity, more preferably peptides having a broad spectrum (for example, they are active against a variety of bacteria of different genera) of antimicrobial activity.
It is also envisioned that anti-sense nucleic acid molecules, e.g., a molecule which is the complement of an avian beta-defensin, a biologically active fragment, variant thereof, are within the scope of the invention. The nucleic acid molecules of the invention, fragments or variants thereof, either DNA or RNA, are useful to prepare probes, primers or expression cassettes which, in turn, are useful to detect, amplify and express other avian beta-defensin genes and related genes.
Therefore, the invention also provides an expression cassette comprising: a DNA sequence which is operably linked to a promoter functional in a host cell, which DNA sequence encodes an avian beta-defensin, a biologically active fragment or a biologically active variant thereof. The host cell may be prokaryotic or eukaryotic in origin. These cassettes may be employed to prepare recombinant peptides. For example, an expression cassette of the invention may be introduced and expressed in a host cell, e.g., an insect cell using a baculovirus vector, so as to yield recombinant avian beta-defensin peptide, a biologically active fragment, or variant thereof. Preferably, the recombinant peptide is recovered from the host cell. It is preferred that a peptide of the invention is active against at least one pathogen including, but not limited to,
Staphylococcus aureus, Escherichia coli, Pasteurella multocida, Bordetella avium, Mycoplasma gallispeticum, Candida albicans, Listeria monocytogenes, Salmonella typhimurium, Salmonella enteriditis
, or
Campylobacter jejuni.
Hence, the invention further provides an isolated and purified avian beta-defensin peptide, a biologically active variant or fragment thereof. Preferred peptides include the prepro form of an avian beta-defensin. More preferably, the peptide is a compound of formula (I):
GCPSX
2
X
3
X
3
X
2
X
3
GSCFGFX
2
SCCX
2
WPWNX
3
, SEQ ID NO:49 wherein X
3
is I, V, M, A, norleucine or L, wherein X
2
is H, N, Q, K or R, and wherein X
1
is I, L or V;
a c
Brockus Charles W.
Harmon Barry G.
Jackwood Mark W.
Portner Ginny Allen
Schwegman Lundberg Woessner & Kluth P.A.
Smith Lynette R. F.
University of Georgia Research Foundation Inc.
LandOfFree
DNA encoding an avian beta-defensin and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with DNA encoding an avian beta-defensin and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA encoding an avian beta-defensin and uses thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3011412