Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 15 to 23 amino acid residues in defined sequence
Reexamination Certificate
2001-08-14
2004-06-22
Smith, Lynette R. F. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
15 to 23 amino acid residues in defined sequence
C530S300000, C424S184100, C424S184100, C514S002600, C514S013800, C514S021800
Reexamination Certificate
active
06753407
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to two families of novel antimicrobial peptides exhibiting therapeutic antimicrobial properties, to antibodies that specifically bind to one of the families of the novel peptides, and to methods of monitoring and improving health in aquaculture species and of preservation of seafood.
BACKGROUND OF THE INVENTION
Infectious diseases are often the most serious impediment to the success of the commercial aquaculture industry in the United States and worldwide. The epithelial surfaces of fish, such as the skin, gills and alimentary tract, provide first contact with potential pathogens. It has been reported that several kinds of endogenous antimicrobial peptides exist in the skin, stomach and blood of amphibians, mammals and insects. Examples include cecropins (insects), defensins (mammals, insects), and magainins (frogs) (See discussion infra, Detailed Description of Preferred Embodiments). These peptides exhibit antimicrobial activity against a broad spectrum of organisms. This ability to exhibit broad-spectrum activity provides the advantages of nonspecificity and rapid response. These advantages enable the host to delay or prevent microbial colonization. Enhancement of nonspecific defenses exhibiting a broad range of activity against numerous pathogens may be a cost-effective method of controlling disease epidemics that inhibit successful aquacultural endeavors.
Therefore, it is an objective of this invention to provide a better understanding of the mechanisms responsible for disease resistance in aquatic animals and to use this knowledge to reduce epidemic disease. Additionally, this invention may aid in the development of peptide antibiotics for the treatment of human diseases. Other features and advantages of the present invention will be more apparent in the detailed description set forth below.
SUMMARY OF THE INVENTION
A first aspect of the present invention is an antimicrobial compound or endobiotic peptide isolated from fish. The compound may be selected from the group consisting of peptides having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3, or SEQ ID NO: 4.
A further aspect of the present invention is a pharmaceutical formulation comprising a compound as described above in a pharmaceutically acceptable carrier.
A further aspect of the present invention is an antibody (e.g., a monoclonal antibody) that specifically binds to a compound as described above.
A further aspect of the invention is a method of treating stress in fish, comprising administering an endobiotic peptide as described above to a fish in an amount effective to treat or combat stress therein.
A further aspect of the invention is a nucleic acid (e.g., a DNA) that encodes a peptide as described above.
A further aspect of the invention is a method of treating stress in fish, comprising administering a nucleic acid to the fish (e.g., by injecting the nucleic acid into muscle of the fish) in an amount effective to treat or combat stress therein.
A further aspect of the invention is a method of monitoring fish health, comprising the steps of: (a) collecting a biological sample from a fish; and (b) detecting the level of at least one endogenous endobiotic peptide in the sample, wherein lower levels of endobiotic peptides indicate decreased health in the fish. Examples of suitable endobiotic peptides include, but are not limited to, the peptides described above.
A further aspect of the invention is a method of monitoring freshness of a fish food product, the method comprising detecting the level of at least one endogenous endobiotic peptide, wherein lower levels of endobiotic peptides indicate decreased freshness in the fish food product. Examples of suitable endobiotic peptides include, but are not limited to, the peptides described above.
A further aspect of the present invention is a method of screening for compounds useful for treating stress in fish, the method comprising the steps of: (a) administering a test compound to a fish; (b) collecting a biological sample from the fish; and (c) detecting the level of at least one endogenous endobiotic peptide in the sample, wherein higher levels of endobiotic peptide in the fish as compared to those found in the absence of administration of the test compound indicate the compound is useful in treating stress in the fish. Examples of suitable endobiotic peptides include, but are not limited to, the peptides described above.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention will now be described more fully hereinafter with reference to the accompanying figures, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
Amino acid sequences disclosed herein are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. Amino acids are represented herein in by single letter code.
“Amino acid sequence” as used herein, refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence, and like terms, are not meant to limit amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
As used herein, the term “antibody” refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Of these IgM and IgG are particularly preferred. The antibodies may be monoclonal or polyclonal and may be of any species of origin including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al.,
Molec. Immunol.
26, 403-11 (1989). Antibodies that bind to the peptides of Endobiotic Family 1 and/or Endobiotic Family 2 can be prepared using intact peptides or fragments containing small peptides of interest as the immunizing antigen. The peptide or oligopeptide used to immunize an animal can be derived from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
The term “antimicrobial”, as used herein, refers to the ability to terminate or inhibit the growth of microorganisms.
The term “biological sample”, as used herein, is used in its broadest sense. A biological sample of a fish may include blood, urine, muscle tissue, skin, gills, viscera, mucosal swab, cell culture, or an aqueous medium housing the fish.
As used herein, the term “endobiotic”, refers to a naturally-occurring, host-produced antibiotic. The vast majority of these endogenous antibiotics are low molecular weight peptides or proteins that exhibit antimicrobial activity against a wide range of microorganisms, including bacteria, viruses, fungi, metazoan and protozoan parasites (Robinette et. al., (1998)
Cell. Mol. Life Sci.
54, 467-475). Examples of endobiotics include cecropins (Bowman, H. (1995)
Ann. Rev. Immunol.
13: 61-92; Steiner et al., (1981)
Nature
292: 246-248), defensins (Selsted et al.,
J. Biol. Chem.
258: 14485-14489; Lehrer et al.
Ann. Rev. Immunol.
11: 105-128), and magainins (Zasl
Noga Edward J.
Silphaduang Umaporn
Myers Bigel & Sibley & Sajovec
North Carolina State University
Smith Lynette R. F.
Zeman Robert A.
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