Serine protease inhibitor nucleic acid molecules and uses...

Details Serine protease inhibitor nucleic acid molecules and uses... Serine protease inhibitor nucleic acid molecules and uses...

1998-01-09

2002-11-12

Pak, Michael (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S235100, C435S325000, C514S04400A, C536S023500

Reexamination Certificate

active

06479253

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to flea serine protease inhibitor nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies, and/or other inhibitors, as well as their use to protect an animal from flea infestation.
BACKGROUND OF THE INVENTION
Hematophagous ectoparasite infestation of animals is a health and economic concern because hematophagous ectoparasites are known to cause and/or transmit a variety of diseases. Hematophagous ectoparasites directly cause a variety of diseases, including allergies, and also carry a variety of infectious agents including, but not limited to, endoparasites (e.g., nematodes, cestodes, trematodes and protozoa), bacteria and viruses. In particular, the bites of hematophagous ectoparasites are a problem for animals maintained as pets because the infestation becomes a source of annoyance not only for the pet but also for the pet owner who may find his or her home generally contaminated with insects. As such, hematophagous ectoparasites are a problem not only when they are on an animal but also when they are in the general environment of the animal.
Bites from hematophagous ectoparasites are a particular problem because they not only can lead to disease transmission but also can cause a hypersensitive response in animals which is manifested as disease. For example, bites from fleas can cause an allergic disease called flea allergic (or allergy) dermatitis (FAD). A hypersensitive response in animals typically results in localized tissue inflammation and damage, causing substantial discomfort to the animal.
The medical importance of hematophagous ectoparasite infestation has prompted the development of reagents capable of controlling hematophagous ectoparasite infestation. Commonly encountered methods to control hematophagous ectoparasite infestation are generally focused on use of insecticides. While some of these products are efficacious, most offer protection of a very limited duration at best. Furthermore, many of the methods are often not successful in reducing hematophagous ectoparasite populations. In particular, insecticides have been used to prevent hematophagous ectoparasite infestation of animals by adding such insecticides to shampoos, powders, sprays, foggers, collars and liquid bath treatments (i.e., dips). Reduction of hematophagous ectoparasite infestation on the pet has been unsuccessful for one or more of the following reasons: (1) failure of owner compliance (frequent administration is required); (2) behavioral or physiological intolerance of the pet to the pesticide product or means of administration; and (3) the emergence of hematophagous ectoparasite populations resistant to the prescribed dose of pesticide.
Prior investigators have described sequences of a few insect serine protease inhibitors:
Bombyx mori
nucleic acid and amino acid sequences have been disclosed by Narumi et al.,
Eur. J. Biochem.,
214:181-187, 1993; Takagi et al.,
J. Biochem.,
108:372-378, 1990; and amino acid sequence has been disclosed by Sasaki,
Eur. J Biochem,
202:255-261, 1991.
Manduca sexta
nucleic acid and amino acid sequences have been disclosed by Kanost et al.,
J. Biol. Chem,
264:965-972, 1989; U.S. Pat. No. 5,436,392, to Thomas et al., issued July 25,-2085, 1990; U.S. Pat. No. 5,196,304, to Kanost et al., issued Mar. 23, 1993; Jiang et al.,
J. Biol. Chem.,
269:55-58, 1994; and
Manduca sexta
peptide sequences have been disclosed by Fox et al.,
Peptides,
12:937-944, 1991.
Locusta migratoria
peptide sequences have been disclosed by Kellenberger et al.,
J. Biol. Chem,
270:25514-25519, 1995.
Rhodnius prolixus
peptide sequences have been disclosed by Van De Locht,
EMBO,
14:5149-5157, 1995.
Lymantria dispar
peptide sequences have been disclosed by Valaitis,
Insect Biochem Molec Biol,
25:139-149, 1995.
Lucilia cuprina
nucleic acid and amino acid sequences have been disclosed by Casu et al.,
Insect Molecular Biology,
3:159-170, 1994. Identification of a serine protease inhibitor of the present invention is unexpected because the most identical amino acid or nucleic acid sequence identified by previous investigators could not be used to identify a flea serine protease inhibitor of the present invention.
In summary, there remains a need to develop a reagent and a method to protect animals from hematophagous ectoparasite infestation.
SUMMARY OF THE INVENTION
The present invention relates to a novel product and process for protection of animals from hematophagous ectoparasite infestation. According to the present invention there are provided flea serine protease inhibitor proteins and mimetopes thereof; flea nucleic acid molecules, including those that encode such proteins; antibodies raised against such serine protease inhibitor proteins (i.e., anti-flea serine protease inhibitor antibodies); and other compounds that inhibit flea serine protease inhibitor activity (i.e, inhibitory compounds or inhibitors).
The present invention also includes methods to obtain such proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, mimetopes, nucleic acid molecules, antibodies, and/or inhibitory compounds, as well as use of such therapeutic compositions to protect animals from hematophagous ectoparasite infestation.
Identification of a serine protease inhibitor protein of the present invention is unexpected because the most identical amino acid or nucleic acid sequence identified by previous investigators could not be used to identify a flea serine protease inhibitor protein of the present invention. In addition, identification of a flea serine protease inhibitor protein of the present invention is unexpected because a protein fraction from flea prepupal larvae that was obtained by monitoring for carboxylesterase activity surprisingly also contained flea serine protease inhibitor molecular epitopes of the present invention.
One embodiment of the present invention is an isolated flea serine protease nucleic acid molecule that hybridizes under stringent hybridization conditions with a
Ctenocephalides felis
serine protease inhibitor gene, including, but not limited to, nucleic acid molecules that hybridize under stringent conditions with a nucleic acid molecule having at least one of the following nucleic acid sequences: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID 35. Particularly preferred flea serine protease inhibitor nucleic acid molecules include nucleic acid sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID 35, and/or nucleic acid sequences encoding proteins having amino acid sequences SEQ ID NO:2, SEQ ID N:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:32, and SEQ ID NO:36, as well as allelic variants of any of the listed nucleic acid sequences or complements of any of the listed nucleic acid sequences.
The present invention also includes an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:32 an

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