Flea chitinase nucleic acid molecules and uses thereof

Details Flea chitinase nucleic acid molecules and uses thereof Flea chitinase nucleic acid molecules and uses thereof

2000-04-07

2002-07-09

Chan, Christina Y. (Department: 1644)

Chemistry: molecular biology and microbiology

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

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

C435S252300, C435S320100, C536S023600, C530S350000

Reexamination Certificate

active

06416977

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to flea chitinase 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
Flea infestation in animals is a health and economic concern because fleas are known to cause and/or transmit a variety of diseases. Fleas 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 fleas 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 fleas. As such, fleas 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 fleas 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 flea infestation has prompted the development of reagents capable of controlling flea infestation. Commonly encountered methods to control flea infestation are generally focused on use of insecticides. While some of these products are efficacious, most, at best, offer protection of a very limited duration. Furthermore, many of the methods are often not successful in reducing flea populations. In particular, insecticides have been used to prevent flea infestation of animals by adding such insecticides to shampoos, powders, collars, sprays, spot-on formulations, foggers and liquid bath treatments (i.e., dips). Reduction of flea infestation on the pet has been unsuccessful for one or more of the following reasons: failure of owner compliance (frequent administration is required); behavioral or physiological intolerance of the pet to the pesticide product or means of administration; and the emergence of flea populations resistant to the prescribed dose of pesticide.
Insect chitinases play an important role in molting and cuticle turnover by hydrolyzing the structural polymer chitin (poly-&bgr;-(1,4)-N-acetyl-D-glucosamine), a principal component of the insect exoskeleton and gut lining. Chitinase activity has been identified in the molting fluids and midguts of several insects; examples include
Bombyx mori, Manduca sexta, Aedes aegypti
, and the wasp Chelonus. Chitinase appears to play an important role in insect growth and development; for example chitinase is essential for the removal and remodeling of old cuticle during metamorphosis of insects from one stage to the next. Inhibition of flea chitinase offers the potential of disrupting flea growth and development, thereby killing the fleas. However, development of flea chitinase inhibitors to disrupt flea growth and development, thereby reducing flea infestation, has been hampered by the lack of the appropriate reagent, i.e. flea chitinase.
Prior investigators have described the following insect chitinase nucleic acid sequences:
Aedes Aegypti
chitinase cDNA, Specht, (1997) et al. direct submission to Genbank, accession number AF02649 1
; Manduca sexta
chitinase precursor mRNA, Muthukrishnan, (1998) direct submission to Genbank, accession number U02270
; Bombyx mori
chitinase mRNA, Kim, M. K.et al. (1997) direct submission to Genbank, accession number BMU86876. Prior investigators have also described the following insect chitinase polypeptides: the endochitinase precursor from
Manduca sexta
, Choi, et al. (1997)
Insect Biochem. Mol. Biol
. 27(1), p 37-47; chitinase-like protein from
Bombyx mori
, Kim, et al. (1997), accession number 18418551; chitinase protein from
Hyphantria cunea
, Kim, et al. (1997), accession number 1841853; chitinase from
Aedes aegypti
, Specht et al. (1997), direct submission to Genbank, accession number 2564719; chitinase protein from
Drosophila melanogaster
, Genbank accession number JC4038; chitinase protein from
Chironomus tentans
, Genbank accession number Y13233; chitinase protein from Chelonus (sp), Genbank accession number A53918; chitinase protein from
Anopheles freeborni
, Genbank accession number AF026495; chitinase protein from
Anopheles gambiae
, Genbank accession number AF008575; and chitinase protein from
Phaedon cochleariae
, Genbank accession number Y 18011.
Identification of flea chitinase proteins and nucleic acids of the present invention is surprising, because overall homology of the flea chitinase to other insect chitinases that have been described is fairly low. The highest identity between a flea chitinase of the present invention and the protein that most closely resembles the flea chitinase in the databases searched is only 64%. Homologies between deduced insect chitinase proteins described to date have typically been greater; for example, amino acid alignment homology of three insect chitinases is 75% between
Bombyx mori
and
Hyphantria cunea
, 80% between
B. mori
and
Manduca sexta
, and 77% between
H. cunea
and
M. sexta
. See Kim, et al., (1998),
Insect Biochemistry and Molecular Biology
, vol 28 pp 163-171.
Identification of a flea chitinase is also surprising, because of the rarity of the mRNA coding for chitinase from the cDNA libraries made from various larval stages. It has been found that in other insects, expression of chitinase is very tightly regulated, and is restricted to short time intervals, such as when molting events are initiating. Since fleas are so small, and have much less RNA, compared to other insects that are traditionally studied, i.e.
Bombyx mori
, the silkworm, and
Manduca sexta
, the tobacco homworm, obtaining sufficient numbers of fleas synchronized to the exact right life stage where chitinase is being expressed is very difficult.
Thus, there remains a need to develop a flea chitinase reagent and a method to protect animals from flea infestation by targeting flea chitinase.
SUMMARY OF THE INVENTION
The present invention relates to a novel product and process for protection of animals from flea infestation. Identification of flea chitinase proteins and nucleic acid molecules of the present invention is surprising, however, due to the low degree of homology of flea chitinase to chitinases described from other insects, and due to the difficulty in obtaining fleas in the proper life stage, such that the fleas are expressing chitinase.
According to the present invention there are provided flea chitinase proteins, and mimetopes thereof; flea chitinase nucleic acid molecules, including those that encode such proteins; antibodies raised against such flea chitinase proteins (i.e., anti-flea chitinase antibodies); and compounds that inhibit flea chitinase 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 a nucleic acid molecule, protein, mimetope, and/or protective compound derived from a protein of the present invention that inhibits the activity of flea chitinase.
One embodiment of the present invention is an isolated nucleic acid molecule that is selected from a group consisting of (a) a nucleic acid molecule that includes one or more of the following sequences: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 1

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