JP2001510324A - Flea protease proteins, nucleic acid molecules, and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to a nomicelin protease protein and a nomicysteine protease protein; a serine protease and a cysteine protease nucleic acid molecule comprising a nucleic acid molecule encoding such a protein; an antibody generated against such protein; a nomicelin protease and / or cysteine protease activity A compound that inhibits The invention also includes methods for obtaining such proteins, nucleic acid molecules, antibodies, and inhibitors. The invention also includes therapeutic compositions containing such proteins, nucleic acid molecules, antibodies, and / or inhibitors, and the use of the compositions to protect a host animal from flea infestation.

Description

DETAILED DESCRIPTION OF THE INVENTION        Flea protease proteins, nucleic acid molecules, and uses thereof [Field of the Invention]   The present invention relates to a novel flea protease protein and the ability of fleas to propagate in animals. Regarding their use to curb. The present invention reduces flea protease activity. Anti-flea protease antibodies and other to reduce fleas propagation in animals The use of the compounds of [Background of the Invention]   Fleas belong to the order Insectoptera, and are genuine parasites of many animals, including birds and mammals It is an ectoparasite. Flea can cause and / or transmit various diseases. Breeding fleas on animals can cause health and economic problems. You. Fleas include, for example, flea allergic dermatitis, anemia, murine typhoid, plague, and And / or transmitting infectious agents that cause them . In addition, fleas are a problem for animals kept as pets. why The owner's home is usually contaminated with fleas that are nourished by pets. Because of this, the breeding of fleas can be a problem for pet owners. This Fleas are not only present in animals but also in the general environment of animals. It is also a problem when it exists.   Because fleas reproduction is of medical and veterinary importance, controlling fleas Has promoted the development of reagents that can Commonly used to control fleas breeding The method generally involves the treatment of sprays, shampoos, powders, disinfectants, or foam. Focus on the use of pesticides in pets or in pet collars It is guessing. Some of these products are effective, but many are at best It only gives short term protection. In addition, many of these methods are: For one or more reasons: (1) the owner's consent was not obtained (frequent administration (2) pets are behavioral or physiological in response to insecticide products or means of administration. (3) Flea populations resistant to prescribed amounts of insecticide Present In many cases, flea populations in pets cannot be reduced successfully. other Anti-flea products include insect growth regulators (IGRs), including methoprene. nontoxic reagents such as growth regulators, which mimic flea hormones. Imitate and affect flea larval growth.   Another method for controlling fleas is to prevent fleas from breeding or breeding. Is to use a flea vaccine that is administered to animals at times. But anti-no Despite considerable interest in the development of the drug, so far, There is no flea vaccine. [Summary of the Invention]   The present invention relates to a flea serine protease protein, a flea aminopeptidase. Proteins and flea cysteine protease proteins; Flea serine protease containing a nucleic acid molecule encoding a protein, aminopeptidic Nucleic acid molecules of dase and / or cysteine protease; such proteins Antibodies raised against; flea serine proteases, aminopeptiders And / or compounds that inhibit cysteine protease activity. The present invention Also includes methods for obtaining such proteins, nucleic acid molecules, antibodies, and inhibitors. No. Therapies comprising such proteins, nucleic acid molecules, antibodies, and / or inhibitors Composition, and such therapeutic composition for protecting a host animal from fleas breeding Is also included in the present invention.   Certain embodiments of the invention provide for use under stringent hybridization conditions. And SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 1 7, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 2 6, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 3 5, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 1 20, SEQ ID NO: 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, Sequence Column No. 121, SEQ ID No. 131, SEQ ID No. 155, SEQ ID No. 114, SEQ ID No. 125, SEQ ID No. 1 18, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, arrangement Column No. 78, SEQ ID No. 158, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 66, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 1 42, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84 and / or Or a serine protease comprising a nucleic acid sequence comprising a nucleic acid molecule comprising SEQ ID NO: 45 A nucleic acid molecule selected from the group consisting of SEQ ID NO: 110 and / or SEQ ID NO: 112; Provided aminopeptidase gene; and SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, Comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO: 76 and / or SEQ ID NO: 94 An isolated nucleus that hybridizes to a gene containing the cysteine protease gene It is an acid molecule.   The present invention provides the ability to use SEQ ID NO: 10 under stringent hybridization conditions. , SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27 , SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44 , SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72 , SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: No. 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, Sequence No. 145, SEQ ID No. 141, SEQ ID No. 123, SEQ ID No. 68, SEQ ID No. 163, SEQ ID No. 162 , SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: No. 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, and / or Is a nucleic acid sequence encoding a protein having an amino acid sequence comprising SEQ ID NO: 95, Or a nucleic acid sequence that is the complement of any of the nucleic acid sequences. Also includes nucleic acid molecules that hybridize. Preferred nucleic acid sequences of the present invention include SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 45, SEQ ID NO: 120, SEQ ID NO: 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, Sequence Column No. 121, SEQ ID No. 131, SEQ ID No. 155, SEQ ID No. 114, Sequence No. No. 125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 66, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 80, SEQ ID NO: 82, Sequence No. 142, SEQ ID No. 138, SEQ ID No. 144, SEQ ID No. 140, SEQ ID No. 122, SEQ ID No. 84 , SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 76, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and And / or alleles thereof comprising a nucleic acid sequence comprising SEQ ID NO: 94 Includes allelic variants. The present invention provides a stringent hybrid SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115 , SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, Sequence No. 161, SEQ ID No. 137, SEQ ID No. 79, SEQ ID No. 159, SEQ ID No. 133, SEQ ID No. 135 , SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: No. 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 10 7, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 Having the amino acid sequence of SEQ ID NO: 89, SEQ ID NO: 92, and SEQ ID NO: 95. A nucleic acid molecule that hybridizes with a nucleic acid molecule having a nucleic acid sequence encoding a protein Thus, the isolated protein encoded thereby is also included.   The present invention relates to recombinant molecules, recombinant viruses, and combinations comprising the nucleic acid molecules of the invention. Also relates to transgenic cells. Such nucleic acid molecules, recombinant molecules, recombinant viruses, and Also included are methods for producing recombinant cells.   Yet another embodiment of the present invention can reduce the growth of blood-sucking ectoparasites. Therapeutic composition. Such therapeutic compositions include stringent agents. Under hybridization conditions, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: No. 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO No. 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, Sequence Column No. 115, SEQ ID No. 126, SEQ ID No. 119, SEQ ID No. 129, SEQ ID No. 153, SEQ ID No. 1 57, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, Sequence No. 135, SEQ ID No. 67, SEQ ID No. 147, SEQ ID No. 149, SEQ ID No. 151, SEQ ID No. 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, Sequence No. 107, SEQ ID No. 111, SEQ ID No. 113, SEQ ID No. 77, SEQ ID No. 2, SEQ ID No. 5, arrangement Amino acid sequence comprising SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, and / or SEQ ID NO: 95 Hybridization with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising The isolated protein encoded by the nucleic acid molecule or its mimetope (m imetope); under stringent hybridization conditions, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 120 , SEQ ID NO: 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, Sequence No. 121, SEQ ID No. 131, SEQ ID No. 155, SEQ ID No. 114, SEQ ID No. 125, SEQ ID No. 118 , SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, Sequence No. 78, SEQ ID No. 158, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 66, SEQ ID No. 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 142, SEQ ID NO: 1 38, SEQ ID NO: 144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84, SEQ ID NO: 110, Sequence No. 112, SEQ ID NO: 76, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO No. 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, and SEQ ID NO: 94 An isolated nucleic acid molecule that hybridizes to a gene comprising a nucleic acid sequence; SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 13, No. 16, SEQ ID No. 19, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, Sequence No. 33, SEQ ID No. 36, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 67, Sequence No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, Sequence No. 96, SEQ ID No. 115, SEQ ID No. 126, SEQ ID No. 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, Sequence No. 79, Sequence No. 159, Sequence No. 133, Sequence No. 135, Sequence No. 67, Sequence No. 147 , SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: No. 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 1 13, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 And a nucleic acid sequence encoding a protein having an amino acid sequence comprising SEQ ID NO: 95 Encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule having a sequence Isolated antibodies that selectively bind to proteins; stringent hybridization SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: No. 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, Sequence No. 143, SEQ ID No. 139, SEQ ID No. 145, SEQ ID No. 141, SEQ ID No. 123, SEQ ID No. 68 , SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: No. 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: No. 89, SEQ ID NO: 92, and a protein having an amino acid sequence comprising SEQ ID NO: 95 A nucleic acid molecule that hybridizes to a nucleic acid molecule having an encoding nucleic acid sequence. Protease activity identified by its ability to inhibit the activity of the loaded protein Sex inhibitors; and protective compounds, including mixtures thereof, and excipients. Flea A method of suppressing reproduction, comprising the step of administering a therapeutic composition of the present invention to an animal. The method of providing is also included in the present invention.   Other embodiments of the present invention identify compounds that can inhibit flea protease activity A method for   (a) In the absence of an inhibitory compound, the protein has proteolytic activity.     SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, sequence     No. 22, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36,     SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:     No. 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96,     Column number 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, arrangement     Column number 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, arrangement     Sequence No. 133, Sequence No. 135, Sequence No. 67, Sequence No. 147, Sequence No. 149, Arrangement     Column No. 151, SEQ ID No. 81, SEQ ID No. 83, SEQ ID No. 143, SEQ ID No. 139, Sequence     No. 145, SEQ ID No. 141, SEQ ID No. 123, SEQ ID No. 68, SEQ ID No. 163, Sequence     No. 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO:     No. 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89,     An isolated amino acid sequence comprising SEQ ID NO: 92, and / or SEQ ID NO: 95     Contacting a flea protease protein with a candidate inhibitory compound     When,   (b) determining whether the candidate inhibitory compound inhibits activity, It is a method provided with. The present invention provides a compound capable of inhibiting the protease activity of flea. Kits for identifying objects are also included.   The present invention uses about 100 μL of about 0.1 μL. Approximately 18 hours at 37 ° C in 2M Tris-HCl Cleaves immunoglobulins when incubated in the presence of Bryn Also includes isolated flea protease proteins. Can cleave immunoglobulins Preferred protease proteins are SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, Group consisting of column number 70, sequence number 71, sequence number 72, sequence number 73, and sequence number 96 An amino acid sequence selected from the group consisting of:   In another embodiment of the present invention, there is provided a flea immunoglobulin proteinase protein. A method for identifying a compound capable of inhibiting an activity,   (a) if no inhibitory compound candidate exists, the protein is an immunoglobulin     Candidate inhibitory compound was isolated under conditions with proteinase activity     Contacting a flea protease protein;   (b) determining whether the candidate inhibitory compound inhibits activity, A method comprising: [Brief description of drawings]   FIG. 1 shows fasted larvae, fed first instar larvae, and fed third instar larvae Scanning showing SDS-PAGE of SDS-PAGE of DFP-labeled larval protease It is an image. [Detailed description of the invention]   The present invention inhibits flea protease activity to protect host animals from fleas breeding. Including the use of harmful compounds. The inventor believes that proteases are an important component of flea midgut And the fleas are present in the host animal and in the immediate (ie, surrounding) environment of the animal. A suitable target for immunotherapy and / or chemotherapy to suppress recruitment It was discovered that. For example, the inventor has found that flea If it contains a compound that reduces rotase activity, Has shown that the survival and / or fecundity of mi This appears to be due to the compound affecting flea digestion and other functions. It is. Flea protease levels and / or activities without substantial harm to the host animal Compounds that reduce the nature are included in the present invention. Such compounds include flea protein An enzyme vaccine, an anti-flea protease antibody, a flea protease inhibitor, and / or Or a compound that inhibits protease synthesis. , Discussed in more detail below.   One embodiment of the present invention relates to a flea that is nourishing from a treated animal Reducing the protease activity of pre-existing and already nourished fleas Treating the animal with a composition comprising the compound protects the host animal from fleas breeding. And thereby reduce the fleaing of animals and their environment.   Note that the word "a" or "an" entity means more than one Must. For example, one compound refers to one or more compounds. This Thus, as used herein, the term "one" refers to "one or more" and "at least". Is also used interchangeably with the word "one." For this reason, one composition of the present invention , One or more agents that target one or more proteases in fleas Compounds may be included.   As used herein, the term "protecting an animal from fleas breeding" refers to the flea Reduce the ability of the population to grow on and around animals (ie, To suppress). Preferably, the size of the flea population is reduced, Most preferably, the object is reduced to such an extent that it no longer suffers from fleas. This specification The host animal used in (1) is obtained by attaching flea and obtaining nutrients from the animal's skin. And fleas are animals that get nourished from them. Fleas and other ectoparasites Settle on the host animal for an extended period of time or temporarily attach to the animal to obtain nutrition. Can be. At any given time, a certain percentage of flea populations While the rest is in the environment around the animal (ie in the animal's environment) May exist. Such environments are not only flea adults, but also flea eggs and / or Can also include flea larvae. The environment is such that fleas present in the environment fly over the host animal. It can be any size that allows it to migrate and fly away from the host animal. this Not only keep fleas from collecting on the animals themselves, but also It is desirable to suppress the gathering.   According to the present invention, the compounds themselves (e.g., protease inhibitors, Animals, depending on the administration of the compound or the compound). Products produced (eg, antibodies produced in response to a flea protease vaccine, Inactive inhibitor "prodrug" converted to active protease inhibitor Is administered to the animal in such a way that it finally enters the midgut of the flea To treat the host animal. The animal is exposed to the compound or its compound in the animal's bloodstream. It is preferable that the treatment is carried out so that the product of This allows fleas to flourish from animals When they are nourished, they come into contact with the compound. For example, flea protea administered to animals The inhibitor is administered such that the inhibitor enters the animal's bloodstream, where the fleas inhibit The agent can be absorbed. In another embodiment, the host animal is a flea protease When the vaccine is administered, the immune response of the treated animal increases, Antibody (anti-flea protease antibody) is produced and circulates in the bloodstream of the animal. And are sucked when the flea takes nutrition. The blood that the fleas sucked is inside the fleas Enters the gut where there are anti-flea protease antibodies, flea protease inhibitors, and And / or a compound of the present invention such as a protease synthesis inhibitor or a product thereof Affects and reduces the proteolytic activity of the midgut. Treatment of the present invention Composition, at least a portion of a compound of the invention, or a product thereof. When fleas nourish from the host animal's blood, they are excreted in flea droppings and continue. The present invention suppresses the reproduction of the flea larvae because the flea larvae ingest this Ability is also included. Flea larvae are not, but not all, nutrients derived from flea dung. It must be noted that you have gained a lot of.   According to the present invention, by reducing the proteolytic activity of flea midgut, A number of consequences have been found to reduce flea accumulation in the treated animal and surrounding environment. Can be These results indicate that (a) nutrition is taken from the treated animal Reduced flea viability; (b) females receiving nutrition from treated animals; Reduced flea production capacity; (c) males receiving nutrition from treated animals; Reduced flea fertility; (d) females receiving nutrition from treated animals; That the viability of the fleas laying eggs is reduced, (e) Changes in the blood intake behavior of fleas that are present (e.g., the amount of fleas Decrease, or reduce the number of times of nutrition intake), (f) The flea larvae nourish from the flea droppings, thereby reducing the viability of the flea larvae And / or (g) altering flea larval growth (eg, reducing feeding behavior) Inhibiting growth, inhibiting molting (e.g., delaying or inhibiting), And / or or by inhibiting adulthood). It is not limited to these.   Direct (eg, anti-flea protease antibodies, or inhibitors of flea proteases) And / or indirectly (eg, a flea protease vaccine) may include one or more flea programs. Compositions comprising one or more compounds that reduce the activity of Rotase may be useful in certain embodiments of the invention. It is an embodiment. Suitable flea proteases to target include flea aminopeptides. Tidase, flea carboxypeptidase, and / or flea endopeptidase Ze is included. Such proteases include cytoplasmic and / or membrane-bound proteases. Rotase may be included. A preferred flea protease to target is serine Proteases, metalloproteases, aspartic proteases and / or Including but not limited to stain proteases. These preferred pairs Step The group of tidases includes aminopeptidases, carboxypeptidases, and It should be noted that endopeptidases are included. Target Suitable flea proteases should include hemoglobin-degrading proteases, blood Peptide form, a protease involved in the liquid coagulation and / or hemolysis (anticoagulation) pathway Proteases, complement, or other host immune response elements involved in For example, proteases that inhibit antibodies) and / or proteases involved in yolk production But not limited thereto. Leucine aminopeptidase, and Aminopeptidases such as aminopeptidase B and aminopeptidase M; Stacin-like metalloprotease; calpain; carboxypeptida Carboxypeptidases, such as A, P and Y; cathepsins B, D, E, G Cathepsins such as, H and L; chymotrypsin; cruzipain Meprin; papain; pepsin; renin; thermolysin and trypsin Numerous proteases, including but not limited to, those known to those of skill in the art, are not limited to these. Particularly preferred proteases for targeting are when targeted in the compositions of the invention. In addition, a protein that suppresses the accumulation of fleas without substantially harming the host animal It is an active protease. For example, such proteases are described herein. Identification can be performed using the methods disclosed herein.   One aspect of the invention is that the significant amount of proteolytic activity found in the midgut of fleas is It is a discovery that it is a serine protease activity. Numerous protease inhibitors The in vitro and in vivo studies used support this finding, the details of which are It is disclosed in the examples. For this reason, particularly preferred proteases for targeting are , A serine protease. Examples of serine proteases include acrosin, bro Melanin, cathepsin G, chymotrypsin, collagenase, elastase, Factor Xa, ficin, kallikrein, papain, plasmin, staphylococcus V 8 Including, but not limited to, protease, thrombin, and trypsin Absent. In one embodiment, suitable flea serine proteases for targeting are: Proteases with trypsin-like or chymotrypsin-like activity are included. " An enzyme with "like" protein activity is the exact structure of a suitable substrate that is cleaved. Although it can be different, that the enzyme has an activity similar to the indicated protease, This It is understood by traders. "~ Sama" protease is usually indicated on the counter It has a tertiary structure similar to the part.   The study of protease inhibitors disclosed in the Examples section has shown that Other proteases include aminopeptidases and / or metalloproteases It also shows that it is included. Examples of such proteases include exo-, and Involved in maturation of endo-metalloproteases, digestive enzymes, and peptide hormones Includes enzymes. One of the aminopeptidases that are also metalloproteases An example is leucine aminopeptidase. Suitable for inclusion in the composition of the present invention. Flea proteases (flea protease vaccines), flea proteases Antibodies that selectively bind to Rotase (anti-flea protease antibodies), flea protea Protease inhibitors (compounds other than vaccines or antibodies that inhibit flea proteases), and And vaccines comprising a mixture of such compounds. Absent. As used herein, these mixtures are combinations of one or more of the described things. Point out. The compositions of the present invention include a protease modulating peptide (protease modul Compounds that inhibit protease synthesis or maturation, such as ating peptides) But not limited to these.   Flea protease vaccines and populations of fleas on and around animals Its use to reduce is a preferred embodiment of the present invention. Flea protection The ase vaccine contains one or more that can elicit an immune response to flea proteases. And other components may be included. Suitable flea protease Kuching includes flea serine protease, flea metalloprotease, and flea Includes spargate protease and / or flea cysteine protease Flea serine protease, flea metalloprotease, and / or flea Minopeptidase vaccines are most preferred. An example of a flea protease vaccine Is a soluble flea midgut preparation of the invention and one or more isolated proteins of the invention Is included.   One embodiment of the present invention is a soluble flea midgut preparation. For such preparations Contains mainly natural components present in the lumen of the midgut of fleas, depending on the preparation method , One or more peripheral mid-mesenteric proteins. Membrane tamper from such preparations K Preferred methods of introducing or excluding quality are known to those skilled in the art. The present invention Such preparations have proteolytic activity (a significant part of which is serine protease activity) Includes the discovery that Preferably, a soluble preparation of soluble flea midgut Serine protease activity is at least about 70% of the proteolytic activity present in Is a 4-aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF) Thus, it may be indicated by the ability to inhibit at least about 70% of the proteolytic activity. You. Serine protease activity can be identified using other known inhibitors or substrates. Can also be. At least the proteolytic activity of the soluble flea midgut preparation of the invention Other suitable inhibitors that can inhibit about 70% include soybean trypsin inhibitor, 1,3 Includes diisopropyl fluorophosphate or leupeptin.   The soluble flea midgut preparation of the present invention includes SDS-PAGE (sodium dodecyl sulfate poly). Acrylamide gel electrophoresis), as determined by about 5-200 kDa (KD or kDa) of the protease contained and determined by SDS-PAGE At least a substantial portion of the serine protease has a molecular weight of about 5-60 kD. is there. A significant portion of the protease activity in the soluble flea midgut preparation of the present invention is pH Optimal pH activity in the range of about 5-10, preferably pH in the range of about 7-9, more preferably Has an optimum activity of about pH 8. Without being bound by theory, such The optimal pH is determined by the majority of the proteases present in the soluble flea midgut preparation of the present invention. Suggests that it is a serine protease. The pH of the midgut of fleas is also about 8. It is also interesting. The protease of the soluble flea midgut preparation of the present invention may be of a certain type. Different inhibition of protease inhibitors (eg, serine protease inhibitors) Pattern, and that the molecular weight and optimal pH of the protease are not uniform These findings suggest that numerous protease isoforms are present in such preparations. Suggests that   Preferably, the soluble flea midgut preparation of the invention comprises (a) disrupting the midgut of the flea, Create a mixture containing a liquid portion and a solid portion, and (b) recover the liquid portion to It is prepared by a method comprising the step of obtaining a midgut preparation. Such person The method is a simplification of the method disclosed in U.S. Pat.No. 5,356,622. You. In the present invention, a soluble midgut preparation having the same proteolytic activity is prepared. To prepare, the method disclosed in U.S. Pat.No. 5,356,622 ibid may also be used. It should be noted that   The midgut of a flea is a fleas that have not been fed or that have just taken blood. Can be obtained from mi (ie, fleas given blood) Therefore). Such midgut is referred to herein as the midgut of the fasted flea and the midgut, respectively. Indicated as the midgut of the food flea. Flea midgut can be obtained from either male or female fleas be able to. As shown in the Examples section, the midgut of a female flea is Shows somewhat higher proteolytic activity than flea midgut. In addition, inside the flea The gut has significantly higher proteolytic activity than the fasted flea midgut. In theory Although not restricted, blood intake is controlled by proteases in the midgut of fleas, And host molecules that can digest the feed blood and damage the fleas (eg, complement, Other factors that are important in neutralizing immunoglobulins, blood coagulation factors (eg, enzymes) , Synthesis, and / or activation of other proteins, cofactors, etc.) available. The midgut of fasted fleas contains important targets not found in the midgut of fed fleas Also understand what you may have and vice versa Will. In addition, the present application focuses primarily on flea midgut proteases However, the present invention provides a suitable marker for suppressing the accumulation of fleas on animals and their environment. Note that it also includes other components of the soluble flea midgut preparation of the present invention that provide a target. Must. See also U.S. Patent No. 5,356,622, ibid.   Methods for disrupting the flea midgut to obtain a soluble flea midgut preparation are described in It is well known to those skilled in the art and, for example, Can be. Such methods include chemical disintegration methods (eg, using midgut as a surfactant). Contact) and mechanical disintegration methods (eg, homogenization, sonication, Use of tissue disruptors or glass beads and freeze / thaw methods) Any technique that facilitates, but is not limited to, these.   Methods for recovering soluble flea midgut preparations are also known to those of skill in the art, and Any method of separating the liquid portion of the midgut from the solid portion can be included (e.g., filtration). Over or centrifugation). In a preferred embodiment, the broken gut midgut is preferably about 10, Centrifuge at an acceleration in the range of 000 to 15,000 g for several minutes (for example, about 1 to 15 minutes). Be killed. The supernatant obtained by such centrifugation is the soluble flea midgut preparation of the present invention. Contains.   The present invention relates to the midgut obtained from a female flea fed with blood, and a male flea fed with blood. Midgut obtained from female, midgut obtained from female flea not giving blood, or blood Not present in the midgut of fleas, such as midgut obtained from male fleas (ie found Nucleic acid molecule encoding a protease (ie, complementary to the coding strand) Nucleic acid molecule that hybridizes with a specific nucleic acid strand) and a stringent hybrid Nucleic acid molecules that are capable of hybridizing under the conditions of Hybridized under unusual hybridization conditions). An isolated protein comprising the amino acid sequence of the present invention is also included. Suitable midgut protea It is present in the lumen of the midgut.   The isolated proteins of the present invention are referred to herein as isolated protease proteins. It is also referred to as a protein, and preferably enhances the immune response to flea midgut proteases. And / or has proteolytic activity. According to the present invention Isolated or biologically pure protein is removed from its original environment Protein. Thus, "isolated" and "biologically pure" Does not necessarily reflect the degree of protein purification. Isolated protease Protein can be obtained from natural sources. Such an isolated tamper The protein can also be produced using recombinant DNA technology or chemical synthesis.   As used herein, an isolated protein of the invention is a full-length protein , Or deletion of amino acids (e.g., truncation of protein such as peptide ), Inserted, inverted, substituted and / or derivatized (eg, glycosylated, Oxidation, acetylation, myristoylation, prenylation, palmitoylation, amidation And / or addition of glycerophosphatidylinositol) proteins. Any homologue of such a protein, the nucleic acid encoding the homologue The sequence, under stringent hybridization conditions, In the complementary strand of the nucleic acid sequence encoding the amino acid sequence of the flea midgut protease ( That is, it can hybridize with the complementary strand) Can be a homolog that is a protein having an amino acid sequence sufficiently similar to You. As used herein, stringent hybridization conditions refer to A similar nucleic acid molecule is identified using a nucleic acid molecule containing an oligonucleotide under the same conditions. Means standard hybridization conditions to be determined. Such standard conditions Is described, for example, in Sambrook et al., “Molecular Cloning: A Laboratory Manual, Cold S pring Harbor Labs Press, 1989; Sambrook et al., ibid. The entirety of which is incorporated herein by reference. Stringent hybridization Conditions are typically used as probes in hybridization reactions. A nucleic acid molecule having at least about 70% nucleic acid sequence identity with the nucleic acid molecule to be obtained. Can be Hybridization tolerating 30% or less nucleotide mismatch Calculate appropriate hybridization and washing conditions to achieve Formula, for example, Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al. , Which are hereby incorporated by reference in their entirety.   The minimum size of a protein homolog of the present invention is the size of the corresponding native protein. Nucleic acid capable of forming a stable hybrid with the complementary sequence of the nucleic acid molecule to be loaded The size is well encoded by the molecule. Because of this, such proteins The size of the nucleic acid molecule encoding the homolog depends on the composition of the nucleic acid and the sequence complementary to the nucleic acid molecule. As well as the hybridization conditions themselves (eg, Temperature, salt concentration, and formamide concentration). The ultimate in such nucleic acid molecules Small size is typically at least about 12-15 nucleic acids if the nucleic acid molecule is GC rich. It is the length of the ochide and, if AT rich, at least about 15-17 bases in length. This Used to encode a protease protein homologue of the present invention. The minimum size of a nucleic acid molecule is about 12-18 nucleotides in length. Nucleic acid molecules A part of the gene, the entire gene, or multiple genes, or The maximum size of such a nucleic acid molecule may be outside of practical limits, Has no constraints. Similarly, the minimum size of the protease protein homologs of the present invention The size is about 4 to 6 amino acids in length, and the preferred size is full-length, multivalent That is, a fusion protein having more than one domain each having a function), or Depending on whether a functional part of such a protein is desired Decided. The protease protein homologs of the present invention preferably have protease activity. And / or may elicit an immune response against flea midgut proteases You. The protease protein homologs of the present invention encode a flea protease. It can be the result of an allelic variation of a native gene. A natural gene is a natural gene Means the form of the gene most often found in Protease protein phase Isozymes can be, for example, random mutagenized or targeted mutations. Coat proteins using classical or recombinant DNA techniques that result in transfection. Production using techniques known to those skilled in the art, including directly modifying the gene But not limited thereto. The isolated homologues of the present invention Protease protein is that the protein can show proteolytic activity, And / or capable of eliciting an immune response to midgut proteases of fleas Thus, it can be identified in a direct manner. Such techniques are known in the art. It is.   Preferred protease proteins of the present invention are flea serine proteases, flea Metalloprotease, flea aspartic protease, flea cysteine A protease, or a homologue of any of these proteases. More suitable Flea serine proteases and flea metalloproteins Ase, or any homolog. Flea aminopeptidase or homology thereof The body is also suitable. Flea cysteine proteases or homologs thereof are also suitable. Flea serine proteases or homologs thereof are particularly preferred.   A preferred protease protein of the present invention has about 5 as determined by SDS-PAGE. A flea protease protein having a molecular weight in the range of ~ 200 kD; Is a homologue of such a protein. Further suitable flea protease proteins are , A flea professional with a molecular weight in the range of about 5-60 kD as determined by SDS-PAGE Thease proteins and homologs of such proteins. More suitable Is a flea serine protease protein, especially with a molecular weight of about 26 kD. Having (notated as PfSP26, but with the flea PrSP26 described in Example 26) For the sake of distinction, it is described here as PafSP-26K), about 24 kD (PfSP24 and To be distinguished from the flea PfSP24 described in Example 27. For this reason, it is described here as PafSP-24K), about 19 kD (described as PrSP19). However, in order to distinguish it from the flea PfSP19 described in Example 32, here PafS P-19K), about 6 kD (described as PfSP6, described in Example 11). (In order to distinguish it from the flea PfSP6, it is written here as PafSP-6K.) (Noted as PfSP28), about 25 kD obtained from the first instar larva (PlfSP-25K1 and About 25 kD obtained from the third instar larva (denoted as PlfSP-25K3), about 28 kD D (PlfSP-28K3) and about 31 kD (PlfSP-31K3) About 95% of flea aminopeptidase proteins, especially as determined by SDS-PAGE those with a molecular weight of kD (denoted as PfAP-95K), and those proteins Homolog.   One preferred embodiment of the present invention provides for stringent hybridization. Flea serine protease gene and flea aminopeptidase gene Nucleic acid molecules that hybridize with offspring or flea cysteine protease genes Isolated flea protease protein comprising the encoded amino acid sequence Quality. As used herein, a flea protease gene is defined as Thus, the regulatory domain that regulates the production of the encoded flea protease protein Regions (such as, but not limited to, transcriptional, translational, or post-translational regulatory regions) and All nuclei associated with the native flea protease gene, such as the Acid sequences.   The inventor has discovered that the large family of serine protease genes A new serine protease family has been discovered. Such gene families are Allelic variants (i.e., similarities present at specific loci in a flea population) But different genes) and / or two or more in the flea genome That it may be due to the presence of the serine protease gene at the locus. discovered. Thus, the present invention relates to the nucleic acid sequences disclosed herein (e.g., , SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 , SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26 , SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35 , SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45 , SEQ ID NO: 120, SEQ ID NO: 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: No. 127, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 155, SEQ ID NO: 114, SEQ ID NO: 125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: No. 136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 66, Column No. 146, SEQ ID No. 148, SEQ ID No. 150, SEQ ID No. 80, SEQ ID No. 82, SEQ ID No. 142 , SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84 Having the amino acid sequence disclosed herein), and / or A nucleic acid sequence encoding a protein (eg, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, Column No. 161, SEQ ID No. 137, SEQ ID No. 79, SEQ ID No. 159, SEQ ID No. 133, SEQ ID No. 13 5, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: No. 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, and / or SEQ ID NO: 96) as well as allelic variants, sequences, Fleas comprising other nucleic acid molecules and amino acid sequences disclosed in the Examples section to Includes serine protease genes (nucleic acid sequencing technology is completely error-free As such, all sequences shown herein are deduced (ie, Note that this is only a deduced) nucleic acid or amino acid sequence. Must. ).   Suitable flea aminopeptidase genes include SEQ ID NO: 107, SEQ ID NO: 111, and And / or amine peptidase protein having an amino acid sequence comprising SEQ ID NO: 113 Includes the nucleic acid sequence of SEQ ID NO: 110 and / or SEQ ID NO: 112, which encodes quality.   Other suitable aminopeptidase genes include SEQ ID NO: 110 and / or SEQ ID NO: 1 Includes 12 allelic variants.   Preferred flea cysteine protease genes include SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 77, and / or SEQ ID NO: 95 A sequence encoding a cysteine protease protein having an amino acid sequence comprising No. 1, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 88, SEQ ID No. 9 0, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76, and / or SEQ ID NO: 94 included. Other suitable cysteine protease genes include SEQ ID NO: 1, SEQ ID NO: No. 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 93, SEQ ID NO: 76, and / or allelic variants of SEQ ID NO: 94 You.   Preferred flea serine protease proteins of the present invention are stringent Under hybridization conditions, the following nucleic acid molecules: nfSP3, nfSP8, nfSP9, nfS P10, nfSP11, nfSP19, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfS P29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSP18, nfS Hybridizer with at least one of P24, nfSP28, nfSP32, nfSP33 and nfSP40 Encoded by a nucleic acid molecule that As used herein, these nucleic acid molecules Are the entire coding region of the flea serine protease gene of the present invention (implementation As described in the examples, at least also indicated by the flea clone number Represents a part of it). A part or other part of the coding region of the corresponding gene Containing nucleic acid molecules are denoted by names that include the size of these nucleic acid molecules (e.g., For example, nfSP40₄₂₆). Contains the estimated full length coding region of known size Nucleic acid molecules are also denoted by names that include the size of these nucleic acid molecules ( For example, nfSP40₈₄₁). Such a nucleic acid molecule, and at least a part of the nucleic acid sequence Is disclosed in the examples.   Particularly preferred serine protease proteins are stringent hybridizers. Under iserization conditions, at least one of the following nucleic acid molecules: nfSP18₅₃₄, NfSP18_{7 75} , NfSP18₂₂₅, NfSP24₄₁₀, NfSP24₁₀₈₉, NfSP24₇₇₄, NfSP24₇₁₁, NfSP28₉₂₃, N fSP32₉₃₃, NfSP32₉₃₃, NfSP32₉₂₄, NfSP32₆₉₉, NfSP33₄₂₆, NfSP33₇₇₈, NfSP33_{1 894} , NfSP33₁₂₀₀, NfSP33₇₂₆, NfSP40₈₄₁, NfSP5₈₀₆, NfSP11₃₀₇, NfSP8₅₁₅, Nf SP8₄₃₆, NfSP12₇₅₈, NfSP26₆₁₀, NfSP27₃₈₆, NfSP23₄₂₃, NfSP34₃₉₀, NfSP36₁₉₇ , NfSP38₃₄₁, NfSP37₂₆₁, NfSP39₂₆₇, NfSP29₆₁₂, NfSP30₆₄₁, NfSP31₆₂₆, NfSP 32₄₃₃, NfSP15₈₁₅, NfSP19₈₅₅, NfSP25₈₆₄, NfSP21₅₉₅And / or nfSP40₇₁₇Horse Hybrid with at least one of the Encoded by a soybean nucleic acid molecule. More preferred serine proteases The protein has the following amino acid sequence: SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, Column number 19, sequence number 22, sequence number 24, sequence number 27, sequence number 30, sequence number 33, Sequence No. 36, Sequence No. 38, Sequence No. 41, Sequence No. 44, Sequence No. 67, Sequence No. 68, arrangement Column No. 69, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 89, Sequence Column No. 92, SEQ ID No. 95, SEQ ID No. 115, SEQ ID No. 126, SEQ ID No. 119, SEQ ID No. 129 , SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: No. 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 1 45, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, Sequence No. 69, SEQ ID No. 85 and / or SEQ ID No. 96 are included. Other serimps that are particularly suitable Rotase proteins encode proteins containing the described amino acid sequences. Encoded by an allelic variant of a nucleic acid molecule to be encoded. As described herein A flea serine protease protein having an amino acid sequence About 50%, preferably at least about 75%, more preferably at least about 90% identical Also preferred are flea serine protease proteins that contain a sex region.   One embodiment of the invention degrades immunoglobulins circulating in a host animal. Flea serine protease (i.e., flea immunoglobulin protein Or IgGase). Examples of flea immunoglobulin proteinases are described in the Examples. It is shown in the section. Preferably, the immunoglobulin proteinase of the present invention comprises about Presence of immunoglobulin in 100 μL of about 0.2 M Tris-HCl at about 37 ° C. for about 18 hours Below, when proteins are incubated, they cleave immunoglobulins You. More preferably, the immunoglobulin proteinase of the present invention comprises about 300 μL Approximately 1 hour at about 37 ° C in 50 mM Tris-HCl, pH 8.0, Disconnect. Suitable immunoglobulin proteinase proteins of the present invention The hinge region of the heavy chain of Roblin can be cut. Immunoglobulin hinge The region is a flexible domain that connects the Fab arm of the immunoglobulin to the Fc portion of the molecule. Inn. More preferred immunoglobulin proteinase proteins include: Molecular weight in the range of about 25-35 kD, more preferably about 31 kD in mature form Are included. Further preferred immunoglobulin proteinases The protein may be encoded by a gene comprising the nucleic acid sequence of SEQ ID NO: 66 SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, Proteins having an amino acid sequence comprising SEQ ID NO: 73 and / or SEQ ID NO: 96 are included. I will. Without being bound by theory, the immunoglobulin protein of the present invention The proteinase activity of the enzyme is determined by immunoglobulin binding to two Fab fragments or one F (a b ')_TwoAn embodiment in which a heavy chain and light chain pair is maintained as one of the fragments Cleaves the immunoglobulin. The Fab fragment used herein is an immunoglobulin. Means the complete immunoglobulin light chain paired with the heavy chain variable region and CH1 domain I do. F (ab ') as used herein_TwoThe fragments are joined by disulfide bonds Mean two Fab fragments as they are. Fab fragment and F (ab ')_TwoAll fragments bind the antigen. Can be combined.   Preferred immunoglobulin proteinase proteins of the present invention have the amino acid sequence D- C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-D (SEQ ID NO: 104), D-C-P-K-C- P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K-S-E-V (SEQ ID NO: 105) And / or D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-D-T-L-S-I-S- Immunoglobulin at a site having an amino acid sequence containing R-T-P-E-V (SEQ ID NO: 106) The hinge region of the heavy chain can be cut.   Preferred flea aminopeptidase proteins of the present invention are stringent. Under hybridization conditions, the following nucleic acid molecules: nfAP and / or nfAP2 (the present invention) The entire flea aminopeptidase coding region of the light flea aminopeptidase gene. Long) is encoded by a nucleic acid molecule that hybridizes to at least one of ing. Particularly suitable aminopeptidase proteins are stringent high Under hybridization conditions, the following nucleic acid molecule nfAP₄₅₃, NfAP₉₀₀, NfAP₇₃₂, NfA P₁₅₈₀, NfAP2₃₈₃And / or nfAP2₅₃₇Hybridizes with at least one of Encoded by the nucleic acid molecule. Stringent hybridization Under the following conditions, the following nucleic acid molecule: nfAP2₃₈₃And / or nfAP2₅₃₇At least of Aminopeptider encoded by a nucleic acid molecule that hybridizes to one Ze proteins are more preferred. Further preferred are SEQ ID NO: 107, SEQ ID NO: No. 111 and / or aminopeptidase protein comprising the amino acid sequence of SEQ ID NO: 113 Quality or allelic variants of a nucleic acid molecule comprising SEQ ID NO: 110 and / or SEQ ID NO: 112 Aminopeptidase protein encoded by As described herein An aminopeptidase protein having an amino acid sequence of at least about 50 %, Preferably at least about 75%, more preferably at least about 90% identity. Flea aminopeptidase proteins containing a region having the same are also preferred.   Preferred flea cysteine protease proteins of the present invention are stringent Hybridizes with nucleic acid molecule nfCP1 under unique hybridization conditions Encoded by a nucleic acid molecule (nfCP1₅₇₃Or nfCP1₁₁₀₉Flea cysteine containing The full length of the coding region of the protease, and its preparation is described in the Examples). Further Are preferably SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 A cysteine protease comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 95, or Are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88, A nucleus containing SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76, or SEQ ID NO: 94 Cysteine protease encoded by an allelic variant of the acid molecule . SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 77, SEQ ID NO: 95 and at least about 50%, preferably at least about 75%, more preferably A flea cysteine protease comprising a region having at least about 90% identity Protein is also preferred.   One embodiment of the present invention relates to a serine protease inhibitor, aminopeptidase Substantially inhibited by inhibitors and / or cysteine protease inhibitors An isolated protein having proteolytic activity. Such an inhibition It can be measured by techniques known to those skilled in the art. For example, in the case of serine protease, Qualitatively inhibited is defined as a protease protein by a serine protease inhibitor. It means that at least half of the proteolytic activity of the protein is inhibited. Good Preferably at least about 70% of the protease protein, more preferably about 90% Proteolytic activity of a protease protein is a serine protease inhibitor Inhibited by Suitable serine protease inhibitors include flea serpin (Serpin) proteins and peptides or analogs thereof.   The isolated proteins of the present invention allow for the recovery of such proteins from flea midgut. Harvesting and recombinantly producing such a protein. It can be produced in various ways. In one embodiment, the flea midgut protease Recovered by the method for obtaining a soluble flea midgut preparation disclosed previously can do. Flea midgut protease protein Chromatography, ion exchange chromatography, filtration, electrophoresis (e.g., standard Semi-electrophoresis, capillary electrophoresis, flow-through electrophoresis ), Hydrophobic interaction chromatography, gel filtration chromatography, reverse-phase chromatography Chromatography, Concanavalin A chromatography, Chromatofocus Known to those skilled in the art, including differential and differential solubilization Can be further purified from destroyed flea midgut However, it is not limited to these methods. In one implementation, the flea midgut protease Is purified using protease inhibitor affinity chromatography. An example is disclosed in the embodiment section.   Another embodiment of the present invention relates to an isolated protein of the present invention using recombinant DNA technology. This is a method for producing a carbonaceous material. Such a method comprises the steps of (a) encoding the protein of the invention. Culturing a recombinant cell having a nucleic acid molecule to be produced to produce the protein And (b) recovering the protein therefrom. Recombinant Details of the production of the vesicles and their culture are provided below. "Protein The term "recover" simply refers to collecting the entire fermentation medium containing the protein. Does not require additional separation or purification steps. No. The protein of the present invention comprises various standard proteins disclosed so far. It can be purified using quality purification techniques.   The isolated proteins of the present invention are preferably recovered in "substantially pure" form. Good. "Substantially pure," as used herein, refers to a protein that is compatible with a vaccine. Means the purity that can be used effectively. For example, vaccines for animals Must not show substantial toxicity and will not produce antibodies in the vaccinated animals. It must be inspiring.   Another embodiment of the present invention provides for the presence of flea midgut under stringent conditions. Isolated nucleus capable of hybridizing to a gene encoding a flea protease It is an acid molecule. Herein, such nucleic acid molecules are referred to as flea protease nucleic acid components. Sometimes referred to as child. Under stringent conditions, flea serine proteases Gene, flea aminopeptidase gene, or flea cysteine protein An isolated nucleic acid molecule that hybridizes to a protease gene is particularly preferred. this Such gene properties are disclosed herein. In the present invention, isolated The isolated nucleic acid molecule is removed from its natural environment (ie, C) a nucleic acid molecule. Thus, "isolated" means that the more purified the nucleic acid molecule is, Does not reflect the degree. An isolated nucleic acid molecule can be DNA, RNA, or DNA or RNA. It can be any derivative.   As mentioned above, the flea protease gene is encoded by the gene. Regulatory regions that regulate the production of flea protease proteins Or post-translational regulatory regions, etc.) and the coding region itself. Contains all nucleic acid sequences related to the native flea protease gene, such as the body You. The nucleic acid molecule of the present invention may be an isolated natural flea protease nucleic acid molecule or the same. May be homologs of The nucleic acid molecule of the present invention may comprise one or more regulatory regions and the entire coding region. Long or partial, or combinations thereof, may be included. Flea protea of the present invention The minimum size of a nucleic acid molecule is determined by stringent hybridization conditions. Below, the minimum that can form a stable hybrid with the corresponding natural gene Size. Flea protease nucleic acid molecules include hybrid proteins, Nucleic acids encoding fusion proteins, multivalent proteins, or truncated fragments Children may also be included.   The isolated nucleic acid molecules of the present invention can be obtained from natural sources by the entire gene (ie, Or a gene that can form a stable hybrid with the entire gene). Can be obtained as part of As used herein, "at least a part of" The term refers to the quantity of an object sufficient to have at least the functional aspects of the object. . For example, as used herein, at least a portion of a nucleic acid sequence refers to a string Stable hybrid with the corresponding gene under eventual hybridization conditions To The amount of nucleic acid sequence that can be formed.   The isolated nucleic acid molecules of the invention can be produced using recombinant DNA technology (eg, polymerase chain Reaction (PCR) amplification, cloning) or chemical synthesis. . Isolated flea protease nucleic acid molecules include native nucleic acid molecules, as well as native Allelic variants and nucleic acid molecules encode the flea protease protein of the invention. Ability to bind to natural nucleic acid molecule isolates under stringent conditions The ability of the nucleotide to be substantially unaffected by its ability to form hybrids A modified nucleic acid molecule that has been modified, such as by insertion, deletion, substitution, and / or inversion. But not limited thereto.   Flea protease nucleic acid molecule homologs can be made using a number of methods known to those of skill in the art. (Eg, Sambrook et al., Ibid.). For example, a nucleic acid molecule Specific mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzymes Cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification, And / or introducing mutations into selected regions of the nucleic acid sequence, Synthesis of oligonucleotide mixtures to "build" and ligation of mixture populations , And classical mutagenesis techniques such as combinations thereof and recombinant DNA Can be modified using various techniques, including, but not limited to, techniques . Nucleic acid molecule homologs screen the function of the protein encoded by the nucleic acid. (E.g., if the homolog is immune to the flea protease) And / or ability to have proteolytic activity), and / or Hybridization of isolated fleas with protease nucleic acids under lingent conditions By redidation, one can select from a mixture of modified nucleic acids .   The isolated flea protease nucleic acid molecule of the present invention includes at least the present invention. Can contain a nucleic acid sequence encoding a single flea protease protein Examples of such proteins are disclosed herein. "Nucleic acid molecule" The term mainly means a nucleic acid molecule as a substance, and the term "nucleic acid sequence" Means a nucleotide sequence on a nucleic acid molecule, but the two terms are used interchangeably A nucleic acid molecule that can encode, in particular, a flea protease protein, or Nucleic acid sequences can be used interchangeably.   One aspect of the present invention is a flea protease nucleic acid molecule of the present invention, comprising a flea protease A nucleic acid strand encoding at least a portion of the enzyme or a homolog thereof, or Under stringent conditions to the complementary sequence of such a nucleic acid strand Is a flea protease nucleic acid molecule of the present invention. Any nucleic acid of the invention A sequence having complementarity to a sequence corresponds to the nucleic acid strand whose sequence is listed herein. (Ie, they form a complete double helix) Form). The double-stranded nucleic acid molecule of the present invention is such that one of the strands is already a nucleic acid. The sequence has been determined and given the sequence number. It should be noted that two of the present invention The chain sequence molecule must also be composed of a sequence complementary to the sequence with the SEQ ID NO. And The nucleic acid molecule of the present invention may itself be double-stranded or single-stranded. And / or any nucleic acid having the given SEQ ID NO: The complementary sequence of the nucleic acid having a number (regardless of whether it is described in this specification) and And nucleic acid molecules having sequences that form defined hybrids. Guide complementary sequences The method of dispensing is known to those skilled in the art. Preferably, low levels of flea protease proteins At least a region (or a plurality of regions) corresponding to the nucleic acid sequence encoding a part Both about 65 percent, preferably at least about 75 percent, more preferably Are at least 85% and even more preferably at least about 95% homologous Flea protease nucleic acid molecule comprising a nucleic acid sequence. Particularly preferably, the flea midgut Flea naturally present in the flea, and preferably contained in the soluble flea midgut preparation of the present invention. A flea protease nucleic acid molecule encoding at least a portion of a protease . Examples of nucleic acid molecules of the invention are disclosed in the Examples section.   Preferred nomiserin protease nucleic acid molecules of the invention are stringent proteins. Hybridizing with at least one of the following nucleic acid molecules under hybridization conditions: Nucleic acid molecules to be raised: nfSP3, nfSP8, nfSP9, nfSP10 , NfSP11, nfSP19, nfSP20, ntSP21, nfSP23, nfSP25, ntSP26, nfSP27, nfSP29, nfSP30, n fSP31, nfSP34, nfSP36, nfSP37, nfSP38, nf SP39, nfSP18, nfSP24, nfSP28, nfSP32, nfS P33 and / or nfSP40. More preferably, stringent high B Hybridizes with at least one of the following nucleic acid molecules under re-dialysis conditions: Nucleic acid molecule, as well as other specific nucleic acid molecules disclosed in the Examples section: P18₅₃₄, NfSP18₇₇₅, NfSP18₂₂₅, NfSP24₄₁₀, NfSP2 4₁₀₈₉, NfSP24₇₇₄, NfSP24₇₁₁, NfSP28₇₁₁, NfSP28_{9 twenty three} , NfSP32₉₃₃, NfSP32₉₂₄, NfSP32₆₉₉, NfSP33₄₂₆, nfSP33₇₇₈, NfSP33₁₈₉₄, NfSP33₁₂₀₀, NfSP33₇₂₆, N fSP40₈₄₄, NfSP5₈₀₆, NfSP11₃₀₇, NfSP8₅₄₅, NfSP8₄₃₆ , NfSP12₇₅₈, NfSP26₆₁₀, NfSP27₃₈₆, NfSP23₄₂₃ , NfSP34₃₉₀, NfSP36₁₉₇, NfSP38₃₄₁, NfSP37₂₆₁, N fSP39₂₆₇, NfSP29₆₁₂, NfSP30₆₄₁, NfSP31₆₂₆, NfS P32₄₃₃, NfSP15₈₁₅, NfSP19₈₅₅, NfSP25₈₆₄, NfSP2 1₅₉₅And / or nfSP40₇₁₇. Still more preferably, nfSP3, n fSP8, nfSP9, nfSP10, nfSP11, nfSP19, nfSP 20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP2 7, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36 , NfSP37, nfSP38, nfSP39, nfSP18, nfSP24, nfSP28, nfSP32, nfSP33 and / or nfSP40, furthermore Preferably nfSP18₅₃₄, NfSP18₇₇₅, NfSP18₂₂₅, NfSP2 4₄₁₀, NfSP24₁₀₈₉, NfSP24₇₇₄, NfSP24₇₁₁, NfSP28_{9 twenty three} , NfSP32₉₃₃, NfSP32₉₃₃, NfSP32₉₂₄, NfSP32₆₉₉, nfSP33₄₂₆, NfSP33₇₇₈, NfSP33₁₈₉₄, NfSP33₁₂₀₀, N fSP33₇₂₆, NfSP40₈₄₁, NfSP5₈₀₆, NfSP11₃₀₇, NfSP 8₅₁₅, NfSP8₄₃₀, NfSP12₇₅₈, NfSP26₆₁₀, NfSP27₃₈₆ , NfSP23₄₂₃, NfSP34₃₉₀, NfSP36₁₉₇, NfSP38₃₄₁, N fSP37₂₆₁, NfSP39₂₆₇, NfSP29₆₁₂, NfSP30₆₄₁, NfS P31₆₂₆, NfSP32₄₃₃, NfSP15₈₁₅, NfSP19₈₅₅, NfSP2 5₈₆₄, NfSP21₅₉₅And / or nfSP40₇₁₇Nucleic acid molecules containing Other specific nucleic acid molecules disclosed in the Examples section.   Particularly preferred nomiserin protease nucleic acid molecules are at least one of the following sequences: And other particular nucleic acid molecules disclosed in the Examples section: No. 9, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 2 5, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32 , SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 120, SEQ ID NO: 130, SEQ ID NO: 15 4, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, Sequence No. 131, SEQ ID No. 155, SEQ ID No. 114, SEQ ID No. 125, SEQ ID No. 11 8, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, Sequence No. 136, SEQ ID No. 78, SEQ ID No. 158, SEQ ID No. 132, SEQ ID No. 134 , SEQ ID NO: 66, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 142, SEQ ID NO: 138, SEQ ID NO: 144, Sequence No. 140, SEQ ID No. 122, SEQ ID No. 84, and / or SEQ ID No. 45, And their complementary sequences. Also preferably, allelic variations of the nucleic acid molecules described above. Body.   Preferred flea aminopeptidase nucleic acid molecules of the present invention are stringent Under hybridization conditions, nfAP and / or nfAP2 A nucleic acid molecule that hybridizes. More preferred flea aminopeptidase of the present invention Nucleic acid molecules are expressed in nfAP under stringent hybridization conditions.₄₅₃ , NfAP₉₀₀, NfAP₇₃₂, NfAP₁₅₈₀, NfAP2₃₆₃And / or Is nfAP2₅₃₇Is a nucleic acid molecule that hybridizes with More preferably, nf AP₄₅₃, NfAP₉₀₀, NfAP₇₃₂, NfAP₁₅₈₀, NfAP2₃₆₃,and/ Or nfAP2₅₃₇An aminopeptidase nucleic acid molecule comprising: Particularly preferred Is the nucleic acid sequence of SEQ ID NO: 110 and / or SEQ ID NO: 112, any of the aforementioned sequences A nucleic acid molecule comprising the complementary sequence thereof, or an allelic variant thereof.   A preferred flea cysteine protease nucleic acid molecule of the invention is a stringent NfCP1 under various hybridization conditions₅₇₃Or nfCP1₁₁₀₉ (The production of which is described in the Examples). More preferably, nfCP1₅₇₃Or nfCP1₁₁₀₉Cysteine protein containing Aase nucleic acid molecule. Particularly preferably, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, Sequence SEQ ID NO: 93 and / or SEQ ID NO: 94, or such a nucleus Nucleic acid molecules that include allelic variants of an acid molecule.   Knowledge of the nucleic acid molecule of the flea protease protein of the present invention will Make a copy of the nucleic acid molecule, as well as a flea protease protein A nucleic acid molecule that includes a portion added to the target gene (eg, a translation initiation site, and / or Or a nucleic acid molecule comprising a transcription regulatory region and / or a translation regulatory region). And / or homologs of the flea protease nucleic acid molecule can be obtained. Book Knowing the amino acid sequence of a portion of the flea protease protein of the invention Those skilled in the art will be able to clone nucleic acid sequences encoding such flea protease proteins. Loaning becomes possible. In addition, the desired flea protease nucleic acid molecule Can be obtained using various means such as the following: Screen the appropriate expression library using an antibody that binds to the ase protein Means: Conventional Clonin Using Oligonucleotide Probes of the Invention Means for screening an appropriate library or DNA using a screening technique; and A suitable library using the oligonucleotide primers of the invention and Means for PCR amplification of RNA or DNA (genomic and / or cDNA libraries) Library available). Preferred for isolating flea protease nucleic acid molecules CDNA libraries include fasted flea individuals, fed flea individuals, fed flea midgut, fasted Includes cDNA libraries created from flea midgut and flea salivary glands. gene Techniques for cloning and amplifying are described, for example, in Sambrook et al. Brook et al., ibid.). The flea protection of the present invention is described in the Examples section. An example of the isolation of a cDNA sequence encoding an ase protein is included.   The present invention also provides a book encoding at least a portion of a flea protease protein. A complementary region of another nucleic acid molecule of the invention (preferably longer) with a stringent Nucleic acids that are oligonucleotides capable of hybridizing under mild conditions Including molecules. The oligonucleotide of the present invention may be RNA, DNA or any of them. It is possible to be a mutant of The minimum size of the aforementioned oligonucleotide is Between the oligonucleotide in question and the complementary sequence of another nucleic acid molecule of the invention And the size required to form a stable hybrid. Minimum size feature The features are disclosed herein. In addition, the size of the oligonucleotide is determined according to the present invention. Must be sufficient to use the oligonucleotide. By this Without limitation, the oligonucleotides of the present invention can be combined with additional nucleic acid molecules. For amplification or extension of nucleic acid molecules. For various applications, including use as primers for flea protea It can also be used in therapeutic applications to inhibit protease production. That's it Therapeutic applications such as the above described oligonucleotides, for example, antisense technology , Triple strand formation technology, ribozyme technology And / or used in technologies based on RNA drugs (RNA drugs). Including The present invention consequently provides for flea use through one or more of such techniques. The aforementioned oligonucleotide for inhibiting the production of a protease protein; and Including methods.   The present invention also includes a recombinant vector, wherein the host cell contains the recombinant vector of the present invention. Of any vector that enables the delivery of a miprotease nucleic acid molecule. It contains a recombinant vector into which the nucleic acid molecule is inserted and contained. The aforementioned The vector contains a heterologous nucleic acid sequence (a nucleic acid sequence not found in nature) Contain adjacent to the Rotase nucleic acid molecule. The vector can be either RNA or DNA And can be either prokaryotic or eukaryotic, and typically Are viruses or plasmids. The recombinant vector is the flea protein of the present invention. Cloning, sequencing, and / or alternative methods of It can be used in operations. Recombination shown herein as a recombinant molecule One type of vector (described in detail below) may be used to express the nucleic acid molecule of the present invention. Can be. Preferred recombinant vectors are capable of replicating in transformed cells. I do. Preferred nucleic acid molecules for inclusion in a recombinant vector of the invention are disclosed herein.   One aspect of the invention disclosed so far is to produce the flea protease protein of the invention. A method of expressing the protein under conditions effective for producing the protein. And culturing cells capable of recovering the protein, This is a method for producing protein. Preferred cells for culture are flea protease proteins A recombinant cell capable of expressing a host cell, wherein the recombinant cell is a host cell of the present invention. It is formed by transformation with one or more of the acid molecules. Transform nucleic acid molecules into cells Converting is accomplished by any method capable of inserting a nucleic acid molecule into the cell. Can be achieved. Although not limited by this, transformation techniques , Transfection, electroporation, microinjection, lipofection, adsorption and Includes protoplast fusion. The recombinant cells may be maintained as single cells, May grow into a tissue, organ or multicellular organ. Transformed according to the present invention Nucleic acids are retained outside the chromosome so that their ability to be expressed is maintained Or in one or more sites within the chromosome of the transformed (recombinant) cell. It is also possible to be included. Preferred nucleic acid molecules for transforming host cells Is disclosed herein.   A host cell suitable for transformation is capable of transformation and is And any cell capable of expressing the selected flea protease protein. The result As a result, after transformation with at least one nucleic acid of the invention, the aforementioned cells Enables production of the flea protease protein of the present invention. Host cells are non-shaped Transformed cells or cells already transformed with at least one nucleic acid molecule. It may be a vesicle. Suitable host cells of the present invention include bacterial cells, fungal cells (such as yeast). ), Insect cells, animal cells and plant cells. preferable Host cells include bacterial cells, yeast cells, insect cells and mammalian cells, Cells (eg, E. coli) and insect cells (eg, Spodoptera; Spodop) tera) is particularly preferred.   Recombinant cells are operably linked to expression vectors, each containing one or more transcription regulatory sequences. Using one or more recombinant molecules comprising one or more nucleic acid molecules of the invention attached, It is preferably produced by transforming a host cell. Operatively The term is used so that the nucleic acid molecule can be expressed when transformed into a host cell. Indicates insertion of the molecule into an expression vector. The expression vector used here is Can transform host cells and result in the expression of specific nucleic acid molecules. Functional DNA or RNA vectors. Preferably, the expression vector is The vector also has the ability to replicate in the host cell. Expression vectors are prokaryotic cells Or eukaryotic cells, typically a virus or plasmid It is. The expression vector of the present invention is a recombinant cell of the present invention (bacterial cell, fungal cell, Function (ie, genetic, including insect, animal, and / or plant cells) Control of offspring expression). The aforementioned nucleic acid molecule of the present invention Motor, operator, repressor, enhancer, termination arrangement , The origin of replication, and other regulatory sequences (consistent with recombinant cells and Operably linked to an expression vector that contains a regulatory sequence (such as a nucleic acid molecule). It is possible to combine. The transcription regulatory sequence used here is used to initiate transcription. And sequences capable of controlling the extension, termination and termination. Particularly important transcription control sequences include promoter sequences, enhancer sequences, and operators. Sequences that control the initiation of transcription, such as transcription and repressor sequences It is. Appropriate transcriptional regulatory sequences are functional in at least one of the recombinant cells of the present invention. It includes any transcription regulatory sequence capable of functioning. The types of transcription regulatory sequences mentioned above are It is known to those skilled in the art. This is not a limitation, but a preferred transcription regulatory arrangement. Rows function in bacterial, yeast, helminth, insect, and mammalian cells , Tac, lac, trp, trc, oxy-pro, omp / lpp, rrn B, bacteriophage lambda (λ) (eg, λp_LAnd λp_RAnd the aforementioned Fusion containing promoter), bacteriophage T7, T7lac, bacteria Ophage T3, bacteriophage SP6, bacteriophage SP01, Tarothionein, alpha fusion factor, PiCia alcoholoxy Enzyme, alphavirus subgenomic promoter (e.g., Sindbis Sirbis subgenome promoter; Sindbis virus subgen omic promoters), baculovirus, heliotis, zea, insects Virus (Heliothis Zea Insect Virus), Vaccini A virus, herpes virus, variola virus, adenovirus, Simian wi Lus40, retrovirus actin, retrovirus long terminal repeat To Rous sarcoma virus, heat shock, phosphate and nitrate transcription regulatory sequences And other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells, etc. Including the sequence of In addition, appropriate transcription control sequences may include tissue-specific promoters and Enhancers, as well as lymphokine-inducible promoters (eg, interferon Or a promoter induced by an interleukin). In addition, the present invention The transcriptional regulatory sequence is naturally associated with the DNA sequence encoding the flea protease protein. It can also include naturally occurring transcriptional regulatory sequences in association.   In addition, the expression vector of the present invention contains the expressed flea protease Secretion signal that allows secretion from the vesicle (producing the protein) (ie, , Signal segment nucleic acid sequence). The appropriate signal segment is , Flea protease protein signal segment, or flea protea of the present invention Any heterologous signal that can regulate secretion of zeoproteins (including fusion proteins) Includes segments. This is not a limitation, but a preferred signal segment Mentions are flea protease, tissue plasminogen activator (t-PA) , Interferon, interleukin, growth hormone, histocompatibility and virus Contains the envelope glycoprotein signal segment.   In addition, the expression vector of the present invention provides a fusion vector that directs the expression of the inserted nucleic acid molecule of the present invention. The fusion sequence may be included as a fusion protein. Flea protease nucleic acid component of the present invention By including a fusion sequence as part of the nucleic acid, the protein encoded by the nucleic acid molecule Increased stability in production, storage and / or use of park. In addition, the fusion Can function as a means to isolate and purify the flea protease protein. (E.g., fusion tags obtained using affinity chromatography). Protein purification). The appropriate fusion segment can be used to provide the desired function (eg, , A function of improving stability and / or a function as a purification means) Domain. Within the scope of the present invention, one or more Can be used. The fusion segment is a flea protection Can bind to the amino terminus and / or carboxy terminus of ase protein Noh. The bond between the fusion segment and the flea protease protein is Proteases can be constructed to be cleavable for direct recovery. Fusion The fusion protein consists of a fusion segment (the flea protease protein has a carboxy terminal and And / or attached to either the amino terminus) Culturing recombinant cells transformed with the fusion nucleic acid sequence It is.   The recombinant molecule of the present invention can be used to transform the nucleic acid molecule (s) in the cell to be transformed. At least one of any transcription regulatory sequences capable of effectively controlling the expression of At least one of any of the nucleic acid molecules described above operably linked to It is a molecule that can be seen. Preferred recombinant molecules are one or more nucleic acid molecules of the invention. And more preferably a nucleic acid component encoding one or more flea protease proteins. And, in particular, one or more nomicerin proteases, aminopeptidases and / or Or a sequence encoding a cysteine protease protein. Similarly, preferred The recombinant cells contain one or more nucleic acid molecules of the invention, more preferably one or more fleas. A nucleic acid molecule encoding a protease protein, in particular, one or more nomicerin proteins Thease, aminopeptidase and / or cysteine protease protein Contains an array that encodes   The expression of transformed nucleic acid molecules can be modified by using recombinant DNA technology. (Eg, the number of copies of the nucleic acid molecule in the host cell, their nuclei) The efficiency with which the acid molecule is transcribed, the efficiency with which the resulting transcript is translated, and Those skilled in the art will appreciate the efficiency of post-translational modifications. By this Without limitation, recombinant techniques effective to increase the expression of the nucleic acid molecules of the invention Surgery involves operably linking a nucleic acid molecule to a high copy number plasmid, Integrate into one or more host cell chromosomes, add vector stable sequences to the plasmid And transcriptional control signals (eg, promoters, operators, enhancers -), Translation control signals (eg, ribosome binding site) Position, Shine-Dalgarno sequence) of the invention Modifying the nucleic acid molecule to correspond to the host cell codon usage; Deleting sequences that destabilize and growing recombinant cells during fermentation This involves using regulatory signals that are temporally separated from recombinant protein production. Book The activity of the expressed recombinant protein of the invention may be determined by fragmentation, modification, etc. of the resulting protein. Or by induction.   According to the present invention, the recombinant cells are transformed into the production of the flea protease protein of the present invention (see above). Are cultured under conditions that effectively produce the flea protease protein of the present invention. And recovering the protein). Although not limited by this, effective conditions for protein production The appropriate media, bioreactor, temperature, pH and And oxygen conditions. An appropriate or effective medium is one that Represents any medium in which the cells of the present invention can produce a flea protease protein. The aforementioned media typically contain assimilable carbohydrate, nitrogen and phosphate sources, Contains other nutrients such as salts, minerals, metals and vitamins suitable for Aqueous medium. The medium may contain complex nutrients or may be limited A minimal medium may be used.   The cells of the invention can be cultured in conventional fermentation bioreactors. , But without limitation, batches, feeding batches, cell recycling, And a continuous fermenter. Culture is performed in shake flasks, test tubes, and microphones. It is also possible to process in rotiter dishes and petri plates. The culturing is performed at a temperature, pH and oxygen content appropriate for the recombinant cells. The aforementioned Culture conditions are sufficient within the scope of one prior art in the art. You.   Depending on the vector and the host system used for production, the resulting fleas The protease protein remains retained in the recombinant cells; Secreted into the ground; secreted between two cell membranes, such as the periplasmic space of E. coli. Or may be retained on the outer surface of the cell or viral membrane. The aforementioned Methods for purifying proteins have been disclosed previously.   The present invention also provides isolated anti-flea protease antibodies, and Including use to reduce fleas in animals and their surroundings . Anti-flea protease antibodies are available in flea midgut (female and male (Including male and male fasted fleas midgut) Antibodies that can be combined. The anti-flea protease antibody is preferably Binds to thease and reduces the proteolytic activity of the protease.   Isolated antibodies are antibodies that have been removed from their original environment. "single The term "isolated" does not denote the aforementioned antibody purification step. As such, isolated Antibodies containing the aforementioned antibodies or antibodies purified in diversified stages Can be included. As used herein, the term “selectively binds” refers to the aforementioned antibody , The ability to preferentially bind to the protease (ie, an unrelated compound in the mixture) That the protease can be identified from this). The binding affinity is typically about 1 0^ThreeM^-1From about 10¹²M^-1Range. Binding was determined by immunoblot assay, immunoprecipitation Down assay, radioimmunoassay, enzyme immunoassay (eg, ELISA), immunofluorescence Various methods known to those skilled in the art, including antibody assays and immunoelectron microscopy, may be used. (For example, see Sambrook et al. (Ibid.)). I want to be illuminated).   The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Is also good. Antibodies of the present invention include antibody fragments, including signal chain antibodies, and genetically engineered Includes those that are functionally equivalent to antibodies, etc. Is capable of selectively binding to at least one of the be able to. Also, the antibodies of the present invention may bind to one or more epitopes. Functional chimeric antibodies. Preferred antibodies are flea protease nucleic acid molecules of the invention ( At least a portion thereof).   The anti-flea antibody of the present invention is an animal to which the flea protease vaccine of the present invention is administered. In which the antibody is increased. The effect of those increased antibodies is that fleas Triggered when fed from the blood of an injected animal (containing the aforementioned antibodies) You. Further, the anti-flea antibody of the present invention comprises one or more flea protease proteins of the present invention. , Or antibodies raised in animals against soluble flea midgut preparations (subsequently Recovered from the animal using known techniques). Furthermore, the antibody of the present invention Describes the technology disclosed so far for obtaining the flea protease protein of the present invention. It is created by the recombination used. Antibodies raised against restricted proteins are: Conveniently, the aforementioned antibodies are not substantially contaminated with antibodies against other substances. The contamination may otherwise interfere with the diagnostic assay, or in the therapeutic composition. It may cause side effects when used for   The anti-flea protease antibody of the present invention has various uses within the scope of the present invention. is there. For example, such antibodies can be used to protect animals from fleas breeding. Can be used in the compositions of the present invention to confer passive immunity to the same. Also, anti-no My antibodies can be used to screen expression libraries and / or Recover the desired protein from a mixture of protein and other impurities Can be used as a tool for Moreover, the antibodies of the present invention kill fleas. Thus, cytotoxic agents can be used to target fleas. Target This means that such antibodies can be conjugated to cytotoxic agents using techniques known to those skilled in the art. (Ie, stably add).   Preferred anti-flea protease antibodies of the present invention are Metalloprotease, flea aspartic protease and / or nomisis Selectively binds to tein protease and preferentially reduces proteolytic activity Can be More preferred anti-flea protease antibodies are anti-flea serine protein antibodies. Ase antibody, anti-flea metalloprotease antibody, anti-flea aminopeptidase antibody, And an anti-flea cysteine protease antibody. Particularly preferred are anti-noise Phosphoprotease antibodies, anti-flea aminopeptidase antibodies, and anti-flea cysteine A nomiserin protease protein of the present invention; For aminopeptidase protein or cysteine protease protein Includes antibodies prepared by   The present invention is directed to reducing flea reproduction in animals and the surrounding environment, Further use of protease inhibitors to reduce the proteolytic activity of Included. The protease inhibitors used here are those that directly interact with proteases This usually results in binding to the active site of the protease or other A compound that acts to inhibit its protease activity. Protea Inhibitors are usually relatively small compounds that interact with the active site of the protease. Different from interacting anti-protease antibodies.   Protease inhibitors treat animals as long as these compounds do not harm the treated animals. Can be used directly as compounds in the compositions of the present invention. Protease Inhibitors can be used to target preferred types of flea proteins using the compositions of the invention. It can also be used to identify ase. For example, the present inventors The priority of serine proteases in the gut, especially in soluble flea midgut preparations, Shown here using a protease inhibitor. Such perceptions are the breeding of fleas on animals Effective reduction of serine protease vaccine, anti-nomicerin protease antibody And other inhibition of serine protease synthesis and activity that can confer resistance to animals Suggest that this can be achieved using agents. For example, the flea disclosed here Immunoglobulin proteinase activity is a target for reducing flea reproduction can do. Other proteases are further present in the flea midgut according to the invention This also suggests targeting such proteases. Protea Methods of using a protease inhibitor are known to those of skill in the art; examples of such methods are provided below. It is disclosed here.   In one embodiment, a protea that can be used to treat an animal with the composition of the present invention. The protease inhibitor is identified by a method comprising the following steps: (a)-or more (I) inhibits flea protease activity in vitro And / or (ii) in a flea feeding assay, eg, Wade Et al., 1988, J. Med Entomol. 25, feeding system as described in 186-190 Flea survival and / or by adding inhibitors to the blood meal of growing fleas Testing for the ability to reduce reproductive quality can result in candidate (ie, putative, possible) inhibition. Identifying the harmful compound; and (b) in the animal infested with the flea, the candidate Testing the efficacy of the inhibitor compound. Which candidate compounds should be administered to animals To determine the in vitro and flea feeding assay data Neither is required, but data from either assay is not necessarily Evaluation of both sets of data is preferred because the resulting data is not expected. For example Candidate Compounds Identified Using In Vitro Assays Function “In Vitro” In vivo, interference in the blood diet inhibits the activity of such compounds It may not work for several reasons, including the presence of components. For example, Protinin can inhibit at least some nomicelin proteases in vitro However, aprotinin is not sufficient in the presence of serum proteins, such as those found in blood. Does not work in minutes. In addition, candidate inhibitor compounds identified by the flea feeding assay The substance is not only the desired compound, but also general (eg, it affects the flea mitochondria) Compounds that reduce the viability and / or fertility of fleas due to May also be included.   In a preferred embodiment, the inhibitor of the flea protease of the invention is:       (A) SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126 , SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, Sequence No. 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 1 43, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, Sequence No. 163, SEQ ID No. 162, SEQ ID No. 69, SEQ ID No. 85, SEQ ID No. 107, SEQ ID No. 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 89 An isolated flea comprising an amino acid sequence such as SEQ ID NO: 92, and / or SEQ ID NO: 95 Protease proteins can be converted to protease proteins in the absence of inhibitory compounds. Contacting the putative inhibitory compound under conditions where has proteolytic activity;       (B) determining whether the putative inhibitory compound inhibits its activity. Identified by the method provided.   To perform this method, a test kit can be used. Preferred Test kits are SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: No. 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: No. 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: No. 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, Sequence No. 126, SEQ ID No. 119, SEQ ID No. 129, SEQ ID No. 153, SEQ ID No. 157, SEQ ID No. 161 , SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: No. 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, Sequence No. 85, SEQ ID No. 107, SEQ ID No. 111, SEQ ID No. 113, SEQ ID No. 77, SEQ ID No. 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, and / or SEQ ID NO: 95, etc. An isolated flea protease protein comprising the amino acid sequence of Means for measuring the degree of inhibition of its activity in the presence of a substance.   In another embodiment, the protease inhibitor is, for example, affinity chromatography. Used in the purification of the corresponding protease by Protease inhibitor under the conditions in which the inhibitor forms a complex with the protease, Incubate with the mixture containing thease. Then, complex the protease Can be recovered from the body. The protease inhibitor can be attached to a solid support, And / or can be used for detection and / or can recover the complex For example, they can be labeled with radioactive, fluorescent or enzyme tags.   Suitable protease inhibitors for use in accordance with the present invention are serine protease inhibitors. Harmful agents (including immunoglobulin proteinase inhibitors and serpins), metalloproteins Thease inhibitor, aspartic protease inhibitor, cysteine protease Inhibitors and aminopeptidase inhibitors. Preferred protease inhibitors are , Serine protease inhibitor, metalloprotease inhibitor, aminopeptidase Inhibitors and cysteine protease inhibitors, especially those that are a wide range of inhibitors including. More preferred are a wide range of serine protease inhibitors.   As is known to those skilled in the art, there is a wide variety of protease inhibitors. As an example AEBSF, aprotinin, bestatin, chloromethylketone TLCK ( Nα-p-tosyl-L-lysine chloromethyl ketone) and TPCK (N-to Sil-L-phenylalanine chloromethyl ketone), chymostatin, cystati 3,4-dichloroisocoumarin, E-64 (trans-epoxysuccinyl) -L-leucylamide- (4-guanidino) butane), EDTA (ethylenediamine Tetraacetic acid), leupeptin, methyl ketone having various residues, oxidized L- Isinthiol, pepstatin, 1,10-orthophenanthroline, phosphora Midon, soybean trypsin / chymotrypsin inhibitor and soybean trypsin Obstruction Contains, but is not limited to, harmful agents. Preferred for use in the present invention New protease inhibitors include AEBSF, bestatin, E-64, leupeptin, Pepstatin, 1,10-orthophenanthroline, phosphoramidon, TLCK And TPCK, including AEBSF (a wide range of serine protease inhibitors), Statins (inhibitors of koisin aminopeptidase) and 1,10-orthofe Nantroline (a wide range of metalloprotease inhibitors) is particularly preferred.   Another preferred inhibitor of the invention is an inhibitor of an immunoglobulin proteinase of the invention. Contains harmful agents. Suitable inhibitors of immunoglobulin proteinase activity include immunoglobulin Interacts directly with the active site of the phosphoproteinase protein, Binds to the active site of an immunoglobulin proteinase or otherwise Interact with or otherwise modify the immunoglobulin protein It is a compound that inhibits the activity of tainase. Immunoglobulin proteinase inhibition The agent also interacts with other regions of the immunoglobulin proteinase protein. For example, by the allosteric interaction, the immunoglobulin proteinase activity May be inhibited. Inhibitors of immunoglobulin proteinase Relatively small compounds, such as anti-immunoglobulin protein It is different from zeta antibody. Preferably, the immunoglobulin proteinase inhibitor of the present invention Binds to a flea immunoglobulin proteinase protein or otherwise The ability to interact and thereby inhibit the activity of flea immunoglobulin proteinases Identified by force.   A preferred immunoglobulin proteinase inhibitor of the present invention is a flea immunoglobulin. Proteinase substrate analogs and the production of flea immunoglobulin proteinases In a way that inhibits the activity of the proteinase, the flea immunoglobulin proteinase Including but not limited to other molecules that bind (eg, to an allosteric site). No. An immunoglobulin proteinase substrate analog is an immunoglobulin proteinase. Interacts with (eg, binds, binds, or modifies) the active site of zeoprotein Refers to a compound. Preferred immunoglobulin proteinase substrate analogs are immunoglobulins. Inhibits the activity of brin proteinase. Similar to immunoglobulin proteinase substrate The body can be any inorganic or organic composition, such as pepti , Nucleic acids, and peptide analogs are possible, but not limited to. Exemption An immunoglobulin proteinase substrate analog is an activity of the proteinase protein. Natural substrates and structures of immunoglobulin proteinases so long as they can interact with the sex site Can be, but need not be. Immunoglobulin proteinase substrates An analog is a computer-generated version of the immunoglobulin proteinase protein of the present invention. Computational structure or computer structure of the natural substrate of an immunoglobulin proteinase Can be designed using Substrate analogs include oligonucleotides, peptides, peptide analogs Random sample of molecules, such as compounds or other inorganic or organic molecules And a corresponding binding partner (eg, a flea immunoglobulin proteinase) The sample is screened by affinity chromatography using Can also be obtained. Preferred immunoglobulin proteinase groups A quality analog is a peptide analog (ie, an immunoglobulin protein of the invention). In the region of natural substrates of inases, especially those that interact with the proteinase active site Structurally and / or functionally similar, but immunoglobulin proteinase activity Inhibits immunoglobulin proteinase activity by interacting with sex sites Compound).   Another preferred flea immunoglobulin proteinase inhibitor of the present invention is an immunoglobulin. Immunoglobulin should be treated in such a way as to inhibit the proteinase activity of Includes antibodies that specifically bind to burin proteinase. Yet another preferred fleas Immunoglobulin proteinase inhibitors are a class of serine proteinase inhibitors Inhibitors from Suitable immunoglobulin proteinase inhibitors are disclosed here. Contains the indicated serine proteinase inhibitors.   Protease inhibitors can be produced using methods known to those skilled in the art. . Protein- or peptide-based protease inhibitors such as cysteine Small peptides containing proteins or protease substrates can be produced recombinantly. It is also possible to modify as necessary.   The present invention relates to an additional protease inhibitor, preferably administered to an animal. To identify protease inhibitor compounds that can be included in the compositions of the present invention The use of the proteolytically active flea protease protein of the invention for included. Methods for identifying flea protease inhibitors include (a) isolated A flea protease protein, with a putative (ie, candidate) inhibitory compound, In the absence of compound, the protein is exposed to conditions that have proteolytic activity. Touching (eg, binding, mixing), and (b) inhibiting the putative To determine whether the compound inhibits the proteolytic activity of the protein. Process is included. Putative inhibitory compounds to be screened include organic molecules, antibodies ( And its functional equivalents) and the substrates themselves. Measure protease activity The methods for doing so are known to those skilled in the art as previously described. Identify inhibitors Particularly preferred for use are the nomiserin proteases of the present invention. Protein, flea aminopeptidase protein and flea cysteine protease It is a protein.   The present invention relates to an inhibitor isolated by such a method, and / or a test kit. Inhibits any flea protease sensitive to such inhibitors Also included are their uses.   The present invention provides the present invention, which can be used in accordance with the methods disclosed for the compounds of the present invention. It should be understood that the mimetope of the light compound is also included. Book In the specification, the mimetope of the proteinaceous compound of the present invention (for example, nomipro Protease, anti-flea protease antibody, protease activity or Often, the mimetope is a proteinase compound Because it has a mimicking structure, it mimics the activity of its proteinaceous compound Any compound. For example, the flea protease protein Has an activity similar to that of the isolated flea protease protein of the present invention. Compound. Mimetopes are modified to make them less susceptible to degradation Reduced peptides; anti-idiotypic and / or catalytic antibodies, or Fragments thereof; non-proteinase immunogenic proteins of the isolated proteins (eg, charcoal Hydrogenated structures); and synthetic or natural organic molecules, including nucleic acids, It is not limited. Such mimetopes are created by computers Designed using the structure of the protein of the present invention. Mimetope, Oligonu Create random samples of molecules such as nucleotides, peptides or other organic molecules Chromatographs using the corresponding binding partners It can also be obtained by screening such samples by fee.   The present invention provides a therapeutic composition comprising a compound of the present invention (ie, at least one). (Herein also referred to as a composition). Contained in the composition of the present invention Preferred compounds to be included include the flea protease vaccines disclosed herein, Flea protease antibodies and / or protease inhibitors are included. Such a cure Therapeutic compositions infest flea by reducing flea protease activity. To prevent flea load on the animal and in the animal's environment. Reduce.   Particularly preferred therapeutic compositions of the invention include at least one of the following compounds: Is: isolated nomicerin protease protein or its mimetope; strict Hybridization with the nomiserin protease gene An isolated nomicelin protease nucleic acid molecule soybean; a nomicelin protein Isolated antibody that selectively binds to zeoprotein; nomicelin protease activity An inhibitor of nomicerin protease activity, identified by its ability to inhibit Isolated flea aminopeptidase protein or its mimetope; Hybridizes with the flea aminopeptidase gene under hybridization conditions Isolated flea aminopeptidase nucleic acid molecule; An isolated antibody that selectively binds to protein; inhibits fleaaminopeptidase activity An inhibitor of flea aminopeptidase activity identified by its activity Flea cysteine protease protein or mimetope thereof; Under the conditions of the hybridization, An isolated flea cysteine protease nucleic acid molecule; An isolated antibody that selectively binds to an enzyme protein; Nomicysteine protein identified by its ability to inhibit the activity of thease Inhibitor of zeal activity.   Another aspect of the present invention relates to a first compound that reduces flea protease activity and a flea A second method that reduces the flea burden by a method other than reducing protease activity 2 is a therapeutic composition containing the two compounds. The present invention includes the use of such a composition. To protect the animal from flea infestation. Method is also included. The first compound of such a composition comprises By effectively reducing the Rotase activity, the activity of the second compound is increased. Reason Without being bound by theory, many anti-flea treatments, especially proteinase Are not very effective because they are broken down in the midgut. Departure Akira reduces proteolytic degradation of such treatments by flea midgut Thus, such an anti-flea treatment can be effectively used.   Preferred first compounds to be included in such compositions include flea protea Vaccine, anti-flea protease antibody and / or flea immunity disclosed in the present invention Includes protease inhibitors that target globulin proteinase activity You.   Preferred therapeutic compositions of the present invention comprise a vehicle comprising the following and mixtures thereof: Contains agent and protective compound: An amino acid sequence comprising the following amino acid sequence under stringent hybridization conditions: Hybridizes with a nucleic acid molecule having a nucleic acid sequence encoding a protein having Isolated protein encoded by a nucleic acid molecule or mimetope thereof: sequence No. 10, Sequence No. 13, Sequence No. 16, Sequence No. 19, Sequence No. 22, Sequence No. 24, Sequence No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 38, SEQ ID No. 41, Sequence No. 44, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 70, SEQ ID No. 71, Sequence No. 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: No. 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, Column No. 149, SEQ ID No. 151, SEQ ID No. 81, SEQ ID No. 83, SEQ ID No. 143, SEQ ID No. 139 , SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: No. 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and / or Is SEQ ID NO: 95; Has a nucleic acid sequence that contains the following nucleic acid sequence under stringent hybridization conditions An isolated nucleic acid molecule that hybridizes to a gene: SEQ ID NO: 9, SEQ ID NO: 11, Sequence No. 12, Sequence No. 14, Sequence No. 15, Sequence No. 17, Sequence No. 18, Sequence No. 20, Sequence No. 21, Sequence No. 23, Sequence No. 25, Sequence No. 26, Sequence No. 28, Sequence No. 29, Sequence Sequence No. 31, Sequence No. 32, Sequence No. 34, Sequence No. 35, Sequence No. 37, Sequence No. 39, Sequence Column number 40, sequence number 42, sequence number 43, sequence number 45, sequence number 120, sequence number 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, SEQ ID NO: No. 131, SEQ ID NO: 155, SEQ ID NO: 114, SEQ ID NO: 125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 66, SEQ ID NO: 146, SEQ ID NO: 148, Column No. 150, SEQ ID No. 80, SEQ ID No. 82, SEQ ID No. 142, SEQ ID No. 138, SEQ ID No. 144 , SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: No. 76, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88 , SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and / or SEQ ID NO: 94; An amino acid sequence comprising the following amino acid sequence under stringent hybridization conditions: Hybridizes with a nucleic acid molecule having a nucleic acid sequence encoding a protein having An isolated antibody that selectively binds to a protein encoded by a nucleic acid molecule: SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 11 9, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, Sequence No. 79, SEQ ID No. 159, SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 67, SEQ ID No. 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 1 39, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, Sequence No. 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and / Or SEQ ID NO: 95; An amino acid sequence comprising the following amino acid sequence under stringent hybridization conditions: Hybridizes with a nucleic acid molecule having a nucleic acid sequence encoding a protein having Identified by its activity that inhibits the activity of the protein encoded by the nucleic acid molecule Inhibitor of protease activity: SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, sequence No. 19, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, Sequence No. 36, SEQ ID No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 67, SEQ ID No. 68, Sequence No. 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, Sequence No. 115, SEQ ID No. 126, SEQ ID No. 119, SEQ ID No. 129, SEQ ID No. 153, SEQ ID No. 157 , SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: No. 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, Column No. 83, SEQ ID No. 143, SEQ ID No. 139, SEQ ID No. 145, SEQ ID No. 141, SEQ ID No. 12 3, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: No. 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, Sequence No. 8, SEQ ID No. 89, SEQ ID No. 92 and / or SEQ ID No. 95.   Suitable second compounds include proteinaceous compounds, insecticides and flea-like (col) lar), including but not limited to No. Preferred second compounds are active immunization (eg, antibody vaccine), passive immunization (Eg, an antibody) or a proteinaceous compound that otherwise Inhibition of mi activity, if inhibited, burden on animals and around animals This can reduce the burden on the user. Examples of second compounds include flea membrane proteins Compounds that inhibit the binding between and a ligand (eg, flea ATPase activity Compound or peptide or steroid hormone binds to its receptor ), Hormones (including peptides or steroid hormones) , Vitellogenesis (production of vitellin and the main yolk protein) Compounds that inhibit fat body function, including its transport into the body and its maturation) Compounds, compounds that inhibit flea muscle movement, compounds that inhibit flea nervous system, flea immune system And / or compounds that inhibit flea feeding.   According to the present invention, the immunoglobulin proteinase of the present invention is Used as a second compound in the composition and is specific for the selected flea protein. The life span of the differentially bound antibodies can also be increased. Immunoglobulin protein The enzyme is administered to an animal and specifically binds to an immunoglobulin proteinase. Thus, the production of antibodies that inhibit the activity of proteinase can be enhanced. Exemption Epidemiological globulin proteinase can be used to administer any desired flea protein to fleas Can be administered to the animal either at the same time or thereafter. Therapeutic group Preferred immunoglobulin proteiner to be included as a second compound in the composition These include: Under stringent hybridization conditions, amino acids SEQ ID NO: 67, SEQ ID NO: 68 and And / or a nucleic acid molecule having a nucleic acid sequence encoding a protein having SEQ ID NO: 69 The isolated protein encoded by the hybridizing nucleic acid molecule or the isolated protein A mimetope; and / or under stringent conditions, a nucleic acid sequence comprising SEQ ID NO: 66 and the invention Other nucleic acids encoding the immunoglobulin proteinases of the invention disclosed in the description An isolated nucleic acid molecule that hybridizes to a gene containing the sequence.   The composition of the present invention comprises a pharmaceutically acceptable excipient, adjuvant and / or carrier. Other components such as can also be included. For example, the compositions of the present invention It can be formulated in a vehicle that the animal to be tolerated can tolerate.   Such excipients include water, saline, Ringer's solution, dextrose solution, And other aqueous physiologically balanced salt solutions. Non-volatile Uses non-aqueous excipients such as oils, sesame oil, ethyl oleate, and triglycerides obtain. Other useful formulations include sodium carboxymethylcellulose, sorbitol And suspensions containing thickening agents, such as dextran or dextran. Excipients contain small amounts of additives such as substances that enhance isotonicity and chemical stability. You can also. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer. And preservatives such as thimerosal, m- or o-cresol, forma Includes phosphorus and benzyl alcohol. Standard prescription is liquid injectable Or a solid, in a liquid suitable as an injection suspension or solution Any of those that can be taken into the device. That is, in non-liquid formulations, May include dextrose, human serum albumin, preservatives, etc., which may be sterile Water or saline can be added prior to administration.   In an embodiment of the present invention, the composition of the present invention comprises an excipient such as an adjuvant or carrier. An immunopotentiator can also be included. Adjuvant Is typically a substance that enhances an animal's immune response to a specific antibody systemically. is there. Suitable adjuvants include Freund's adjuvant; other bacterial cells Wall components; aluminum-based salts; calcium-based salts; silica; Otide; Toxoid; Serum protein; Viral coat protein Preparations from other bacteria; gamma interferon; Hunter's Titermax Juxant (Vaxcel in Norcross, Georgia)^TMBlock copolymers Adjuvant; Ribi adjuvant (Ribi ImmunoChem R, Hamilton, MT) available from esearch); and QuilA (from Superfos Biosector A / S in Denmark) (Available from Co., Ltd.) and their derivatives. Not something. The carrier will typically comprise the therapeutic composition in the treated animal. Compounds that increase half-life. Suitable carriers include polymeric controlled release formulations, Biodegradable implants, liposomes, bacteria, viruses, oils, esters and Includes, but is not limited to, recalls.   One aspect of the present invention is that the composition of the present invention can be slowly released to an animal. Formulation with limited release. Controlled release formulations used herein Contains the compositions of the present invention in a controlled release vehicle. release Controlled vehicles include biocompatible polymers, other polymer matrices, Puser microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, Liposomes, lipospheres, and transdermal delivery systems are included However, the present invention is not limited to these. Other controlled release formulations of the present invention include: Upon administration, liquids that form solids or gels in situ are included. Preferred release Controlled release formulations are biodegradable (eg, bioerodable).   A preferred controlled release formulation of the present invention is a flea protein feeding animal. A constant rate sufficient to achieve a therapeutic dose level of the composition that reduces ase activity The present invention provides the blood of an animal to be treated over a period ranging from about 1 to about 12 months in degrees. Can be released. Controlled release formulations of the present invention are preferred. <Is at least 1 month, more preferably at least 3 months, even more preferably less At least 6 months, more preferably at least 9 months, more preferably at least 1 month Treatment can be given for 2 months.   To protect animals from flea infestation, the therapeutic compositions of the present invention may be treated with the compositions. The effect of reducing the protease activity of fleas sucking from the bloodstream of the treated animal Is administered to animals in an appropriate manner. Thus, the animal to be treated is a flea protein Or by reducing flea protease activity and at least one To reduce the fleas suffering by reducing the activity of one other flea A suitable animal. Preferably, the protease activity is about 50 percent, more Preferably reduced to about 70 percent, more preferably to about 90 percent . The method by which the composition is administered to the animal and the animal becomes compatible depends on the nature of the composition and the nature of the composition. Depends on dosing regimen. Protease activity with at least one booster shot Animals administered tin would generally be expected with any vaccine treatment It becomes compatible in about the same time as the time. For example, about 4 to 6 weeks after the initial dose Animals receiving inter-boost shots become compatible for about another 3 to 4 weeks. Administered a composition containing an anti-flea protease antibody or a protease inhibitor The animals are allowed to reach the proper serum level of the compound of the invention (usually 1 to 3 days). It will be compatible soon.   In a preferred embodiment, the compositions of the present invention cure flea when administered to a host animal. Within at least about 21 days after sucking blood from the treated animal, Flea viability can be reduced to cents. (Fleas are usually one or more animals Note that it will survive for about 40 to 50 days. A) a more preferred composition is a host animal At least 14 days after the flea has been sucked from the treated animal Flea viability can be reduced by at least about 65 percent within. Further A preferred composition, when administered to an animal, bleeds from the animal where the fleas were treated Later reduces flea viability by at least 90 percent within at least 7 days be able to.   In another preferred embodiment, the composition of the present invention, when administered to a host animal, Within at least 30 days after sucking blood from the treated animal, Percent, preferably at least about 70 percent, and more preferably at least Also reduce flea fertility (ie, egg-laying ability) by about 90 percent. (Fleas are Note that spawning does not usually start until about 7 days after sucking blood. )   In accordance with the present invention, the composition comprises an animal that has become compatible (ie, the animal has been treated). Become an animal. )) To reduce the flea protease activity of the fleas sucked from The animal is administered in such a way that the animal is compatible. For example, the nomip of the present invention Rotase vaccines, when administered to animals in an effective manner, Produce sufficient titer of antibodies in the bloodstream of the animal to reduce the activity An immune response can be elicited (ie, stimulated). Similarly, the antinoise of the present invention Miprotease antibodies, when administered to animals in an effective manner, Move the titer in an amount sufficient to reduce its activity and in amounts that are present in the animal's bloodstream. To be administered. The protease inhibitor compounds of the present invention can be used in animals in an effective manner. When administered, the blood of the animal is at a concentration sufficient to reduce flea protease activity. It is administered in such a way as to be in the stream. The oligonucleotide nucleic acid molecules of the present invention also Administered in an effective manner, thereby reducing flea protease expression. Can be.   The compositions of the present invention may be administered to animals prior to or during flea infestation. The present invention If the vaccine is administered to an animal, the animal will receive the flea pro The animal develops an immune response before being capable of responding to inhibit the activity of thease. It should be noted that time is required to allow Obtaining an animal immune response Methods are known to those skilled in the art.   Acceptable protocols for administering the compositions in an effective manner include individual Includes dosage size, number of doses, frequency of dosing, and mode of administration. Such a professional The determination of the protocol is performed by a person skilled in the art. Appropriate single doses should be given in the appropriate Dose that can protect animals from flea infestation when given more than once across It is. For example, a protease vaccine or a preferred dose of these mimetopes The dose is 1 microgram (μg or ug) per kilogram body weight of the animal. Also note. ) To about 10 milligrams (mg). Booster vaccine connection The species can be administered from about two weeks to several years after the initial administration. Booster vaccine injection Feeding is preferably insufficient to protect the animal from flea infestation Is administered. A preferred dosing schedule is about From 10 μg to about 1 mg of vaccine from about 1 to about 2 weeks to about 12 months Administer about twice. In one aspect, the booster dose of the composition of the invention is the initial dose Administered about 4 to 6 weeks after dose, additional booster administered about once or twice a year Is done. Modes of administration include oral, nasal, topical, transdermal, rectal, and parenteral routes. It can be, but is not limited to. Parenteral routes include subcutaneous, intradermal, intravenous, and And intramuscular routes, but are not limited to these.   In other embodiments, the anti-flea protease antibody compositions or the mimetopes thereof are preferred. A single dose can be from about 1 μg to about 10 mg per kilogram of animal body weight. It is a range of products. Anti-flea antibodies are given for about one hour to about every two weeks for several weeks after the first dose. Can be re-administered. Booster treatment is preferably performed with antibody titers in animals. It may be administered if it is sufficient to protect the animal from invasion of the mi. preferable The dosing schedule is from about 10 μg to about 1 mg of anti-flea Thease antibody is administered every two to four weeks. Proper administration The method is as disclosed herein and is known to those skilled in the art.   According to one aspect, the nucleic acid molecule of the present invention is used in an animal that can be protected from a disease. The nucleic acid molecule is used as a protective protein (eg, a flea protease vaccine, an anti-flea vaccine). Protease antibodies or protein-like protease inhibitors) or protected RNA Administered to animals in such a manner that they can express antisense RNA, ribozymes or RNA drugs). Can be. Nucleic acid molecules can be obtained by direct injection (e.g., with "naked" DNA or RNA components). Child. For example, as taught in Wolff et al., 1990, Science 247, 1465-1468. . ) Or (b) a recombinant virion vaccine or a recombinant cell vaccine (ie Vehicle selected from the group consisting of recombinant vaccine particles and recombinant cell vaccines That can be transported more cells) It is not limited to them. ) Can be delivered to animals in various ways, including   The recombinant virion vaccine of the present invention is packaged in a viral membrane. Recombinant molecules of the invention, and recombinant molecules of the invention that can be expressed in animals after administration Is included. Preferably, the recombinant molecule is packaging deficient. Alf Virus, poxvirus, adenovirus, herpes virus, and letovirus A large number of recombinant virions, including but not limited to Can be used. When administered to animals, the recombinant virus particle vaccine of the present invention Is a disease that infects cells in the immunized animal and is caused by the parasites of the invention. Indicate the production of a protective protein or RNA nucleic acid molecule that can protect animals You. A preferred single dose of the recombinant virion vaccine of the invention is About 1 × 10 per gram weight^FourFrom about 1 × 10⁷Virus plaque forming unit ( pfu). Dosing protocols are unambiguous for protein-based vaccines. This is the same as that disclosed in the detailed description.   The recombinant cell vaccine of the present invention expresses at least one protein of the present invention. The presently disclosed recombinant cells of the present invention are included. Preferred recombinant cells include Salmonella (Salmonella), E. coli, Mycobacterium, S . S. frugiperda, baby hamster kidney, myoblast G8, CO S, MDCK and CRRF recombinant cells are included, but Salmonella recombinant cells are more preferred. Is more preferable. Such recombinant cells can be administered in various ways, but these Per kilogram body weight, preferably 10⁸From 10¹²Bacterial range throwing It is advantageous to administer orally in dosages. The protocol for administration is Similar to those disclosed herein for cytoplasm-based vaccines. Recombinant Cell vaccines can include whole cells or cell lysates.   The composition of the present invention is administered to any animal susceptible to flea infestation, including warm-blooded animals. Can be given. Preferred animals to treat include mammals and birds, Cat, dog, human, cattle, chinchilla, polecat, goat, mouse, mink, rabbit, Raccoon, rat, sheep, squirrel, pig, chicken, ostrich, quail and turkey, average And other furry animals, pets and / or economical edible animals are more preferred. Security Particularly preferred animals for protection are cats and dogs.   The present invention includes a composition for treating flea infestation by any fleas. You. Thus, the compositions of the present invention can be derived from any flea species. target Preferred fleas include fleas of the following genera: Ctenocephal ides), Cyopsillus (Cyopsyllus), Diamanus (Diamanus) (E (Oropsylla), chicken flea (Echidnophaga), chicken flea (Echidnophaga), (European flea (Neospsyllus), human flea (Pulex) , Genus Tunga, and Xenopsilla, but steno Separidis Canis (Ctenocephalides canis), Stenocephalidis ferris (Ctenocephalidesf) elis), Diamanus montanus, Echidnophag a gallinacea), Nosopsyllus faciatus, prepress Pulex irritans, Pulex simulan s), Tunga penetrans and Xenopsila keopis (Xen opsylla cheopis) species are preferred. When protecting animals from fleas, Particularly preferred of the mi are Stenocephahlide (Ctenocephahlide) s felis), Ctenocephalides canis and press Pulex species (eg, Pulex irritans and Rex Simulans (Pulex simulans) species are included.   The invention also relates to the use of a composition of the invention for reducing invasion by other ectoparasites, And a composition comprising a protease vaccine, an anti-protease antibody and any Inhibits the synthesis and / or activity of proteases derived from ectoparasites Compositions that reduce the invasiveness of foods, in particular controlled release containing such compositions Includes the use of prescription agents. Preferred ectoparasites to target include arachnids, insects and And hills are included. More preferred ectoparasites to be targeted are fleas; ily Ixodidae hard ticks (eg, Ixodes and Kira Soft ticks of the genus Ambloymma and family Argasidae ticks) (eg, Parker's mite (O. parkeri) and O. turicata) Ticks, including both Ornithodoros, as well as midges (midge s) (e.g., Culicoides), mosquitoes, snow flies, black flies, horse flies, and Fly flies (horn flies), Mekurabu, Tsetse flies, flies and fly sickness Ants; spiders; lice; Includes Hemiptera insects such as bed bugs and assassin bugs, including . More preferred target ectoparasites are fleas, mosquitoes, chironomids, snub flies, Includes fly, tick and Rhodius.   The following examples are provided for illustrative purposes and limit the scope of the invention. It is not intended to be.                                  Example   The examples here are intended to cover a number of molecular biology which would be known to those skilled in the art. It should be noted that this includes microbiological, immunological and biochemical techniques. this Such techniques are disclosed, for example, in Sambrook et al., Supra (ibd.), Borov. sky, Arch Insect Biochem. and Phys., 7:18 7-210, 1988, and related literature. On April 25, 1996 Examples 1-21 of published PCT Publication No. WO 96/11706, And the sequence information provided in the sequence listing are incorporated herein by reference. Incorporated in the present application. Example 1   This example describes a method for determining the internal amino acid sequence of flea aminopeptidase. are doing.   The gastrointestinal tract of flea fed from the blood of about 10200 cats was Gut Dissection Buffer (50 mM Tris- HCl, pH 8.0 and 100 mM CaCl_Two) Cut in. Flea digestive tract Sonicate and centrifuge it at about 14000 rpm for about 20 minutes. Produced an extract of the flea digestive tract. 2 ml digestion of the resulting pellet Wash in tube analysis buffer, sonicate briefly, and again at about 14000 rpm For about 20 minutes. The resulting pellet is washed with 20 mM NaAc (pH 6. 0), 0.1% breeze, complete protease inhibition cocktail (Piers ce)) and 0.25 mM bestatin in 4 ml buffer And sonicated. Sonicate at about 14000 rpm for about 20 minutes Centrifuged. Both the pellet and the supernatant were collected. The pellet is sonicated again. And centrifuged as above, and the resulting supernatant was combined with the original supernatant .   The storage supernatant is applied to a polyCAT cation exchange HPLC column and  With a NaCl gradient ranging from 0M to 1M NaCl in NaAc (pH 6.0) Ta The protein was eluted. The fraction collected from the column was used for H-Leu-AMC fluorescence. Analyze by light and pool the active fractions and use a C-1 reverse phase HPLC column (TM S250, Toso Haas). 0.1% in water Use a gradient of acetonitrile in TFA, between 20% and 100% acetonitrile The protein was eluted from the column in the range. Included in both fractions from column Proteins were analyzed by SDS-PAGE gel electrophoresis and silver staining. The results of the gel electrophoresis showed that some of the fractions contained approximately 95 kDa protein. Showed the presence of quality. This protein has the relevant PCT Publication No. WO 96/11. No. 706 correlates with the approximately 95 kDa protein described in Example 12. And it was identified using membrane pellets obtained from flea midgut lysate.   To determine the internal amino acid sequence of the 95 kDa protein, a 95 kDa protein was used. The fractions containing the protein are pooled, dried, and BNPS-stained at room temperature for about 72 hours. Digested with Kator. BNPS-skatole digest was converted to 18% Tris-glycine Separated by PAGE gel electrophoresis and soaked in PVDF membrane. Related PCT release Using the technique described in Example 7 of WO 96/11706, about 28 The major band of kDa was excised and sequenced at the N-terminus. Internal pepti The partial N-terminal amino acid sequence of LADQFQATHARSAFP CFDEPAM (shown here as SEQ ID NO: 107) was obtained. Example 2   This example involves the cloning and isolation of another flea aminopeptidase nucleic acid molecule. And sequencing are described.   Manduca sexta and rat aminope Nucleic acid sequence 5 ′ CCC AAA TTT TCC corresponding to the conserved region in petittase ATW GON CCN GC 3 ′ (n indicates any nucleotide, And the primer APN3 represented by SEQ ID NO: 108) 3 In combination with the reverse primer (SEQ ID NO: 87), the relevant PCT publication number Cattle blood as described in Example 8 of WO 96/11706 with feed Flea Aminopeptidase Inheritance from Selected Flea cDNA Expression Library One of the children The portion was PCR amplified. The product resulting from the PCR amplification is diluted about 1:50, And using SEQ ID NO: 107 (described in Example 1) and nucleic acid sequence 5 'CAA TTY CAA GCT ACY CAT GC3' (SEQ ID NO: 109 In combination with a degradation primer APN1C designed to Second semi-nested using primer APN3 ) Used as template in PCR amplification. The resulting PCR product, nf AP2₃₈₃Is approximately 383-bp when visualized on a 1% agarose gel. Met. PCR product nfAP2₃₈₃Was gel purified and the TA vector And subjected to standard DNA sequencing techniques. nfAP2₃₈₃Nucleotide sequence of Is shown as SEQ ID NO: 110. The translation of SEQ ID NO: 110 corresponds to the amino acid sequence SEQ ID NO: 111 is shown, and PfAP2₁₂₇Of about 127 amino acids A putative flea aminopeptidase protein was generated.   PCR product nfAP2₃₈₃Is³²Labeled with P and standard hybridization Phage expression library of whole fleas fed on bovine blood using application technology Was used as a probe for screening. 2100 nucleotide insert And nfAP2₂₁₀₀A single plaque purified clone corresponding to was isolated. nf AP2₂₁₀₀The partial nucleic acid sequence is obtained using standard techniques from the 5 'end of the NfAP2 indicating SEQ ID NO: 112 in column₅₃₇Flea aminopeptide core called flea An acid molecule was obtained. The translation of SEQ ID NO: 112 translates into the nucleic acid molecule nfAP2₅₃₇But PfA P₁₇₈And about 178 showing the amino acid sequence SEQ ID NO: 113 Suggests that it encodes a non-full length flea aminopeptidase of amino acids, Changes the first codon from about nucleotide 2 to about nucleotide of SEQ ID NO: 112. It shows that it extends to C4. SEQ ID NO: 113 contains SEQ ID NO: 107 .   SEQ ID NO: 112 of flea aminopeptidase nucleic acid sequence is characterized from another organism And compared with the additional nucleic acid sequence. The nucleic acid sequence is the corresponding Manduka sexta aminopeptida between regions Approximately 50% match with N-N-nucleotides. Example 3   This example includes the cloning of a flea cysteine protease nucleic acid molecule. And sequencing has been described.   nfCP1₅₇₃Cysteine protease nucleic acid molecule referred to herein as Was produced by PCR amplification using the following method. Nucleic acid sequence 5'TTG GGA TAC ACT TTG ACT GTT AAC C3 ' Primer Cal3F represented by SEQ ID NO: 97 (to obtain calreticulin gene Was designed in conjunction with the M13 universal primer to use standard techniques. As described in Example 8 of related PCT Publication No. WO 96/11706. From a flea cDNA expression library using bovine blood as feed The A fragment was PCR amplified. Surprisingly, the isolated DNA fragment is Correlated with thease nucleic acid sequence. The sequence obtained from this DNA fragment was used And here represented as SEQ ID NO: 98, combined with the M13 reverse primer Nucleic acid sequence used for CR amplification 5 'GTG AGC AAC CAT TAT TT Primer Cys1R with C CAT ATC 3 'was constructed. Array here Nucleic acid sequence 5 'CTT TCC TCA CAA TAC represented as No. 74 Using primer Cys1F representing CAC CAA GGA AGC 3 ', M A third PCR amplification was performed in combination with 13 universal primers. Where SEQ ID NO: Nucleic acid sequence 5'CTT GTA CGA TTG TCT CAA represented as 76 Using the primer Cys2F with CAG GC 3 ', the M13 universal primer was used. A fourth PCR amplification was performed in combination with the mer. The resulting PCR products are each Each gel was purified and cloned into the A vector system, and standard DNA sequencing was performed. Lancing technology. Represents the coding region for the flea cysteine protease The complex nucleic acid sequence is identified as nfCP1₅₇₃SEQ ID NO: 7 It is estimated and shown here as 6. The translation of SEQ ID NO: 76 translates into the nucleic acid molecule n fCP1₅₇₃Shows the amino acid sequence SEQ ID NO: 77, where PfCP1₁₉₁To Non-full length flea cysteine protease protein of about 191 amino acids that applies Quality coding, whereby the first codon is About It shows that it ranges from nucleotide 1 to about nucleotide 3.   nfCP1₅₇₃Nucleic acid molecules and PfCP1₁₉₁Nucleic acid and amino acid sequences of proteins Using known homology searches in Genebank, respectively. Compared to acid and amino acid sequences. SEQ ID NO: 77 is a P. Sachibum (P.s. ativum) cysteine protease Do you get it. SEQ ID NO: 77 and p. Cis of P. sativum The most conserved regions of continuous similarity between the amino acids of the tein protease are SEQ ID NO: 77 from about amino acid 71 to about amino acid 165 and p. Sachibum (P . sativum) cysteine protease from about amino acid 17 to about amino acid 168 and there is about 42% homology between the two regions. nfCP1₅₇₄ Comparison of nucleic acid sequences encoding from about 205 to about 492 amino acids of This indicates that the regions are about 54% homologous. Example 4   This example includes the cloning of a flea serine protease nucleic acid molecule and Sequencing is described.   Additional serine protease cDNA nucleic acid molecules are assigned to the relevant PCT Publication No. W Isolated in a manner similar to that described in Example 8 of O96 / 11706. Flea cDNA expression library on bovine blood feed (Related PCT Publication No. (Produced as described in Example 8 of WO 96/11706). The actual primers used in the PCR amplification of the serine protease nucleic acid molecule M13 reverse primer (SEQ ID NO: 87) or H57 primer (SEQ ID NO: 99) in combination with cat-try No. 2 (sequence No .: 86). The resulting PCR product is gel-purified and Cloned in Coutre. A recombinant TA vector clone was isolated and It was found to correspond to the cloned serine protease gene. these The newly cloned nucleic acid molecule is described in Sambrook et al., Supra. As described above, nucleic acid sequencing was performed using the Sanger dideoxy chain termination method. Kensing. A. Flea clone 5 (primer cat-try 2 and M13 retrograde Nucleic acid sequence of a flea serine protease nucleic acid molecule corresponding to That is, nfSP5₈₀₆Is represented herein as SEQ ID NO: 114. SEQ ID NO: 116 and SEQ ID NO: 117 are both nucleic acid molecules nfSP5₈₀₆Contained in an array of It is. Translation of SEQ ID NO: 114 translates into the nucleic acid molecule nfSP5₈₀₆Is the amino acid sequence Shows SEQ ID NO: 115, and shows PfSP5₂₄₅About 245 amino acids shown here as Noic acid suggests that full-length flea encodes near a serine protease protein This means that the first codon is from about nucleotide 2 to about nucleotide of SEQ ID NO: 114. It spans Reotide 4 and has a stop codon at about nucleotide 7 of SEQ ID NO: 114. It exhibits an open reading frame ranging from 37 to about nucleotide 739. Gene Bank (G homology search of SEQ ID NO: 114 with Gals Galus (Gal). luc galus) reveals most homology between trypsin genes Thus, there is about 52% homology between the corresponding regions of the two nucleic acid molecules. B. Flea clone 11 or nfSP11₃₀₇(Primer cat-tiger Flea serine protein corresponding to (a) made using No. 2 and M13 retrograde The nucleic acid sequence of the zea nucleic acid molecule is represented herein as SEQ ID NO: 118. SEQ ID NO: 1 20 and SEQ ID NO: 121 represent the nucleic acid molecule nfSP11₃₀₇In the array. Arrangement Translation of SEQ ID NO: 118 translates to the nucleic acid molecule nfSP11₃₀₇But PfSP11₁₀₂As As applied herein, about 102 amino acids having the amino acid sequence of SEQ ID NO: 119 Encoding a non-full-length flea serine protease protein. Thereby, the first codon is from about nucleotide 1 to about nucleotide of SEQ ID NO: 118. Ranges to Pride 3. C. Flea clone 39, nfSP39₂₆₇(Primer cat-tiger Flea serine protein nucleus corresponding to (No. 2 and H57) The nucleic acid sequence of the acid molecule is represented herein as SEQ ID NO: 122. SEQ ID NO: 12 2 translates into the nucleic acid molecule PfSP39₂₆₇But nfSP39₈₉Apply here as In other words, a non-full-length flea of about 90 amino acids having the amino acid sequence of SEQ ID NO: 123 Suggests that it encodes a serine protease protein, thereby The dong extends from about nucleotide 1 to about nucleotide 3 of SEQ ID NO: 122. Example 5   This example includes the cloning of a flea serine protease nucleic acid molecule and Sequencing is described.   A. Library using bovine blood as feed   Certain flea serine protease cDNA nucleic acid molecules are fed from bovine blood. Flea cDNA expression library (Related PCT Publication No. WO 96/117) No. 06 (produced as described in Example 8) Two nucleic acid molecules, Cat-Tri # 1 (SEQ ID NO: 124) and Using Cat-Try No. 2 (SEQ ID NO: 86), the relevant PCT Publication No. Isolated in a manner similar to that described in Example 8 of WO 96/11706. . Two clones that strongly hybridize to the probe were isolated, and As described in Sambrook et al., Supra, Sanger. Nucleic acid sequencing was performed using the dideoxy chain termination method.   1. Flea clone 8 or nfSP8₄₃₆Flea serine protein corresponding to The nucleic acid sequence of thease nucleic acid molecule is represented herein as SEQ ID NO: 125. Array number No. 127 is the nucleic acid molecule nfSP8₄₃₆Included in the array. SEQ ID NO: 125 Translation of PfSP8₁₄₅And the amino acid sequence of SEQ ID NO: 126 This gives rise to a protein of 145 amino acids, whereby the first codon is SEQ ID NO: Ranging from about nucleotide 2 to about nucleotide 4 of 125. Geneba Genbank homology search was performed using SEQ ID NO: 125 with Anofereres. Trypsin precursor gene of Anopheles gambiae Between the corresponding regions of two nucleic acid molecules. Has about 48% homology.   2. Flea clone 12 or nfSP12₇₅₈Flea serine corresponding to The nucleic acid sequence of the Rotase nucleic acid molecule is represented herein as SEQ ID NO: 128. Array No. 130 and SEQ ID No. 131 are both nucleic acid molecules ntSP12₇₅₈Array of Contained within. Translation of SEQ ID NO: 128 translates to the nucleic acid molecule PfSP12₂₄₆Indicates that Producing a protein of about 246 amino acids having the amino acid sequence of SEQ ID NO: 129, Thereby, the first codon is from about nucleotide 1 to about nucleotide of SEQ ID NO: 128. It spans Otide 3 and has a stop codon at about nucleotide 73 of SEQ ID NO: 128. It exhibits an open reading frame ranging from 9 to about nucleotide 741. Gene Bank (Ge nbank) homology search was performed using SEQ ID NO: 128 and rat trypsinogen. Reveals the greatest homology between the genes, which allows the correspondence of the two nucleic acid molecules There is about 57% homology between the regions.   B. Library using cat blood as feed   Certain flea serine protease cDNA nucleic acid molecules were (SEQ ID NO: 124) and Cat-Try No. 2 (SEQ ID NO: 86) probes The cat blood is fed to the feed by screening the library using Isolated from a flea cDNA expression library. Cat feed Feed the cat's blood, except for the flea library, which was fed cat blood. Flea library (see PCT Publication No. WO 96/11706) (Described in Example 8). Powerfully probed with the probe Two clones were isolated and the nucleic acid sequence was determined using the methods described above. Singed.   1. The nucleic acid sequence of one of the flea serine protease nucleic acid molecules, ie, nfS P26₆₁₀Is represented herein as SEQ ID NO: 132. Translation of SEQ ID NO: 132 The translation is PfSP26₁₈₅And shows SEQ ID NO: 133 of the amino acid sequence. Giving rise to a non-full length sequence of 185 amino acids, whereby the first codon is SEQ ID NO: : 132 from about nucleotide 1 to about nucleotide 3. Gene Genbank homology search revealed SEQ ID NO: 133 with Aedes a. The sequence between the tryptic protein sequences of Aedes aegypti Reveals a large degree of homology, which results in an approximation between the corresponding regions of the two amino acid molecules There is 48% homology.   2. Nucleic acid sequence of flea serine protease nucleic acid molecule, ie nfSP27₃₈₆ Is represented herein as SEQ ID NO: 134. The translation of SEQ ID NO: 134 is Pf SP27₁₂₈And the amino acid sequence of SEQ ID NO: 135 is shown. Giving rise to a protein of the amino acid whereby the first codon is Ranging from about nucleotide 1 to about nucleotide 3. Example 6   This example includes the cloning of certain flea serine protease nucleic acid molecules. And sequencing are described.   Certain flea serine protease cDNA nucleic acid molecules can be used to feed cat blood MRNA isolated from whole fleas isolated from reverse transcriptase PCR amplification. cat After the breeding was started with blood of m., MRAN was simply collected from fleas collected over 72 hours. Released. Thus, mRNA was isolated from mRN isolated at various time points over a 72 hour period. A mixture. Using ground flea, mRNA is isolated and the Tris reagent ( Molecular, Cincinnati, Ohio ・ Research Center (Molecular Research Center) From Invitrogen) and Invitrogen Fast Track (Invitro) gen fast track^TM) RNA Isolation Kit (Sande, CA) Invitrogen (San Diego, CA) ) Was used to extract total flea RNA. Stratagene RT-P CR Kit (San Diego, CA) (Available from Stratagene) was used to synthesize cDNA. First-chain cD Primers used for NA synthesis were: 5'dT-2VT3 'and 5'dT-2VC3 '(Alameda, CA) Peron Technologies, Inc. (Operon Technologies, I nc.) available on Differential Display Kid And a molar mixture such as   The actual primers used for PCR amplification of the cDNA described above were H5 Cat-Try No. 2 (used in combination with 7 primers (SEQ ID NO: 99) SEQ ID NO: 86). The resulting PCR product is gel-purified and Khutor^TMWas cloned. Isolate the recombinant TA vector clone and Acid molecules were subjected to nucleic acid sequencing using assays as described above. .   A. The nucleic acid sequence of one of the flea serine protease nucleic acid molecules, ie, nfS P23₄₂₃Is represented here as SEQ ID NO: 136. The translation of SEQ ID NO: 136 is , Nucleic acid molecule nfSP23₄₂₃But here, PfSP23₁₄₁And amino A non-full-length flea serine protein of about 141 amino acids that shows the acid sequence SEQ ID NO: 137 Encoding the ase protein, whereby the first codon is SEQ ID NO: 136 Ranging from about nucleotide 1 to about nucleotide 3. Gene Bank (G homology search of SEQ ID NO: 136 with Homo sapiens (Ho). Mo sapiens) has the greatest homology between the plasminogen precursor genes. Reveals that there is about 51% homology between the corresponding regions of the two nucleic acid molecules. You.   B. Another nucleic acid sequence of flea serine protease nucleic acid, nfSP24₄₁₀ Is represented here as SEQ ID NO: 78. The translation of SEQ ID NO: 78 translates into the nucleic acid molecule nf SP24₄₁₀But PfSP24₁₃₆And applies to SEQ ID NO: 79. A non-full length flea serine protease tan of about 136 amino acids having a amino acid sequence Suggests that it encodes a protein, whereby the first codon is SEQ ID NO: 78. Ranging from about nucleotide 1 to about nucleotide 3. Gene Bank (Genbank) homology search was performed with SEQ ID NO: 79 and Anopheles gumbi Chymotrypsin protein sequence of Anopheles gambiae Between the corresponding regions of two nucleic acid molecules. Have about 38% homology.   C. Another nucleic acid sequence of a flea serine protease nucleic acid molecule, nfSP33₄₂₆ Is represented herein as SEQ ID NO: 82. The translation of SEQ ID NO: 82 is a nucleic acid Molecule nfSP33₄₂₆But PfSP33₁₄₂Shown here as the amino acid sequence A non-full length flea serine protease of about 142 amino acids, which shows SEQ ID NO: 83. Suggests that it encodes a protein, whereby the first codon is SEQ ID NO: 8 It is shown to range from about nucleotide 1 to about nucleotide 3 of 2. Gene Van Genbank homology search revealed SEQ ID NO: 83 with Drosophila (Dr. osophila) serine protease strain protein Reveals the greatest homology between the two amino acid sequences, There is about 45% homology between the active regions.   D. Another nucleic acid sequence within one of the flea serine protease nucleic acid molecules, ie, n fSP36₁₉₇Is represented herein as SEQ ID NO: 138. SEQ ID NO: 138 Is a PCR amplified nucleic acid molecule ntSP36₅₀₀Represents a partial sequence of SEQ ID NO: 13 8 translates into the nucleic acid molecule nfSP36₁₉₇But PfSP36₆₅As shown here , An amino acid sequence SEQ ID NO: 139, a non-full length flea cell of about 65 amino acids Coding for a protease protein, which results in the first codon Extends from about nucleotide 1 to about nucleotide 3 of SEQ ID NO: 138. Is done. A homology search for Genebank gave SEQ ID NO: 139 And Drosophila melanogaster ter) to determine the greatest homology with the ester protein sequence, Thus, there is about 42% homology between the corresponding regions of the two amino acid sequences.   E. FIG. Another nucleic acid sequence of the flea serine protease nucleic acid sequence, ie, ntSP3 8₃₄₁Is represented herein as SEQ ID NO: 140. Translation of SEQ ID NO: 140 , Nucleic acid molecule nfSP38₃₄₁But PfSP38₁₁₃Shown here as an amino acid A non-full-length flea serine protein of about 113 amino acids having SEQ ID NO: 141 Suggests that it encodes an ase protein, so that the first codon is No. 140 from about nucleotide 3 to about nucleotide 5. The Genbank homology search identified SEQ ID NO: 141 with rat Revealed the greatest homology with the trypsinogen protein sequence, And there is about 30% homology between the corresponding regions of the two amino acid sequences.   F. The nucleic acid sequence of one of the flea serine protease nucleic acid molecules, ie, nfS P34₃₉₀Is herein represented by SEQ ID NO: 142. Translation of SEQ ID NO: 142 , Nucleic acid molecule nfSP4₃₉₀But PfSP34₁₃₀Shown here as the amino acid sequence Non-full length flea serine protea of about 130 amino acids, SEQ ID NO: 143 Coding for a proteinase, so that the first codon is No. 142 is shown to range from about nucleotide 1 to about nucleotide 3. The Genbank homology search revealed that SEQ ID NO: 143 and Drosov Of Drosophila melanogaster Reveals the greatest homology between the delta precursor protein sequence, There is about 33% homology between the corresponding regions of the two amino acid sequences. Example 7   This example includes the cloning of a flea serine protease nucleic acid molecule and Sequencing is described.   The serine protease cDNA nucleic acid molecule is replaced with the relevant PCT Publication No. WO96 / Isolated in a manner similar to that described in Example 8 of US Pat. Cat blood A cDNA expression library of all fleas bred in solution (as described in Example 5) Actual) used in the PCR amplification of the serine protease nucleic acid molecule The primer was Cat-in combination with the M13 reverse primer (SEQ ID NO: 87). Trial No. 2 (SEQ ID NO: 86) is included. The resulting PCR product was diluted 1:25 And the forward vector primer T3, represented herein as SEQ ID NO: 100 Nucleic acid sequence 5 'ATT CCT CGT GGT TCA GTC GCT C 3' The reverse primer shown (nfSP33₇₇₈Derived from the nucleic acid sequence of Used as a template in a second RCR reaction Was. The resulting PCR product was gel purified and the TA vector^TMCloned into Was. Clones were subjected to nucleic acid sequencing as described above.   Nucleic acid sequence of one of the flea serine protease nucleic acid molecules, ie, nfSP3 3₇₇₈Is represented here by SEQ ID NO: 84. SEQ ID NO: 8, as expected 4 includes a portion of SEQ ID NO: 82. The translation of SEQ ID NO: 84 translates into the nucleic acid molecule nfS P33₇₇₈But here is PfSP33₂₅₉And the sequence of the amino acid sequence No. 85, a non-full length flea serine protease protein of about 259 amino acids Quality coding, whereby the first codon is about the same as SEQ ID NO: 84. Ranging from nucleotide 2 to about nucleotide 4. Gene Bank (Ge nbank) homology search was performed with SEQ ID NO: 84 and Drosophila (Drosop). Hira) Serine protease maximal phase between the stubble genes Homosexuality was determined, which resulted in nucleotides 23-778 of SEQ ID NO: 84. A strain of serine protease of Drosophila (stubb) le) There is about 54% homology between nucleotides 2324-3064 of the gene. Example 8   This example includes the cloning and cloning of another flea serine protease nucleic acid molecule. And sequencing has been described.   Using the method described in Example 5, the cDNA for the flea serine protease Clones were cloned using mRNA isolated from whole mino fed bovine blood. Obtained. The resulting cDNA was combined with H57 primer (SEQ ID NO: 99). Using Immercat-Tri # 2 (SEQ ID NO: 86), PCR amplification Used as a plate. The resulting PCR product is gel-purified and the TA vector^TM(T A Vector^TM). One recombinant TA vector clone Isolated and Flea Serine Protease Nucleic Acid Molecules and Illustrative nFS37₅₀₀ Can be used for nucleic acid sequencing as described in Sambrook et al., Supra. I did.   Flea serine protease nucleic acid molecule nFS37₅₀₀Some of the nucleic acid sequences Wachi nfSP37₂₆₁Is represented here as SEQ ID NO: 144. SEQ ID NO: 14 4 translates into the nucleic acid molecule nfSP37₂₆₁But here, PfSP37₈₇Is shown The non-full-length flea sequence of about 287 amino acids shown in SEQ ID NO: 145 of the amino acid sequence Suggests that it encodes a phosphoprotease protein, thereby Is from about nucleotide 1 to about nucleotide 3 of SEQ ID NO: 144. Present. Genebank homology search yielded SEQ ID NO: 145. Reveals the greatest homology between the chicken and chicken trypsinogen protein sequences Thus, there is about 31% homology between the corresponding regions of the two amino acid sequences. Example 9   This example includes the cloning of certain larval flea serine protease nucleic acid molecules. And sequencing have been described.   Certain serine protease cDNA nucleic acid molecules are amplified by PCR in cats. Mixed-age juveniles made using first, second and third-instar larvae reared on blood Isolated from a raw cDNA library. Actual primer used for PCR amplification Is the H57 primer (SEQ ID NO: 99) or the M13 reverse primer (sequence No. 87), any primer cat-try 2 in combination with any of No. (SEQ ID NO: 86). The resulting PCR product is gel purified and TA Khutor^TM(TA Vector^TM). Three recombinant TA vectors Clone with PrimerCat-Tri 2 and M13 reverse primer Used as a primer to isolate and contain the PCR product, and one clone Isolated using Primer Cat-Tri # 2 and H57 primers Contains PCR products. These new cloned nucleic acid molecules have been described above. Nucleic acid sequencing was performed as described.   A. Infants isolated using Cat-Trie # 2 and M13 reverse primers Nucleic acid sequence of one of the raw flea serine protease nucleic acid molecules, ie, nfSP2 9₆₁₂Is represented herein as SEQ ID NO: 146. Translation of SEQ ID NO: 146 is nuclear Acid molecule nfSP29₆₁₂But here is PfSP29₂₀₄Shown as and amino Flea serine near the full length of about 204 amino acids showing the acid sequence SEQ ID NO: 147 Suggests that it encodes a Rotase protein, whereby the first codon is Open reading spanning from about nucleotide 10 to about nucleotide 12 of SEQ ID NO: 146 Present a frame. Genebank homology search yielded SEQ ID NO: The greatest homology between 146 and the rat trypsinogen gene was determined, Thereby, there is about 50% homology between the corresponding regions of the two nucleic acid molecules.   B. Infants isolated using Cat-Trie # 2 and M13 reverse primers Another nucleic acid sequence of one of the raw flea serine protease nucleic acid molecules, ie, nfS P30₆₄₁Is represented herein as SEQ ID NO: 148. The translation of SEQ ID NO: 148 is , Nucleic acid molecule nfSP30₆₄But here is PfSP30₂₁₃Shown as Non-full length flea serine of about 213 amino acids, which shows the amino acid sequence SEQ ID NO: 149 Suggests that it encodes a Rotase protein, whereby the first codon is Ranging from about nucleotide 3 to about nucleotide 5 of SEQ ID NO: 148 . A homology search for Genebank revealed that SEQ ID NO: 148 and Ano Trypsin remains of Anopheles gambiae Reveals the greatest homology between the genes, and thereby the corresponding regions of the two nucleic acid molecules of There is about 52% homology between them.   C. Infants isolated using Cat-Trie # 2 and M13 reverse primers Another nucleic acid sequence of one of the raw flea serine protease nucleic acid molecules, ie, nfS P31₆₂₆Is represented here by SEQ ID NO: 150. Translation of SEQ ID NO: 150 , Nucleic acid molecule nfSP31₆₂₆But here is PfSP31₂₀₈And amino A non-full length flea serine protein of about 208 amino acids, which shows the acid sequence SEQ ID NO: 151 Suggests that it encodes an ase protein, so that the first codon is No. 150 from about nucleotide 3 to about nucleotide 5, or about nucleotide Ranging from 6 to about 8 nucleotides. Gene Van Genbank homology search was performed using SEQ ID NO: 150 with Anopheles Phases between the trypsin genes of Anopheles gambiae Reveals homology, which results in approximately 52% homology between the corresponding regions of the two nucleic acid molecules There is.   D. Larvae isolated using Cat-Trie No. 2 and H57 primers Nucleic acid sequence of mi serine protease nucleic acid molecule, nfSP32₄₃₃Here And SEQ ID NO: 80. Translation of SEQ ID NO: 80 translates into the nucleic acid molecule nfSP32.₄₃₃ But here, PfSP32₁₄₄And the amino acid sequence SEQ ID NO: A non-full length flea serine protease protein of about 144 amino acids And the first codon is about the nucleotide of SEQ ID NO: 80. Ranging from otide 1 to about nucleotide 3. Gene Bank nk) homology search was performed using SEQ ID NO: 80 and Anopheles gumbiae (Ano). phenes gambiae) reveals the greatest homology between trypsin genes Thus, there is about 52% homology between the corresponding regions of the two nucleic acid molecules. Example 10   This example includes the cloning and cloning of another flea serine protease nucleic acid molecule. And sequencing has been described.   A cDNA library of all fleas fed from bovine blood (relevant PCT publication number) No. WO 96/11706, described in Example 8), and fspN (1996). "A New Ectoparasite Salivary Protein and Such" published April 18, 2008 PCT Publication No. WO 96/1170 entitled "Apparatus for Collecting Various Proteins" Immunized with a collection of flea salivary gland products designated as described in US Pat. Immunoscreening was performed with antisera collected from Saki. Immunity screen as follows Training. New Zealand outbreak on fspN flea saliva products White rabbit antiserum was purified from Stratagene, Inc. (Stratagene, Ind.). c.) available from picoBlue^TM) Immune screening key Used in the immunoscreening protocol described in the kit's instruction manual. Was. CDNA expression library for immunoscreening, ie, cDNA Methods for clonal expression and immunoscreening of lambda phage plaques The means for transitioning to membranes for logging is available from La Jolla, CA allfornia), Stratagene, Inc. (Stratagene, I nc.) available in the instruction manual of the ZAP-cDNA synthesis kit. Have been.   nfSP32₈₁₅Nut about flea serine protease nucleic acid molecule called The reotide sequence is set forth as SEQ ID NO: 152 and corresponds to SEQ ID NO: 154 I do. Translation of SEQ ID NO: 152 translates into the nucleic acid molecule nfSP15₈₁₅But here PfS P15₂₅₄As about 25 and showing the amino acid sequence SEQ ID NO: 153 Suggests encoding a four amino acid full length flea serine protease protein Thus, the first codon is from about nucleotide 1 to about nucleotide 1 of SEQ ID NO: 152. It spans nucleotide 3 and the stop codon is about the nucleotide of SEQ ID NO: 152. It exhibits an open reading frame ranging from 763 to about nucleotide 765. Gene Bank ( Genbank) homology search found SEQ ID NO: 152 with Anopheles gumbi Homology between the trypsin genes of Anopheles gambiae Which results in about 52% homology between the corresponding regions of the two nucleic acid molecules. is there. Example 11   In this example, additional flea serine protease nucleic acid molecule cloning And sequencing are described.   Certain flea serine protease cDNA nucleic acid molecules are derived from undeveloped flea cD As a probe for screening an NA expression library, two nucleic acid molecules n fSP8₂₉₉(SEQ ID NO: 127) and nfSP19₃₅₉(SEQ ID NO: 155) As described in Example 8 of relevant PCT Publication No. WO 96/11706. It was isolated by a method similar to that performed. A probe that is strongly crossed The clones were isolated and subjected to the nucleic acid sequencing described above.   A. Nucleic acid sequence of flea serine protease nucleic acid molecule, ie nfSP19₈₅₅ Is represented herein by SEQ ID NO: 156. SEQ ID NO: 155 is a nucleic acid molecule nfS P19₈₅₅In the array. The translation of SEQ ID NO: 156 is PfSP19₂₅₃Called And an apparent full length of about 253 amino acids representing the amino acid sequence of SEQ ID NO: 157. The apparent start codon, which gives rise to the protein and is therefore the first codon, has the sequence No. 156, ranging from about nucleotide 1 to about nucleotide 3. The Genbank homology search found SEQ ID NO: 156 with Aedes Maximum homology between trypsin of Aedes aegypti Which results in approximately 53% homology between the corresponding regions of both nucleic acid molecules. is there.   B. Nucleic acid sequence of another flea serine protease nucleic acid molecule, nfSP25₈₆₄ Is represented herein by SEQ ID NO: 158. The translation of SEQ ID NO: 158 is Pf SP25₂₆₀And about 260 which shows the amino acid sequence SEQ ID NO: 159 Giving rise to a protein of amino acids whereby the first codon is SEQ ID NO: 158 It ranges from about nucleotide 2 to about nucleotide 4 and the stop codon is SEQ ID NO: : 159 extends from about nucleotide 782 to about nucleotide 784 . A homology search for Genebank revealed that SEQ ID NO: 159 and ano Chymotrypsis of Anopheles gambiae Reveals the greatest homology between the two protein sequences, which There is about 34% homology between the corresponding head regions of the acid sequences. Example 12   This example includes the cloning and cloning of another flea serine protease nucleic acid molecule. And sequencing has been described.   Flea serine protease cDNA nucleic acid molecules are derived from flea CDNA Expression Library (Related PCT Publication No. WO96 / 11706) Produced as described in Example 8) nfSP11₂₅₂(SEQ ID NO: 121) using the relevant pct publication no. Isolated by a method similar to that described in Example 8 of O96 / 11706. Was. The clones that were strongly crossed with the probe were isolated and Sambro Nuclei using the Sanger dideoxy chain termination method as described in Ok et al., supra. Subjected to acid screening.   Nucleic acid sequence of flea serine protease nucleic acid molecule, nfSP21₅₉₅Is Here, this is represented by SEQ ID NO: 160. The translation of SEQ ID NO: 160 is PfSP21₁₉₈ And about 198 amino acids that represent the amino acid sequence SEQ ID NO: 161 Resulting in the first codon having about the nucleotide sequence of SEQ ID NO: 160 Extending from nucleotide 2 to about nucleotide 4 and the putative stop codon is about Ranging from about 596 to about 598 nucleotides. Gene Bank (G Enbank) homology search was performed with SEQ ID NO: 161 and Tachypleus triden Tartus tridentatus aggregation factor G tamper Reveals the greatest homology between the two amino acid sequences, There is about 45% homology between the corresponding regions. Example 13   This example describes the isolation and characterization of a 31 kD serine protease. Have been.   Gastrointestinal tract obtained from about 1500 fleas fed cat blood for about 24 hours, Gut Digestion Buffer (Gut Detection Buffer) (50 mM Squirrel (8.0), 100 mM CaCl_Two). For digestive tract, freeze and thaw 4 Twice, followed by disruption by sonication. The resulting extract takes 20 minutes By centrifugation in a microfuge at 4 ° C. at 14000 rpm. Was made transparent. The supernatant was collected.   The digestive tract supernatant was separated with Sepharose beads (Sigma). ma)), and previously cross-linked with a benzamidine column buffer (Benzamidin). e Column Buffer) (50 mM Tris (8.0), 100 mM CaCl_Two 3 ml containing p-aminobenzamine equilibrated with 400 mM NaCl) Column. The supernatant was incubated on the column for about 10 minutes. Tampa The quality is determined by the Bradford Assay (Bio-Rad). Unbound protein is removed until undetectable by BioRad Benzamidine Column Buffer (Benzamidine Column Buffer) The column was slowly drained using r).   Then, the protease bound to the benzamidine column was added to 10 mM p-amid. 10 ml Benz supplemented with Novenzamidine (pH 8.0 with NaOH) Elution was performed using amidine column buffer. Protease in the eluent is concentrated Using a Microcon 3 concentrator (Amicon). . Filtered completely to a volume of 3 ml digestive tract cutting buffer.   About 120 μl of the concentrated eluent was further concentrated to a volume of about 30 μl. This concentrate Was contained in a 14% Tris-glycine electrophoresis gel (per lane). Gel electrophoresis on 15 μl (approximately 300 digestive tract equivalents per lane) Separated. After the electrophoresis, the separated protease was added to a CAPS buffer (10 mM Impregnate the PVDF membrane using CAPS (pH 11), 0.5 mM DTT) Was. The membrane is coated with Coomassie Brill. (L. Blue). Major protein band of about 31 kDa is possible Was visualized. Thereafter, the membrane was subjected to automatic N-terminal sequencing (related PCT publication no. (Produced as described in Example 7 of WO 96/11706). The partial N-terminal amino acid sequence of the flea protease was compared to IVGGEDVDIST. CGWC (shown as SEQ ID NO: 68). Example 14   This example includes IgG ace activity (ie, immunoglobulin G protein 31kD flea serine protein contained in a formulation having proteolytic activity The isolation and characterization of thease has been described.   A gastrointestinal extract of fleas fed with cat blood was used as described in Example 13. Manufactured and selected on a benzamidine column. Protein A Sepharose With feline immunoglobulin G protein (IgG) purified on Incubating the amidine eluate at 37 ° C. overnight resulted in IgG pro Thease activity was analyzed. Flea gastrointestinal benzamidine eluent replaces cat IgG The digesting activity was analyzed by gel electrophoresis through a 14% SDS-PAGE gel. The sample by silver staining the gel using standard methods. Was detected. On a silver stained gel, a 50 kDa bar representing cat IgG heavy chain Is that benzamidine elution contains IgG protease activity Indicates that   After that, 0.1M KPO_Four(PH 6.5) and 2M (NH_Four)_TwoSO_Fourincluding The benzamidine eluent is purified by applying the eluent to the column with buffer. On a propylaspartamide hydrophobic interaction chromatography (HIC) column Purified. Use a 2M to 0M ammonium sulfate gradient in HIC column buffer Then, the protease bound to the column was eluted. Using the method described above, Column fractions were tested for IgG protease activity. IgG protease activity The fractions containing the acid are pooled and polyCat in 20 M sodium acetate (pH 6). Loaded on a cation exchange column. 0M to 1M NaC in 20M sodium acetate The protein was eluted using a 1 g sodium chloride gradient. Eluted from the column The fractions tested were tested for IgG protease activity and each fraction was subjected to SDS -Separated by electrophoresis using PAGE. The highest level of IgG protea The two fractions exhibiting the protease activity were transferred to a tandem migrated to about 31 kDa on an SDS-PAGE gel. Included a parka band. Weak protease activity corresponds to an approximately 28 kDa band. Was.   Using the plot method described above, the 31k present on the SDS-PAGE gel Da protein was used for N-terminal amino acid sequencing. Partial N-end The terminal amino acid sequence was converted to IVGGEDVDIST (C) GWQI (S) FQ (S) E Determined as NLHF (C) GG (S) IIAPK (shown here as SEQ ID NO: 69) Specified. By comparison of SEQ ID NO: 69 with SEQ ID NO: 68 (described in Example 13) Is a single residue difference between the two amino acid sequences at residue 15 of each sequence (ie, , Q and C), respectively. SEQ ID NO: 69 is compatible with IgG ace activity. As correlated, the data indicate that the larval protein comprising SEQ ID NO: 68 It indicates that it shows G-ace activity. Example 15   This example includes a 31 kDa compound contained in a formulation with IgG ace activity. Cloning and sequencing of mi serine protease is described .   Flea protease nucleic acid molecule is fed to cat blood by PCR amplification Feed whole flea library (described in Example 6) and bovine blood Whole flea library (Example 8 of related PCT Publication No. 96/11706) ). The actual primers used for PCR amplification are SEQ ID NO: 68, including FP31A designed using the N-terminal amino acid sequence; The primer is a nucleic acid sequence 5′GAA used in combination with the M13 universal primer. GAT GTW GAT ATT TCW ACA TGT GG 3 ′ (SEQ ID NO: 1 01). The resulting PCR product was gel purified and the TA vector^TMNiku Loaned and subjected to nucleic acid sequencing as described above.   DNA sequence of DNA fragment obtained from cDNA library using bovine blood as feed Using the row, the FP31B primer (5 'GAA AAT GAA ATC CAC  TTA AAC ATT ACG 3 ′), (here represented as SEQ ID NO: 102) Was designed. Flea protease cDNA nucleic acid molecule is amplified by PCR amplification A whole flea library and cattle blood fed cat blood as described in It was isolated by PCR amplification of a whole flea library as feed. PCR amplification FP31B primer combined with M13 reverse primer. So After that, the resulting PCR product was diluted 1:25 and the sequence 5 'CTC TTA TTG TAC GAG GGA TGC 3 '(shown herein as SEQ ID NO: 103) ) With primer FP31C in combination with T3 primer of Used as a template for the PCR reaction. Nested (nest ed) PCR product is converted to TA vector^TMCloned into DNA sequence On the phone.   The nucleic acid sequence of the resulting flea serine protease nucleic acid molecule, ie, nfSP28₉₂₃ Is represented here as SEQ ID NO: 66. The translation of SEQ ID NO: 66 is Pf SP28₂₆₇267 having the amino acid sequence SEQ ID NO: 67 Producing a protein of amino acids whereby the putative start codon is From about nucleotide 8 to about nucleotide 10, or from about nucleotide 11 to about nucleotide It spans nucleotide 13 and has a stop codon at about the nucleotide position of SEQ ID NO: 66. Presents an open reading frame extending from nucleotide 803 to about nucleotide 805. SEQ ID NO: 6 7 is SEQ ID NO: 68 except that Q is used instead of C, and SEQ ID NO: 6 9 inclusive. Genebank homology search yielded SEQ ID NO: 66 And Bombyx mori Vitalin Degradation Protein Reveals the greatest homology between the two genes, which allows the corresponding There is about 53% homology between the regions. Example 16   This example includes larval serine proteases.^ThreeH-DFP label is described You.   About 100 unbred larvae, 100 first instar larvae, and 100 third instar larvae, 100 μl of digestion tract cutting buffer (Gut Dissection Buffer) ) (50 mM Tris (8.0), 100 mM CaCl_Two). About 400μl Of water was added to the collected larvae, which were then sonicated. The sonicated product was 15000 By centrifugation at 4 ° C. for 30 minutes in an SS-34 rotor at RPM. And made it transparent. The supernatant from each larval sonicate is collected and about 120 μl (1.2 μl per larval equivalent).   A sample containing about 25 larval equivalents (about 30 μl) was prepared by adding about 2.5 μCi of 3H Isopropyl fluorophosphate (DFP; New England Nuclear (Available from New England Nuclear), and Incubated at 4 ° C for 18 hours. Following the incubation period, each larva The 5 larvae equivalents of the steps (about 6 μl) were transferred to a 14% Trisglycine SDS-PAGE gel. To The gel was then used for tritium signal for autoradiography. Enhancement (New England) Immerse in Nuclear (available from New England Nuclear) And dried. After that, the dried gel was subjected to X-ray (Kodak XO-mat).   The results indicate that gastrointestinal tract extracts from unbred larvae were The first-instar larvae reared (FIG. 1, lane A) without thease (FIG. 1, lane A) Both lane B) and the reared third instar larvae (FIG. 1, lane C) are serine pro It shows that it produces thease. In particular, the reared first instar larva is approximately 25 kD Third-instar pups that initially produced a serine protease with a molecular weight and were bred Raw is approximately serine with molecular weights of about 25 kD, 28 kD and 31 kD. Produces Rotase. The approximate size of a standard molecular weight protein marker is As shown in FIG. Example 17   In this example, the partial N-terminal access for several larval serine proteases Methods for determining the amino acid sequence are described.   About 10300 third instar larvae were digested with a digestive tract cutting buffer (50 mM Tris, pH 8). . 0), 100 mM CaCl_Two). Larvae are homogenized by sonication And centrifuged at 15000 rpm for 30 minutes at 4 ° C. in an SS-34 rotor. Separated and clarified. The supernatant was collected. Transfer the third instar supernatant to a benzamidine column Buffer (50 mM Tris (pH 8.0), 100 mM CaCl_Two, 400 mM N Sepharose beads (available from Sigma) equilibrated with aCl) ) Was mixed with 5 ml of p-aminobenzamidine crosslinked. Supernatant at 4 ° C overnight And shook with the beads. Wash beads in approximately 45 ml benzamidine column buffer Purification removed unbound proteins. Thereafter, the beads were placed at 4 ° C. for 2 hours. 100 mM p-aminobenzamidine (pH 8.0 adjusted with NaOH) Including Mixed with about 10 ml of benzamidine column buffer and bound to the beads The protein was eluted. Thereafter, the eluted proteins were collected. The elution step And repeated it again. The eluted protein was centrifuged by Centriprep 10 Concentrator (Amico (Available from Amicon) by ultrafiltration. Digest concentrate Diluted with tube cutting buffer to a final volume of about 5 ml.   Example 1 shows the partial N-terminal amino acid sequence of the protein eluted from the beads. Obtained using the method described in 1. Shows molecular weight of about 25 kDa and about 26 kDa Coomassie Brilliant Blue (Coomassie B) illiant Blue) stained membrane. Has a molecular weight of about 25 kDa The partial N-terminal amino acid sequence obtained as a protein is IVGGVSVNIND YGYQLSLQSNGR, shown here as SEQ ID NO: 162. about Partial N-terminal amino acid sequence obtained as a protein having a molecular weight of 26 kDa Is IVGGHDTSIKQHPYQV, wherein SEQ ID NO: 163 Is shown. Example 18   This example includes the production of certain flea serine protease proteins of the present invention. The synthesis method is exemplified.   A. Flea serine protease protein PfSP1₂₄₆In the following way Was. In a related PCT Publication No. WO 96/11706, described in Example 20. Flea serine protease nucleic acid molecule nfSP1 produced as described above₆₇₀To X digested with hol restriction endonuclease, gel purified, and digested with Xhol. Expression vector λP_R/ T^Twoori / S10HIS-RSET-A9 (1995 Et al., PCT Publication No. WO 95/2419, published September 14, 1998. No. 8, produced subcloned specifically in Example 7), and Decomposed by phosphoric acid. Here, pHisCro-nfSP1₆₇₀Arose as shown Recombinant molecules are available from E.I. E. coli HB101 competent cells (jib Transformed into GOBRL (available from Gibco BRL) Transformed cells e. Coli: pHisCro-nfSP1₆₇₀Was formed. Recombinant The cells were described in Example 20 of Related PCT Publication No. WO 96/11706. The culture was performed as follows. Flea serine protease protein PfSP1₂₁₆Ni Kelchelation chromatography followed by reversed-phase high-performance liquid chromatography (HPLC) for purification. Purified PfSP1₂₁₆Immunoblot analysis of Rabbit anti-flea protease anti-produced as described in Example 14. Serum selectively PfSP1₂₁₆Was shown to be bound.   B. Flea serine protease protein PfSP2₂₃₃By the following method Was generated. Related to PCT Publication No. WO 96/11706, Example 20 Flea serine protease nucleic acid molecule nfSP2 produced as described_{71 Five} Was digested with XhoI restriction endonuclease, gel purified and purified as in Example 39A. Expression vector λP as described_R/ T^Twoori / S10HIS-RSET- Subcloned into A9. Here, pHisCro-nfSP2₇₁₅Called The resulting recombinant molecule is designated E. coli HB101 competent cells (Jibgo Via (Available from Gibco BRL) to obtain the recombinant cells. -. Coli: pHisCro-nfSP2₇₁₅Was formed. Recombinant cells Culture as described in Example 20 of PCT Publication No. WO 96/11706 did. Flea serine protease protein PfSP2₂₃₃The nickel chelate Purification by chromatography followed by reverse phase HPLC. Purified Pf SP2₂₃₃Immunoblot analysis of PCT publication No. WO 96/1170 Rabbit anti-flea protein produced as described in Example 14 of No. 6. Ze antiserum selectively binds to PfSP2₂₃₃Was shown to be bound.   C. Flea serine protease protein PfSP13₂₂₅To the following method Thus generated. Example 20 of Related PCT Publication No. WO 96/11706 Flea serine protease nucleic acid molecule nfSP1 produced as described in 3₇₀₀Was digested with XhoI restriction endonuclease, gel purified, and Expression vector λP as described in A._R/ T^Twoori / S10HIS-RSE It was subcloned into T-A9. Where pHisCro-nfSP13₇₀₀age The resulting recombinant molecule represented by E. coli HB101 competent cells (jib (Available from Gibco BRL), Recombinant cells e. Coli: pHisCro-nfSP13₇₀₀Was formed. Recombinant Cells were described in Example 20 of Related PCT Publication No. WO 96/11706. Cultured as Flea serine protease protein PfSP13₂₂₅To Purify by nickel chelation chromatography followed by reverse phase HPLC. Made. Purified PfSP13₂₂₅Immunoblotting analysis of the relevant PCT publication no. Rabbits produced as described in Example 14 of WO 96/11706 Anti-flea protease antiserum selectively binds PfSP13₂₂₅Indicates that the did.   D. Flea serine protease protein PfSP20₂₂₂To the following method Thus generated. Where nfSP20₆₆₉As shown and clearly matured About 669-bp DN designed to encode a protease protein The A fragment was prepared using the XhoI site-containing primer F27-S (sense) 5′GAG CTC.  TCG AGA ATC GTA GGA GGA CAC GAT AT 3 '(sequence No .: 164) and EcoRI site containing primer F20-A (antisense ) 5'G GAC GAA TTC TTA AAC ACC AGA CAC TTC Using CTT G 3 '(SEQ ID NO: 165), the flea serine protease PCR amplification was carried out from run 20. PCR product nfSP20₆₆₉With XhoI and Digested with EcoRI restriction endonuclease, gel purified, and treated with Example 18A Expression vector λP as described in_R/ T^Twoori / S10HIS-RSET -Subcloned into A9. Where pHisCro-nfSP20₆₉₉As The resulting recombinant molecule shown is designated E.I. E. coli HB101 competent cells (Dibgo Transformation into recombinant (available from Gibco BRL) The cells were described in Example 20 of Related PCT Publication No. WO 96/11706. Cultured as described. Recombinant PHISCRO-PfSP20₂₂₂Fusion protein T7-tagged monoclonal antibody directed against the quality fusion moiety (Novagen ( Novagen, Inc.). Kori: pHisCro-nfSP20₆₉₉For immunoblot analysis of lysates, And a protein of about 31-kD was identified. Flea pickling Protease protein PfSP20₂₂₂The nickel chelated chroma Purified by chromatography followed by reverse phase HPLC. Purified PfSP20₂₂₂ Immunoblot analysis of the relevant PCT Publication No. WO 96/11706. Rabbit anti-flea protease anti-blood produced as described in Example 14 Kiyoshi selectively PfSP20₂₂₂Was shown to be bound. Example 19   This example includes the various flea serine proteases described in the previous examples. It is described that nucleic acid molecules can be obtained from multiple sources.   The nucleic acid molecule corresponding to flea clone 4 is the total flea lactose fed on bovine blood. Library (described in Example 8 of PCT Publication No. WO 96/11706). ), A library of whole fleas fed on cat blood (described in Example 5). ), A library of all unfed fleas (Related PCT Publication No. WO 96/1170) No. 6, Example 8) and a library of mixed-age whole fleas (Example 9). The nucleic acid molecule corresponding to flea clone 5 is bovine Library of all fleas feeding on cat blood and all fleas feeding on cat blood From the library. The nucleic acid molecule corresponding to flea clone 6 is bovine A library of all fleas that feed on blood, and a library of all fleas that feed on cat blood. Obtained from a library of whole and unbred fleas. Flea clone The nucleic acid molecule corresponding to No. 7 is a library of all fleas fed from bovine blood, and And obtained from a library of all fleas fed cat and blood. Flea clone The nucleic acid molecule corresponding to No. 8 is a library of all fleas fed from bovine blood, and And from unobtained libraries of all fleas. Corresponds to flea clone 12 Nucleic acid molecules can be obtained from a whole flea library fed from bovine blood, and from cat blood. Was obtained from a library of all fleas fed. Corresponds to flea clone 13 Nucleic acid molecules can be obtained from cat fetal blood, a library of whole fleas fed from bovine blood. From the library of all fleas used as feed and the unfed Was. The nucleic acid molecule corresponding to Flea Clone 20 is derived from bovine blood feed. A library of fleas, a library of all fleas fed on cat blood, and Obtained from a library of all grown fleas. Nucleus corresponding to flea clone 28 Acid molecules can be found in whole flea libraries feeding on bovine blood and in cat blood. Obtained from a library of all flea feeds. Example 20   This example demonstrates the flea cysteine protease of the present invention described in Example 3. Provides additional nucleic acid and deduced amino acid sequences for nucleic acid molecules encoding proteins I do. This embodiment is described in e. Cysteine protease protein in E. coli cells It also provides for the production of   A. Additional cysteine protease nucleic acid molecules   Obtaining the nucleic acid sequence of another part of the coding region of the cysteine protease gene To this end, the PCR product described in Example 3 was subjected to another nucleic acid sequence analysis. This Where nfCP1₁₁₀₉Of the flea cysteine protease shown as The complex nucleic acid sequence representing the coding region is deduced and shown herein as SEQ ID NO: 1. . SEQ ID NO: 76 is a nucleic acid molecule, nfCP1₁₁₀₉Included in the array. SEQ ID NO: 1 translates into the nucleic acid molecule nfSP1₁₁₀₉But here, PfCP1₃₂₇Shown as The full-length flea system of about 327 amino acids shown in SEQ ID NO: 2 of the amino acid sequence Encoding an inprotease protein, thereby indicating an initiation codon Extends from about nucleotide 126 to about nucleotide 128 of SEQ ID NO: 1, and And the stop codon is from about nucleotide 1107 to about nucleotide 11 of SEQ ID NO: 1. 09 open reading frames. The complement of SEQ ID NO: 1 is the SEQ ID NO: Shown as 3. PfCP1₃₂₇Is SEQ ID NO: 4. Having the nucleic acid sequence represented by and the coding strand and the nucleic acid sequence having SEQ ID NO: 6 Nucleic acid molecule nfCP1 having a complementary chain₉₈₄It is expressed as PfCP1₂₂₆As shown here The designated mature protein is identified as about 22 represented herein as SEQ ID NO: 8. Contains 6 amino acids. PfCP1₂₂₆The nucleic acid molecule encoding_{6 81} And represented by SEQ ID NO: 7. PfCP1₃₂₇Amino acid sequence of That is, SEQ ID NO: 2) is PfCP1₃₂₇Has an approximate molecular weight of about 42 kD and about It is estimated to show an estimated pI of pI8.84.   SEQ ID NO: 1 of the nucleic acid sequence is the nucleic acid reported in Genbank By comparison with the sequence, SEQ ID NO: 1 has three genes: Drosophila ( Drosophila cysteine protease gene, Bombix (Bo) mbyx) and the cysteine protease gene and Sarcophaga (Sarcop haga), which has the greatest homology, ie, about 55 It is indicated to show% homology. SEQ ID NO: 2 of the amino acid sequence (ie, P fCP1₃₂₇Amino acid sequence) was reported in Genebank. By comparison with the amino acid sequence shown, SEQ ID NO: 2 has the following three proteins: A Drosophila cysteine protease protein, Bombyx cysteine protease proteins and monkeys Sarcophaga cysteine protease protein It is indicated to show a high degree of homology, ie about 42% homology.   B. E. Production of cysteine protease protein in E. coli cells   Where nfCP1₆₆₀(Apparently mature cysteine protease protein About 660-bp nucleic acid molecule, designated as First age of breeding, first age using bovine blood, second age using bovine blood And third-instar flea larvae fed on bovine blood (this combination of tissues (Shown here as mixed-age larvae tissue of interest in the Examples). PCR amplification was performed from a mixed-age cDNA library. Total RNA is Chom czynski et al., Anal. Biochem. (Anal. Biochem.), 16 2, 156-159, 1987. Extracted from mixed-aged tissues using the nidinium-phenol-chloroform method . Approximately 5,164 mixed-age larvae were used for each RNA production. Ask the manufacturer According to the recommended method, Poly (A) Quick (R) (Poly (A) Q uCA) Stratagene Cloning Systems (Stratagene) Oligo-dT cells using Cloning Systems) By coarse chromatography, poly A + selected RNA was converted to each total RNA standard Separated from Using the Stratagene ZAP-cDNA synthesis kit protocol Lambda (λ) Uni-ZAP^TMXR vector (Stratagene Kroni From Stratagene Cloning Systems (Available from Co., Ltd.) to construct a mixed-age cDNA expression library. About 6.34μ g mixed-age poly A + RNA was used to generate a mixed-age library. Raw Library of about 2.17 × 10 7 with about 97% recombinants^Tenp Amplified to a force value of fu / ml. Primers used for PCR amplification were 5 'GAT AAG GAT CCG TTA CCA GAT TCT TTC GAC TGG 3 '(including BamHI site, shown as SEQ ID NO: 64) Sense primer CysBS 'and nucleotide sequence 5'TTA TCA AGC TTC CAT TTA CAT GCC GTA AAA ATC 3 '(Hi (shown as SEQ ID NO: 65, including an ndIII site) It was Lymer CysHA. Resulting PCR product nfCP₆₆₈For nucleic acid sequence analysis To obtain the nucleic acid sequence of the coding strand represented here as SEQ ID NO: 94. Was. The translation of SEQ ID NO: 94 is nfCP1₆₉₆But PfCP1₂₂₀Called an array It was shown to encode a protein of about 220 amino acids having the number: 95. This sequence analysis indicates that the stop codon is downstream from that predicted by SEQ ID NO: 1. Indicating that it was actually about 36 base pairs; alone, nfCP1₆₆₈To Thus, the encoded protein is about about less than that predicted by SEQ ID NO: 1. Note that it is 12 amino acids shorter. Nucleic acid molecule nfCP1₆₆₀Is PfC P1₂₂₀Including the coding region.   Recombinant cells e. Coli: pCro-nfCP1₆₆₀Was produced by the following method. . Nucleic acid molecule nfCP1₆₆₀With BamHI and HindIII restriction endonuclei Digested with gelase, gel purified, and expressed vector Lambda P_R/ T^Twoori / S 10HIS-RSET-A9 (Tripp et al., Published September 14, 1995). Generation as described in International PCT Publication No. WO 95/24198; And subcloned it into BamHI and HindII. Digested with I and dephosphorylated. pCro-nfCP1₆₆₀Occurrences shown as The recombinant molecule obtained is E. coli BL-21 competent cells (Wisconsin, Inc.) and transfected with recombinant cells. Coli: pCro- nfCP1₆₆₀Was formed. Related to PCT Publication No. WO 95/24198 Real Recombinant cells were cultured as described in Example 20. Before introduction, about 1 ml medium The nourishment was collected and about 1 ml of culture was collected with less than about 60 minutes of introduction. So After that, the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE) Dissolve in loading buffer and add 14% Tris-glycine Dissolved in acrylamide gel and T7 (tag) antibody (Novag en)) and analyzed by immunoblotting. Example 21   This embodiment is described here and in related PCT Publication No. WO 96/11706. Encoding the serine protease proteins of the invention described in the Examples section of 3 provides another nucleic acid and deduced amino acid sequence of the nucleic acid molecule.   A. nfAP2₂₁₀₀Including nucleotides obtained from³²DNA labeled with P (Related US patent application Ser. No. 08/63, filed Apr. 24, 1996). 9,075) (described in Example 23). CDNA library of all fleas fed from bovine blood using (Described in Example 8 of PCT Publication No. WO 96/11706). Learning. Here nfSP18₄₅₉About 459-nucleotides shown as Clones with the insert were isolated.   nfSP18₅₃₄And nfSP18₄₅₉Manufactured using nucleic acid sequences obtained from The nucleic acid sequence of the complex nucleic acid molecule is referred to herein as nfSP18.₇₇₆Shown here and distributed here It has a nucleic acid sequence having the nucleic acid sequence of the coding strand shown as SEQ ID NO: 9. Arrangement Translation of SEQ ID NO: 9 translates into the nucleic acid molecule nfSP18₇₇₅But here is PfSP18₂₂₈age And about 228 amino acids of amino acid sequence SEQ ID NO: 10 Suggests that it encodes a long flea cysteine protease protein, Thus, the first codon is from about nucleotide 1 to about nucleotide 3 of SEQ ID NO: 9. And the stop codon is from about nucleotide 685 to about nucleotide of SEQ ID NO: 9. 687. The complement of SEQ ID NO: 9 is designated as SEQ ID NO: 11. It is represented here. PfSP18 coding coding region₂₂₈Is the nucleic acid molecule n fSP18₂₂₈Nucleic acid sequence represented by SEQ ID NO: 12 And a complementary strand having the nucleic acid sequence SEQ ID NO: 14. I do. PfSP18₂₂₈Has the amino acid sequence of SEQ ID NO: 10 (ie, SEQ ID NO: 10) SP18₂₂₈Shows an approximate molecular weight of about 25 kD and an estimated pI of about 9.09. Is estimated.   The nucleic acid sequence SEQ ID NO: 9 is the nucleic acid reported in Genbank By comparison with the sequence, SEQ ID NO: 9 shows that Anopheles stephensi (A nophases trypsin 1 gene It is shown to be homologous, ie, about 51% homologous. Sequence number of amino acid sequence No .: 10 (that is, PfSP18₂₂₆The amino acid sequence of by comparing with the amino acid sequence reported in 0 is SEQ ID NO: 10 and Vespa crabro tampa It is shown that they showed the greatest homology between the proteins, ie, about 59% homology.   B. Rest of flea serine protease nucleic acid molecule clone 24 (described in Example 6) From a cDNA library of all fleas fed from bovine blood. NfSP24 that amplifies₄₁₀Were determined using primers designed from. In the first PCR reaction, the nucleotide sequence 5′GGA CAA ACT GTT CA With a sense primer having T TGC AG 3 '(shown as SEQ ID NO: 46) One flea 24F was used in combination with the M13 universal primer. Second PCR In the reaction, the nucleotide sequence 5 'CCC TCA TTT GTC GTA ACT C Flea 2 which is an antisense primer having C 3 '(shown as SEQ ID NO: 47) 4R was used in combination with the M13 reverse primer. The resulting PCR product is Each gel was purified and cloned into the TA Vector® system. And subjected to standard DNA sequencing techniques.   A composite nucleic acid sequence representing the flea serine protease coding region was identified. And nfSP1₁₀₈₉As estimated here, and as SEQ ID NO: 15. It is shown here. SEQ ID NO: 78 is a nucleic acid molecule, nfSP1₁₀₈₉Contained in an array of . Translation of SEQ ID NO: 15 translates into the nucleic acid molecule nfSP24₁₀₈₉But here, PfSP24₂₅₈ And about 258 amino acids, which is shown in SEQ ID NO: 16 of the amino acid sequence. Suggests that it encodes a full-length flea serine protease protein. Thereby, the initiation codon is from about nucleotide 33 to about nucleotide of SEQ ID NO: 15. And the stop codon is from about nucleotide 807 of SEQ ID NO: 15. It exhibits an open reading frame spanning about nucleotide 809. The complement of SEQ ID NO: 15 is , Here shown as SEQ ID NO: 17. PfSP24₂₅₈Code to code Region is the nucleic acid molecule nfSP24₇₇₄And represented by SEQ ID NO: 18 Coding strand having a nucleic acid sequence and a phase having the nucleic acid sequence SEQ ID NO: 20 It has a complementary chain. PfSP24₂₃₇Nominated mature protein shown here as The quality includes about 237 amino acids, represented herein as SEQ ID NO: 22. PfSP 24₂₃₇The nucleic acid molecule encoding₇₁₁And SEQ ID NO: 21. PfSP24₂₅₈Amino acids (ie, SEQ ID NO: 16) ) Is PfSP24₂₅₈Has an estimated molecular weight of about 28 kD and an estimated pI of 6.70 It is estimated to show pi.   The nucleic acid sequence SEQ ID NO: 15 is the nucleus reported in Genbank By comparing with the acid sequence, SEQ ID NO: 15 is different from SEQ ID NO: 15 by Anofere Trypsin 1 from Anopheles stephensi It is shown that they showed the greatest homology between the genes, ie, about 51% homology. A SEQ ID NO: 16 of the amino acid sequence (ie, PfSP24₂₅₈Amino acid sequence) By comparing to the nucleic acid sequences reported in Genbank , SEQ ID NO: 16, SEQ ID NO: 16 and Anopheles gumbiae (Anoph) maximal homology between the chymotrypsin II proteins of C.eles gambiae) , Ie, about 59% homology.   C. Remaining flea serine protease nucleic acid molecule clone 32 (described in Example 20) DNA) from a cDNA library of all fleas fed cat blood NfSP32 that amplifies A₄₃₃Determined using primers designed from . In the first PCR reaction, the nucleotide sequence 5′GGC TAG GTT AGT G Sense primer indicating GA TTC TGG 3 '(shown as SEQ ID NO: 48) Was used in combination with the M13 universal primer. Second PC In the R reaction, the nucleotide sequence 5 ′ GCA AAT CAG TTC CAG AAT Anti-sense ply showing CCA CTA ACC 3 '(shown as SEQ ID NO: 49) The flea 32R, a mer, was used in combination with the M13 reverse primer. The resulting P Each CR product is gel purified and applied to the TA Vector® system Cloned and subjected to standard DNA sequencing techniques.   A composite nucleic acid sequence representing the flea serine protease coding region was identified. And nfSP32₉₂₄As estimated here, and as SEQ ID NO: 23. Shown here. SEQ ID NO: 80 is a nucleic acid molecule, nfSP32₉₂₄Included in the array I will. Translation of SEQ ID NO: 23 translates into the nucleic acid molecule nfSP32.₉₂But here is PfSP 32₂₆₈About 268 having the amino acid sequence SEQ ID NO: 24 Full length amino acid suggests encoding a serine protease protein , Whereby the start codon is from about nucleotide 6 to about nucleotide of SEQ ID NO: 23. It spans tide 8 and the stop codon is from about nucleotide 810 of SEQ ID NO: 23. It exhibits an open reading frame spanning about nucleotide 812. The complement of SEQ ID NO: 23 is , Here shown as SEQ ID NO: 25. PfSP32₂₆₈Code to code The carrying region contains the nucleic acid molecule nfSP32₆₉₉And represented by SEQ ID NO: 26. Coding sequence having the nucleic acid sequence to be sequenced, and nucleic acid sequence SEQ ID NO: 28 Having a complementary strand. PfSP32₂₆₈Amino acid sequence (ie, SEQ ID NO: 2) 4) PfSP32₂₆₈Has an estimated molecular weight of about 28.6 kD and about pI 7.36. Is estimated.   The nucleic acid sequence SEQ ID NO: 23 is the nucleus reported in Genbank By comparing with the acid sequence, SEQ ID NO: 23 is different from SEQ ID NO: 23 by Fusarium. ・ Preprotripp of Fusarium oxysporum It is shown to show the greatest homology between the syn genes, ie, about 52% homology . Amino acid sequence SEQ ID NO: 24 (ie, PfSP32₂₆₈Amino acid sequence of ) Is compared to the amino acid sequence reported in Genbank. And SEQ ID NO: 24, SEQ ID NO: 24 and Bombix Mori (Bomb yx mori), the largest among the vataline-degrading protease precursor proteins It is shown to show homology, ie about 63% homology.   D. The remainder of the flea serine protease nucleic acid molecule clone 33 was used in Example 20. Amplify DNA from a mixed-age flea cDNA library described above nfSP33₇₇₈Were determined using primers designed from. First PC In the R reaction, the nucleotide sequence 5′CAG GGC GCT CTG CAG AAC A sense primer having GCA AC 3 '(shown as SEQ ID NO: 50) Flea 33F was used in combination with the M13 universal primer. Second PCR reaction In response, the nucleotide sequence 5 'ATT CCT CGT GGT TCA GTC GC An antisense primer having TC 3 ′ (shown as SEQ ID NO: 51) Mi32R was used in combination with the M13 reverse primer. The resulting PCR product is Each gel was purified and cloned into the TA Vector® system And subjected to standard DNA sequencing techniques.   A composite nucleic acid sequence representing the flea serine protease coding region was identified. And nfSP33₁₈₉₄Examined and estimated here, and as SEQ ID NO: 29 Shown here. SEQ ID NO: 84 and SEQ ID NO: 82 represent the nucleic acid molecule nfSP3 3₁₈₉₄Included in the array. The translation of SEQ ID NO: 29 translates into the nucleic acid molecule nfSP33_{18 94} But here is PfSP33₄₀₀And SEQ ID NO: 3 of the amino acid sequence Encodes a full-length flea serine protease protein of about 400 amino acids that represents 0 And the initiation codon is therefore approximately the nucleotide of SEQ ID NO: 29. 335 to about nucleotide 337, and the stop codon is SEQ ID NO: 29. Exhibits an open reading frame ranging from about nucleotide 1535 to about nucleotide 1537 You. The complement of SEQ ID NO: 29 is designated herein as SEQ ID NO: 31. PfSP3 3₄₀₀Coding region is the nucleic acid molecule nfSP33₁₂₀₀Is expressed as And a coding strand having the nucleic acid sequence represented by SEQ ID NO: 32, and the nucleic acid It has a complementary strand having the sequence SEQ ID NO: 34. PfSP33₂₄₂As here The designated mature protein shown is about the amino acid sequence represented herein as SEQ ID NO: 36. Contains 242 amino acids. PfSP33₂₄₂The nucleic acid molecule encoding SEQ ID NO: Where nfSP33 represented by 35₇₂₆As shown. PfSP33₄₀₀ Has the amino acid sequence of PfSP33 (ie, SEQ ID NO: 30).₄₀₀Is about 44kD And an estimated pi of about pI 7.59.   The nucleic acid sequence SEQ ID NO: 29 is the nucleus reported in Genbank By comparing with the acid sequence, SEQ ID NO: 29 was compared with SEQ ID NO: 29 by Drosophila. La Melanogaster (Drosophila melanogaster) Maximum homology with the phosphoprotease strain gene, about 48% homology What is shown is shown. Amino acid sequence SEQ ID NO: 30 (ie, PfSP3 3₄₀₀Amino acid sequence) was converted to the amino acid sequence reported in Genbank. By comparison with the amino acid sequence, SEQ ID NO: 30 was compared to SEQ ID NO: 30 by Drosov. Of Drosophila melanogaster Maximum homology between the serine protease strain proteins, ie, about 63% phase It is shown that the same was shown. Example 22   This example demonstrates another nucleic acid encoding a serine protease protein of the invention. 1 provides the nucleic acid and deduced amino acid sequence of the molecule.   The serine protease cDNA nucleic acid molecule is replaced with the relevant PCT Publication No. WO96 / Isolated in a manner similar to that described in Example 8 of US Pat. Cat blood Fluid cDNA Flea cDNA Expression Library (Related PCT Publication No. WO 96/11706), prepared as described in Example 8 of Ser. The actual primers used in the PCR amplification of thease nucleic acid molecule were H57 primers. -(SEQ ID NO: 99) in combination with Cat-Try No. 2 (SEQ ID NO: 86) I do. The resulting PCR product was gel purified and the TA vector^TMCloned into I let you. A clone of the recombinant TA vector was isolated and subjected to nucleic acid sequencing .   Flea clone 40, nfSP40₄₂₈Flea protea corresponding to The complex nucleic acid sequence of the zea nucleic acid molecule has been deduced, and Show. Translation of SEQ ID NO: 37 translates into the nucleic acid molecule nfSP40₄₂₈But here is PfSP 40₁₄₂And a non-full length of about 142 amino acids represented by SEQ ID NO: 38 Suggests encoding a flea serine protease protein. SEQ ID NO: The complement of 37 is shown herein as SEQ ID NO: 39.   Feed the rest of flea serine protease nucleic acid molecule clone 40 and cat blood NfSP40 to amplify DNA from a cDNA library of all fleas₄₂₆Or Were determined using the primers designed by the company. In the first PCR reaction, Peptide sequence 5'GGC AAG TTT CGT TTC ACA ATA GG 3 ' Flea 40F, which is a sense primer having Used in combination with 13 universal primers. In the second PCR reaction, the nucleotide sequence Row 5 'TCC AAC CCT AAC TTT TAA ACC TTC 3' (sequence number No. 53), which is an antisense primer with M13 Used in combination with retrograde primer. Each of the resulting PCR products is And subjected to standard DNA sequencing techniques.   A composite nucleic acid sequence representing the flea serine protease coding region was identified. And nfSP40₈₄₁Investigated here and estimated, and as SEQ ID NO: 40 Shown here. SEQ ID NO: 37 is a nucleic acid molecule, nfSP40₈₄₁Contained in an array of It is. Translation of SEQ ID NO: 40 translates into the nucleic acid molecule nfSP40₈₄₁But here is PfSP 40₂₄₂And about 242 amino acids which are shown in SEQ ID NO: 41 of the amino acid sequence Suggests that it encodes a non-full-length acid flea serine protease protein. Thereby, the first codon is from about nucleotide 2 to about nucleotide of SEQ ID NO: 40. The stop codon extends from about nucleotide 728 to about nucleotide 728 of SEQ ID NO: 40. It exhibits an open reading frame spanning nucleotide 730. The complement of SEQ ID NO: 40 is Here, SEQ ID NO: 42 is shown. PfSP40₂₄₂Coding to code The region is the nucleic acid molecule nfSP40₇₁₇And represented by SEQ ID NO: 43 Coding strand having the nucleic acid sequence and complement having the nucleic acid sequence SEQ ID NO: 45 With chains. PtSP40₂₄₂(Ie, SEQ ID NO: 41) is Pf SP40₂₄₂Shows an estimated molecular weight of about 26 kD and an estimated pI of about pI 6.5. Is estimated.   The nucleic acid sequence SEQ ID NO: 40 is the nucleus reported in Genbank By comparing with the acid sequence, SEQ ID NO: 40 is different from SEQ ID NO: 40 by Dermatov Agoides pteronisinus (Dermatophagoides ptero) nyssinun), the greatest homology between the Der P3 allergen genes, That is, it was shown that about 57% homology was exhibited. SEQ ID NO: 41 of amino acid sequence That is, PfSP40₂₄₂The amino acid sequence of Genbank ) Was compared to the amino acid sequence reported in No .: 41 and Bombyx mori's Vitalin degradation Shows the greatest homology between the Rotase precursor proteins, ie, about 40% homology Is shown. Example 23   This embodiment is described in e. E. coli serine protease protein of the invention Illustrate production.   A. Flea serine protease protein PfSP24₂₅₈In the following way Produced. Where nfSP24₇₁₄About 714 bp nucleic acid molecule shown as Designed to encode a more mature serine protease protein) With the nucleotide sequence 5 ′ CAC AGG ATC CAA TAA TTT GTG GTC AAA ATG C 3 '(including BamHI site; shown as SEQ ID NO: 54) Flea 24EF, a sense primer having the nucleotide sequence 5 ′ AAA AAG AAA GCT TCT TTA ATT TTC TGA CAT T GT CGT G 3 '(including HindIII site; shown as SEQ ID NO: 55) NfSP24 using flea 24ER, an antisense primer indicating₁₀₈₉From PCR amplified. The resulting PCR product nfSP24₇₁₄With BamHI and H digested with indIII restriction endonuclease, gel purified, and digested with BamHI And Lambda P digested with HindIII and dephosphorylated_R / T^Twoori / S10HIS-RSET-A9. pCr o-nfSP24₇₁₄The resulting recombinant molecule, designated as Coli BL-2 One competent cell (formerly Madison, Wis.) )) (Available from Novagen, Inc.) Recombinant cells e. Coli: pCro-nfSP24₇₁₄Was formed. Related PCT Recombinant cells as described in Example 20 of Publication No. WO 95/24198 Was cultured. Prior to introduction, about 1 ml of culture is collected and no more than about 60 minutes. Approximately 1 ml of culture was collected at the time of transfer. Thereafter, the sample was subjected to sodium dodecyl sulfate. Polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer And a 14% Tris-glycine acrylamide gel . Using a T7 (tag) antibody (available from Novagen) Immunoblot analysis of the protein showed that the transfected sample expressed about 36 kD protein. But not in the unintroduced sample.   B. Flea serine protease protein PfSP32₂₆₈In the following way Generated. Where nfSP32₆₉₈(Apparently mature serine protease tan About 698-bp nucleic acid molecule (designed to encode protein) With the nucleotide sequence 5 ′ GCG GGA TCC TAT TGT GGG TGG TGA AGC AGT 3 '(including BamHI site; set forth as SEQ ID NO: 56) ) Which is a sense primer having the nucleotide sequence 5′GA C GGT ACC ATG TAT AAA ATA ATA ATA TTA AAC TCC  GG 3 ′ (including KpnI; shown as SEQ ID NO: 57) NfSP32 using flea 32ER which is an immersion₉₃₃Was amplified by PCR. The resulting PCR product nfSP32₆₉₈With BamHI and HindIII restriction Digest with nuclease, gel purify, and quench with BamHI and KpnI. And dephosphorylated expression vector 1 pTrcHisB (California InVigen Inc. of San Diego, Calif. trogen Corp.)). pTrc- nfSP32₆₉₈The resulting recombinant molecule, designated as Coli BL-21 The transfected cells were transduced into recombinant cells. Coli: pTrc-nfSP3 2₆₉₈Was formed. The recombinant cells are cultured, and the protein product is sectioned. Separation by SDS-PAGE as described above in Example A. T7 (tag ) Immunoflot analysis of proteins using antibodies requires approximately 38 kD Protein expression was shown, but not in untransfected samples.   C. Flea serine protease protein PfSP33₄₀₀In the following way Produced. Where nfSP33₁₂₀₀(Apparently mature serine protease Approximately 1200 bp nucleic acid molecule (designed to encode protein) , Nucleotide sequence 5 'CCG GGA TCC TAT GTT AGC GAT CGT CCC GTC AAAC 3 '(including BamHI site; SEQ ID NO: 58) Flea 33EF, which is a sense primer having Row 5 'CCG GAA TTC TTA TCC CAT TAC TTT GTC GA Anti-sense having T CC 3 '(including EcoRI; shown as SEQ ID NO: 59) NfSP33 using the sprimer flea 33ER₁₈₉₄PCR amplified from I let you. The resulting PCR product nfSP33₁₂₀₀With BamHI and EcoRI restriction Digested with endonuclease, gel purified, and BamHI and EcoRI Expression vector lambda P digested with and dephosphorylated_R/ T^Twoori / S10H It was subcloned into IS-RSET-A9. pCro-nfSP33₁₂₀₀When The resulting recombinant molecule shown as Formed into E. coli BL-21 competent cells To the recombinant cells. Coli: pCro-nfSP33₁₂₀₀Was formed. Recombinant cells were cultured using the methods described above in Section A.   D. Flea serine protease protein PfSP40₂₄₂In the following way Produced. Where nfSP40₇₁₆About 716 bp nucleic acid molecule shown as Designed to encode a more mature serine protease protein) With the nucleotide sequence 5 ′ GCG GGA TCC AAT AGT AGG AGG AGG TGA AGA TGT AG3 '(including BamHI site; shown as SEQ ID NO: 60) Flea 40EF, a sense primer having the nucleotide sequence 5 ′ CCG GAA TTC TTC TAA CAA ATT TTA TTT GATT Antibodies with CC TGC 3 '(including EcoRI; shown as SEQ ID NO: 61) NfSP40 using flea 40ER which is a sense primer₈₄₁From PCR Width. The resulting PCR product nfSP40₇₁₆With BamHI and EcoRI Digested with restriction endonucleases, gel purified, and digested with BamHI and Eco Expression vector lambda P digested with RI and dephosphorylated_R/ T^Twoori / S1 Subcloned into 0HIS-RSET-A9. pCro-nfSP40_{71 6} The resulting recombinant molecule, designated as E. coli BL-21 competent cells Into a recombinant cell. Coli: pCro-nfSP40₇₁₆Form Was. The recombinant cells are cultured and the protein products are described above in Section A. Separated using the method described. Exemption of protein using T7 (tag) antibody Epidemic blot analysis showed that the transfected sample expressed about 38 kD protein, but not yet. It was shown that there was no in the introduced sample. Example 24   This embodiment is described in e. Another serine protease protein of the invention in E. coli cells 1 illustrates the production of quality.   A. Flea serine protease protein PfSP28₂₃₇In the following way Produced. Where nfSP28₇₁₁About 711 bp nucleic acid molecule shown as Designed to encode a more mature serine protease protein) With the nucleotide sequence 5′GGA TCC AAT CGT TGG AGG TGA AGA TG 3 '(including BamHI site shown in bold; shown as SEQ ID NO: 62) Flea 28F, a sense primer having the nucleotide sequence 5 ′ GAA TTC GAA ATC CAC TTA AAT ATT AGC 3 '(bold (SEQ ID NO: 63). NfSP28 using flea 28R, a primer₉₂₃Was amplified by PCR. The resulting PCR product nfSP28₇₁₁With BamHI and EcoRI restriction ends Digested with nuclease, gel purified and digested with BamHI and XbaI. And dephosphorylated expression vector lambda P_R/ T^Twoori / S10HIS-R It was subcloned into SET-A9. pCro-nfSP28₇₁₁Called The resulting recombinant molecule is designated E. coli BL-21 competent cells (Wisconsin) Novagen, I. of Madison, Wis. nc.)) to obtain recombinant cells. Coli: pCro-n fSP28₇₁₁Was formed. Using the method described above in Example 20, The cells were cultured and the protein product was degraded. Protein using T7 antibody Quality immunoblot analysis showed expression of the approximately 38 kD protein in the transfected sample. , But not in the unintroduced sample. Rabbit anti-flea midgut protea Zepolyclonal antibodies (the preparation of which is described in relevant PCT Publication No. WO 95/24). 198, as described in Example 14). An approximately 38 kD protein was identified in the transfected sample. Example 25   This example demonstrates the production of another serine protease protein of the invention in eukaryotic cells. The structure is exemplified.   Flea serine spanning from about 11 to about 802 nucleotides of SEQ ID NO: 66 Contains a protease nucleic acid molecule and is used as a baculovirus polyhedron transcription control sequence Operatively linked recombinant molecule pBv-nfSP28₇₉₂Is manufactured by the following method. did. The flea serine protease nucleic acid molecule is added to the baculovirus expression vector. For cloning, the fragment containing the flea serine protease nucleic acid molecule is fSP28₉₂₃Was amplified by PCR. Nucleic acid sequence 5'-GCG GGA TTC T AT AAA TAT GAA ACT TTT GGT AGT TTT TGC-3 Primer flea 28F3 (SEQ ID NO: 62; BamHI And the nucleic acid sequence 5'-GCT CTA GAC CAC TTA AAC Anti-sense primer flea having ATT AGC ATA TTT TTC-3 ' Using 28R3 (SEQ ID NO: 63; XbaI shown in bold), nfSP2 8₇₉₂The 792 nucleotide PCR fragment, referred to as nfSP28₉₁₃Amplified from I let you. N-terminal primer modified to enhance expression in baculovirus system , Designed from the pol h sequence of baculovirus.   PfSP28₂₆₄Baculovirus recombinant molecules capable of directing the production of To make, an approximately 792 base pair PCR product (Bv-nfSP28₇₉₂Shown as ) Were digested with BamHI and XbaI, and digested with BamHI. By subcloning into XbaI, pVL-nfSP28₇₉₂As shown here The resulting recombinant molecule was produced.   The resulting recombinant molecule, pVL-nfSP28₇₉₂Is properly inserted by the restriction map. The direction of entry was confirmed. The recombinant molecule is Frugiperda (S. frugi) perda) Sf9 cells (available from InVitrogen) No.), linear baculogold baculovirus DNA (Pharmingen (P) co-infected with S. haringen). Frugiperda (S . frugiperda): pVL-nfSP28₇₉₂Form recombinant cells indicated by did. Flea serine protease protein PfSP28₂₆₄To generate S. Frugiperda: pVL-nfSP28₇₉₂Was cultured.   Flea serine protease protein PfSP28₂₆₄S to generate full Gibelda: pVL-nfSP28₇₉₂Immunoblot of supernatant from cell culture With the feline anti-fSP flea 28 polyclonal antibody produced as follows. Performed using. E. described above in Example 24. E. coli produced recombinant Using mi28 protein (shown here as rSP flea 28 protein) Immunized the cat. rSP flea 28 protein was added at about 1 mg / ml in PBS. To an appropriate concentration, and an equal volume of TiterMax Site Ruhr, Norcross, GA (Available from CytRx Crop.). One line of cats About 50 μg each of rSP flea 28 protein in adjuvant by subcutaneous injection Immunized with quality. A second dose of the same dose of rSP flea 28 protein in adjuvant Was administered 32 days later. Blood samples are given 32 and 47 days after the first immunization before immunization. Pull was taken (pre-bleed). Serum samples obtained from pre-immunization and blood collection on day 47 were Used for continuous immunoblot experiments. The latter is anti-fSP flea 28 polyclonal Shown as an antibody.   Immunoblot analysis identifies about 33 kD protein and about 36 kD protein did. Example 26   This example includes the production of a 31 kD flea midgut serine protease peptide. And the generation of internal sequence data.   The midgut obtained from fleas fed with blood of about 30,000 cats was used for digestive tract cutting. Buffer (50 mM Tris (8.0), 100 mM CaCl_TwoU.S. Pat. It cut | disconnected as described in 56,622 (the said literature). Digestive tract (about 10,000 Was broken up by freeze-thaw cycles followed by sonication . The resulting extract is placed in a swinging bucket centrifuge at 4 ° C. for 20 minutes. Clarified by centrifugation at 3050 rpm for minutes. Collect the supernatant, And 400mM NaC with benzamidine column chromatography product Adjusted to l.   For each batch, the digestive tract supernatant was washed with the buffer for the benzamidine column (50 mM). Tris (pH 8.0), 100 mM CaCl_Two, 400 mM NaCl) Equilibrate and place on Sepharose beads incubated at 4 ° C with shaking overnight. 5 ml disposable column containing bridged p-aminobenzamidine (Missouri Sigma, St. Louis, MO Hand possible). Protein in Bradford Assay (California) Bio-Rad Laboratories, Hercules, CA (Available from Bio-Rad Laboratories). Unbound protein using Benzamidine column buffer Was slowly washed off the column.   Protease bound to the benzamidine column was converted to 100 mM p-aminobenzene. 4 ml of benzamidine supplemented with benzoamidine (pH 8.0 with NaOH) Elution was performed using a gin column buffer. About 21 ml additional benzamidine Residual bound protease was washed out with column buffer. After that, -(Ultrafree) 20 10-kD centrifugal concentrator (Massachusetts, Mass.) Millipore from Bedford, MA The recovered protease was concentrated to a volume of about 2 ml. concentrated Later, it was obtained from three standards in order to make the digestive tract equivalent to about 30,000 in about 6 ml. The protease stocks were combined. Bradford assay for protein concentration And found to be about 0.5 mg / ml.   Approximately 150 μg of the isolated protease stock was dispensed into a 14% preparative well. Squirrel-Glycinegel (San Diego, CA) CA) (available from Novex) Separation by electrophoresis (PAGE). Approximately 30 minutes after electrophoresis, Coomassie Brilliant blue dye (0.1 (w / v) Coomassie blue R, 40% (v / V) methanol, 10% (v / v) acetic acid) and stained for about 2.5 hours. 5 By destaining in 0% (v / v) methanol, the proteins in the gel Visualized. The band corresponding to the 31-kD protease was analyzed with a Razole blade. Resected. After electroelution and concentration, except for adding a small amount of acetic acid to the sample, The protein is electroeluted, concentrated, and cyanogen bromide (CNBr) For 24 hours (Silver et al., J. Biol. Chem. . Biol. Chem.), 270, 13010-13016, 1995), Lower the pH of the sample, thus reducing auto-digestion by 31-kD protease I let it. CNBr shows that methionine (M) remains under the conditions used for this digestion. It is known to cleave after After CNBr has been digested, peps in the sample Tide was separated by PAGE on an 18% Tris-glycine gel. Electric swimming After the operation, a CAPS buffer (10 mM CAPS (pH 11), 0.5 mM DTT, 0% (v / v) methanol) to separate the isolated protease peptide from PVD Electroblotted on F membrane. The membrane is stained with Coomassie Brilliant Blue and And destained with 50% (v / v) methanol. Identify three stained peptide bands Showed apparent molecular weights of about 14 kD, 21 kD and 22 kD, respectively. . The 21 kD and 22 kD bands were cut separately. Using standard techniques, 4 73A Protein Sequencer (Foster City, CA (F oster City, CA) Applied Biosystems (Applied)  Biosystems) (available from Biosystems). The peptide was subjected to N-terminal amino acid sequencing. Results obtained from automated sequencers were difficult to analyze due to overlapping sequences. However, the analysis of the chromatogram showed that the N-terminal amino acid sequence of the 21-kD peptide was Here, H / R-V / P-G / A / S-Y / G-E / N-D / K-V / R-D / A-D-Y-D- represented by SEQ ID NO: 70 F-D / P-V-A, and the N-terminal amino acid sequence of the 22-kD peptide is I / Q-V-G-Y / G-E / N / T-D / M / P-V-K / D-I-N / S-M / T / N-F / C represented by SEQ ID NO: 71 It was shown that. The N-terminal amino acid sequence of the intact 31-kD protease is I-V-G-G-E-D-V-D-I-S-T-C-G-W-C (SEQ ID NO: 59, co-pending U.S. Patent Application No. No. 08 / 639,075 as disclosed in the example of IVGGEDVDIST (C) GWQI (S) F Q (S) ENLHF (C) GG (S) IIAPK (SEQ ID NO: 69, Disclosed in Example 35 of co-pending US patent application Ser. No. 08 / 639,075. ). SEQ ID NO: 68 contains cysteine and the sequence These sequences vary at residue 15 in that number: 69 includes glutamine. 2 Even more potent with the 2-kD peptide, these sequences The sequence of both (SEQ ID NO: 70) and 22-kD (SEQ ID NO: 71) peptides , Whereby SEQ ID NO: 71 is the N-terminal of a 31 kD protease. It comes to the conclusion that it represents an edge. This sequence is a 21 kD (SEQ ID NO: 70) pepti The sequence that results for the 21 kD peptide when removed from the sequence of H / R-P-A / S-Y-N-K-R-A-D-Y-D-F-D-V-A shown here with the number 72. this The sequence of the amino acid sequence comprises the stretchable putative amino acids from about residue 107 to about residue 121. Aligns with the acid and immediately aligns with the methionine residue in SEQ ID NO: 67. These data are nfSP28₉₂₃(SEQ ID NO: 66, co-pending US Patent Application No. 08/6) (As described in Example 36 of No. 39075) Confirm that it actually encodes a 31 kD protease. Example 27   This example shows that the 31 kD flea midgut serine protease contained in the formulation example is , Cat immunoglobulin G, A and M proteins and bovine, canine, human, And proteolysis of rabbit immunoglobulin G protein Is exemplified.   A 31 kD flea midgut serine protease is fed to cat blood as follows. Purified from fleas. Producing a flea midgut extract fed on cat blood; and Selected on a benzamidine column as described above in Example 26. afterwards , PolyCAT A Cation Exchange Chromatography (Colon, MD) (Available from PolyLC, Inc.). U.S. Patent Application Serial No. 08 / 639,075, assigned to The benzamidine eluate was further purified as described above and analyzed by silver-stained SDS-PAGE gel. A protein band that migrated to approximately 31 kD was isolated.   A. Flea midgut serine fed cat's blood to degrade immunoglobulin The activity of the Rotase is described in co-pending US patent application Ser. No. 08 / 639,075. Using a method similar to that described in Example 35, the immunoglobulin heavy chain Indicated by measuring digestion. In particular, cat IgG, cat IgA and And a 1 μg sample of cat IgM substrate (Besyl Laboratories, Inc. Laboratories, Inc.) at 37 ° C. for 18 hours In the meantime, purified 3M with a total volume of 27 μl 0.1 M Tris-HCl (pH 8.0) Flea midgut fed with 500 cat blood of 1 kD flea midgut serine protease Incubate with equivalent volume. The reaction mixture was purified with 14% Tris-glycine SDS- Separated by PAGE and gels were silver stained using standard methods. 31k D-protease treated cat IgG, IgA and IgM in the lane containing silver stain The bands migrating at approximately 50, 60 and 80 kD on the gel run are generally disappearing. , A 31-kD flea midgut serine protease, This indicates that the heavy chain of ip was degraded.   B. 31k fed cat blood to degrade IgG from several species The activity of the D flea midgut serine protease was determined at 37 ° C. for 18 hours at 27 μl of 0. Purified cat or bovine IgG (Ta) with a total volume of 1 M Tris-HCl (pH 8.0). Purified from cat and bovine blood on protein A Sepharose), or purified Dogs, rabbits or hide IgG (Sigma Chemical Co., Ltd.) ical Co. Incubating a 1 μg sample (available from Thus illustrated. The reaction mixture was subjected to 14% Tris-glycine SDS-PAGE. Thus, they were separated and the gels were silver stained using standard methods. 31kD protea Lane containing cat, cow, dog, rabbit and human IgG heavy chains The band migrating at about 50-55 kD on the colored gel generally disappears (purification 31). (compared to a control sample lacking the addition of kD protein) is the 31 kD flea midgut Serine proteases have been shown to be able to degrade IgG from various species. Example 28   This example involves the proteolysis of feline immunoglobulin G at specific sites. The activity of the 31 kD flea midgut serine protease contained in the formulation example is described. .   A 31 kD flea midgut serine protease was described in Examples 26 and 27. Purified from flea extract fed cat blood as described.   To study the cleavage site specificity of purified 31kD flea midgut serine protease , 10 μg of cat immunoglobulin obtained from cat blood on Protein A Sepharose Bryn G was added to 100 μl of 0.2 M Tris-HCl (pH 8.0) Purification of midgut-equivalent amount of flea fed from 200 cat blood in total amount 3 Incubated with 1 kD flea midgut serine protease. The reaction mixture was added to 14 % Tris-Glycine separated by SDS-PAGE and soaked in PVDF membrane , Stained with Coomassie R-250 and standardized Destained. Cut out a band of about 33 kD, and use techniques known to those skilled in the art. N-terminal amino acid sequencing was performed using surgery. About 28 amino acids The N-terminal amino acid sequence was determined, and here, SEQ ID NO: 73: X-P-P-P-E- Expressed as M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K. SEQ ID NO: 73 Genebank homology search using Orytragus caniculus (Ory max for the gamma heavy chain constant region 2 of E. ctolagus caniculus And thus there was about 71% homology over 28 amino acids. Further Have the amino acid sequences of sheep, rat, rabbit, monkey, cow, and human IgG. The sequence of SEQ ID NO: 73 is a 31 kD flea midgut serine fed from purified cat blood Protease can be found in cat IgG just before the putative C-terminus of the IgG hinge region. It was shown to break the heavy chain. Putative first amino acid cysteine and second Of the amino acid proline occurs in the putative hinge region, while the third amino acid proline The remaining 26 amino acids of SEQ ID NO: 73 beginning with phosphorus correspond to the predicted constant heavy chain-2. Occurs in the area.   In a further study, a purified 31 kD flea midgut serine protein for feline IgG was The specificity of the cleavage site of the enzyme and the cleavage site were Compared to the Cat purified from cat blood on Protein A Sepharose Of immunoglobulin G (100 mg) at room temperature for 4.5 hours in a final volume of 150 100 mM sodium acetate (pH 5.5), 50 mM cysteine, 1 mM Incubated with 1 mg papain in EDTA. 14% Tris reaction mixture − Glycine Separated on SDS-PAGE gel, soaked on PVDF membrane, Coomassie (Coomassie) R-250 and destain according to standard procedures Colored. A band of approximately 33 kD was cut out and used with techniques known to those skilled in the art. And subjected to N-terminal amino acid sequencing. Partial N-terminal amino acid of about 25 amino acids The amino acid sequence was deduced, and SEQ ID NO: 96: X-P-P-P-E-M-L-G-G-P-S-I-F-I- Expressed here as F-P-P-K-K-K-D-D-L-L-I. This sequence is a purified 31 kD flea Is a 33kD cat IgG cleavage product produced by midgut serine protease? Almost identical to that obtained, so that the only difference is that the amino acid 19 Phosphorus (SEQ ID NO: 96) was replaced with lysine (SEQ ID NO: 73). Example 29   This example demonstrates the cat's IgG degrading activity in the midgut of fleas fed by live cats. The dynamics are illustrated.   A. To determine the kinetics of IgG degradation in cats in the midgut of continuously fed flea , The female flea contained in the chamber is disclosed in co-pending US patent application Ser. No. 08 / 63,083. 9075 cats (ie, cats) as described in Example 21 of U.S. Pat. One chamber per animal) was fed. Flea chamber for 15 minutes, 30 minutes, At 1 hour, 2 hours, 4 hours, 6 hours, 8 hours and 17 hours, for cutting Moved to After feeding with cats, the midgut of the flea is removed from the US Pat. Removed as described in No. 22 (cited above), homogenized by freezing and thawing, And 50 mM Tris (pH 8.0) and 100 mM CaCl_TwoTris containing Sonicated with buffer. The extract is centrifuged at about 14000 xg for 20 minutes. And the soluble material was recovered. Then, add 1 microliter of soluble material (Μl) diluted to a final final concentration of approximately 1.2 midgut equivalents per Tris buffer . Thereafter, the protein contained in one midgut equivalent was converted to SDS-PAG under reduced pressure. E and proteins were visualized by silver staining. The result is Ig G heavy chain levels are more apparent at 17 hours than at 8 hours and earlier Low and light chain levels are reduced and not in the same range as the heavy chains Was. Thereafter, the protein contained in the equivalent amount of 5 gastrointestinal tracts at each time point was removed under reduced pressure. In S Goats separated by DS-PAGE gel and labeled with alkaline phosphatase Anti-cat IgG (heavy and light chain) antibodies (Gaisersburg, MD Itersburg, MD) 's Kirke Girld and Perry Laboratory (Available from Kirkegaard and Perry) Western blot analysis was performed. The results show that the cat IgG heavy chain is at least 8 It is present in the midgut of fleas fed continuously for 17 hours, but is continuously fed to cats for 17 hours. This indicates that no fleas were detected in the midgut. Visualized with 17-hour sample Although the amount performed was clearly less than that visualized in the 8 hour sample, the light chain Visualized in all samples. These results indicate that fleas continue to feed on cats. Even when the IgG degradation protein was introduced into the midgut of the flea at 17 hours, Zezy levels are sufficient to degrade all detectable cat IgG ingested. You. These results indicate that if the fleas continuously feed on cats, they will be transmitted to the midgut of the fleas. The level of IgG-degrading protease entered was at least 8 hours ingested. Is not sufficient to degrade detectable cat IgG.   B. Feline Ig in the gastrointestinal tract of flea fed for a specified time and then released from the cat To measure the kinetics of G degradation, chisels (in the chamber) are run for 1 hour or 24 hours. Cats were fed as in Section A for a period of time. 1 or 24 hour breeding period Then, move the flea chamber and grow ink at 28 ° C and 75% relative humidity. Placed in the incubator. Fleas are cut at 0, 1, 2, 4, and 8 hours Was. Homogenize the midgut and remove midgut contents as described in Section A , Silver stained SDS-PAGE and immunoblot analysis. 1 hour contact Fleas fed were cat IgG detectable in the midgut at 0 and 1 hour cuts. Had high molecular weight proteins, including certain heavy and light chains, while The midgut of the flea evaluated at more time points has no detectable IgG heavy chain band. won. The result is that if the fleas were fed in cats and then removed, Within 2 hours, the cat's IgG heavy chain was almost completely degraded. 24 hours Fleas fed in cats have no detectable IgG weight in midgut extract at any time point. No chain or light chain protein. These results indicate that fleas were released from feeding. As in the case where no new cat IgG is fed, I g G-degrading protease sufficiently degraded all cat IgG heavy chains in less than 2 hours Suggest that. Example 30   This example involves the proteolysis of feline immunoglobulin G at specific sites. The potential of the 31 kD flea midgut serine protease contained in the formulation was described. I have.   A 31 kD flea midgut serine protease is described above in Examples 14 and 15. Cat blood was purified from midgut extract of fed fleas as described.   Further investigation of cleavage site specificity of purified 31kD flea midgut serine protease 1.5 mic purified from cat blood on Protein A Sepharose Of cat immunoglobulin G at 37 ° C. in 300 microliters of 5 Purified 31 kD flea midgut serine with a total volume of 0 mM Tris-HCl (pH 8.0) Incubate with 200 equivalents of midgut flea fed with cat blood from protease I did it. Approximately 10 microliter aliquots of the incubation mixture 1, 2, 4, 6, 8, 12, 16 and 24 hours after the start of the incubation Removed. Following removal, each aliquot was added to approximately 1 microliter of 20 mM  Mixed with p-aminobenzamidine and stored at -80 ° C. Incube Of each aliquot from the perfusion mixture was applied to 14% Tris-glycine SDS. -Separated by PAGE, blotted onto PVDF membrane, Coomassie (Coomas) sie) Stained with R-250 and destained according to standard procedures. About 34 The kD band was identified by the amount of alicode excised after 1 hour incubation. Was. Cut the band and use the techniques known to those skilled in the art to Sequencing. The partial N-terminal amino acid sequence of about 25 amino acids was determined. And here, SEQ ID NO: 104: D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F- Expressed as P-P-K-P-K-D. Another 10 amino acids are also amino acids prior to SEQ ID NO: 104. Obtained by acid. A mixed amino acid sequence of about 35 amino acids was determined. : 105: D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R- Expressed as K-S-E-V. Gene bank homology using SEQ ID NO: 105 Is immunoglobulin gamma of Homo sapien. Triplex constant region 2 exon hinge IGHG3 gene (GeneBank (GenBa nk) accession number X99549) and show most homology to it. And about 69% homology over 35 amino acids. In addition, cats, Usaki, cattle And the human IgG amino acid sequence and the sequence of SEQ ID NO: 105 were obtained from purified cat blood. A 31 kD flea midgut serine protease that feeds on the putative C in the IgG hinge region It was shown to cleave the feline IgG heavy chain 6 amino acids just before the end. the first 6 amino acids, aspartic acid, cysteine, proline, lysine, cysteine and And proline occur in the putative hinge region while the seventh amino acid proline The remaining 29 amino acids of SEQ ID NO: 105 beginning with the deduced constant heavy chain-2 region Occurs within.   SEQ ID NO: 105 and a cat IgG (shown here by SEQ ID NO: 106 Amino acid comparisons for the hinge and CH2 region portions of It is. SEQ ID NO: 105: D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K- SEQ ID NO: 106: D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K- SEQ ID NO: 105 (continued) D-D-L-L-I-K-R-K-S-E-V SEQ ID NO: 106 (continued) D-T-L-S-I-S-R-T-P-E-V   The mismatch between SEQ ID NO: 105 and SEQ ID NO: 106 is shown in bold. application The difference between SEQ ID NO: 105 and SEQ ID NO: 106 is that We believe it may be due to a sequencing error of the last 10 amino acids.                                  Sequence listing   The following sequence listing has been submitted under 37 CFR §1.821. Con A copy in computer readable form is also provided here.   Applicant has determined that the sequence identification numbers 1-165 are based on 37 CFR §1.821 (f). And the contents of the computer-readable copy submitted here Are the same.   Although various embodiments of the present invention have been described in detail, modifications and adaptations of these embodiments are not It will be apparent to those skilled in the art that this is the case. However, such changes and Suitability is clearly within the scope of the invention as described in the following claims. It should be understood.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 33/00 171 C07K 16/40 C07K 14/81 C12N 1/21 16/40 7/00 C12N 1/21 9/50 7/00 C12P 21/02 C 9/50 G01N 33/566 C12P 21/02 33/573 G01N 33/566 C12N 15/00 ZNAA 33/573 A61K 37/02 (31) Priority claim number 60 / 042,945 (32) Priority date April 4, 1997 (1997.4.4) (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), A P (GH, KE, LS, MW, SD, SZ, UG), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA , BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK , TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU (72) Inventor Hunter, Sealy Woo United States, 80525, Colorado, Fort Collins, Tanglewood Drive 2325 (72) Invention Frank Frank Glenn United States of America, Raleigh, 80549, Wellington, 10317 North County Road 13 (72) Inventor Steigler, Gary El, United States, 80526, Colorado, Fort Collins, Zent Street 2934 (72) Inventor Gaines, Patrick Patrick Jay United States, 80525, Colorado, Fort Collins, 1200 eStuart Street Number 62 (72) Inventor Silver, Gully, United States, 80521, Colorado, Fort Collins, Fleetwood 1424

Claims (1)

[Claims]   1. An isolated nucleic acid molecule that is a stringent hybridization SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, sequence No. 15, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 23, Sequence No. 25, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 32, Sequence No. 34, SEQ ID No. 35, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 42, Sequence No. 43, SEQ ID No. 120, SEQ ID No. 130, SEQ ID No. 154, SEQ ID No. 116, SEQ ID No. 117 , SEQ ID NO: 127, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 155, SEQ ID NO: 114, Sequence No. 125, SEQ ID No. 118, SEQ ID No. 128, SEQ ID No. 152, SEQ ID No. 156, SEQ ID No. 160 , SEQ ID NO: 136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: No. 66, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 80, SEQ ID NO: 82, Column No. 142, SEQ ID No. 138, SEQ ID No. 144, SEQ ID No. 140, SEQ ID No. 122, SEQ ID No. 8 Serine protease containing a nucleic acid molecule selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 45 Gene; an isolated nucleic acid selected from the group consisting of SEQ ID NO: 110 and SEQ ID NO: 112 Aminopeptidase gene containing molecules; and SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, Cysteine containing a nucleic acid molecule selected from the group consisting of SEQ ID NO: 76 and SEQ ID NO: 94 Isolated that hybridizes with a gene selected from the group consisting of protease genes Nucleic acid molecule.   2. An isolated nucleic acid molecule that is a stringent hybridization SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: No. 22, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, Sequence No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, Sequence No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 96, SEQ ID No. 115, Column No. 126, SEQ ID No. 119, SEQ ID No. 129, SEQ ID No. 153, SEQ ID No. 157, SEQ ID No. 1 61, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, Sequence No. 67, SEQ ID No. 147, SEQ ID No. 149, SEQ ID No. 151, SEQ ID No. 81, SEQ ID No. 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: No. 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, Column number 111, sequence number 113, sequence number 77, sequence number 2, sequence number 5, sequence number 8, Column No. 89, SEQ ID NO: 92 and SEQ ID NO: 95. Hybridizes with a nucleic acid molecule having a nucleic acid sequence encoding a protein An isolated nucleic acid molecule.   3. An isolated nucleic acid molecule that is a stringent hybridization SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: No. 22, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, Sequence No. 38, SEQ ID No. 41, SEQ ID No. 44, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, Sequence No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 96, SEQ ID No. 115, Column No. 126, SEQ ID No. 119, SEQ ID No. 129, SEQ ID No. 153, SEQ ID No. 157, SEQ ID No. 1 61, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, Sequence No. 67, SEQ ID No. 147, SEQ ID No. 149, SEQ ID No. 151, SEQ ID No. 81, SEQ ID No. 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: No. 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, Column number 111, sequence number 113, sequence number 77, sequence number 2, sequence number 5, sequence number 8, An amino acid sequence selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95 Hybridizes with a nucleic acid molecule having a nucleic acid sequence encoding the containing protein Isolated nucleic acid molecule.   4. A therapeutic composition that reduces flea infestation when administered to an animal, , The composition can be used under stringent hybridization conditions. Column No. 10, SEQ ID No. 13, SEQ ID No. 16, SEQ ID No. 19, SEQ ID No. 22, SEQ ID No. 24, Column No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 38, SEQ ID No. 41, arrangement Sequence No. 44, Sequence No. 67, Sequence No. 68, Sequence No. 69, Sequence No. 70, Sequence No. 71, Sequence Sequence No. 72, Sequence No. 73, Sequence No. 96, Sequence No. 115, Sequence No. 126, Sequence No. 119 , SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, Sequence No. 79, SEQ ID No. 159, SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 67, SEQ ID No. 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 1 39, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, Sequence No. 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and And a protein comprising an amino acid sequence selected from the group consisting of Encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule having a nucleic acid sequence Proteins or their mimetopes; stringent hybrids SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: No. 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO No. 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: No. 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: No. 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, Sequence No. 83, SEQ ID No. 143, SEQ ID No. 139, SEQ ID No. 145, SEQ ID No. 141, SEQ ID No. 123 , SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: No. 8, SEQ ID NO: 89, SEQ ID NO: 92 and nucleic acid sequence selected from the group consisting of SEQ ID NO: 95 An isolated nucleic acid molecule that hybridizes to a gene containing the sequence; stringent SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 6 SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115 , SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, Sequence No. 161, SEQ ID No. 137, SEQ ID No. 79, SEQ ID No. 159, SEQ ID No. 133, SEQ ID No. 135 , SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: No. 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 10 7, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8 , SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95, an amino acid selected from the group consisting of Nucleus that hybridizes to a nucleic acid molecule that has a nucleic acid sequence that encodes a protein An isolated antibody that selectively binds to a protein encoded by an acid molecule And; under stringent hybridization conditions, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: No. 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, Sequence No. 143, SEQ ID No. 139, SEQ ID No. 145, SEQ ID No. 141, SEQ ID No. 123, SEQ ID No. 68 , SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: No. 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: No. 89, including an amino acid sequence selected from the group consisting of SEQ ID NO: 92 and SEQ ID NO: 95 A nucleic acid component that hybridizes to a nucleic acid molecule having a nucleic acid sequence that encodes a protein Pros identified by their ability to inhibit the activity of the protein encoded by the offspring A protected compound selected from the group consisting of a protease inhibitor and a mixture thereof A composition containing   5. A method of reducing flea infestation comprising treating with a therapeutic composition. Thus, the composition may be used under stringent hybridization conditions. And SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: No. 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 1 47, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, Sequence No. 139, SEQ ID No. 145, SEQ ID No. 141, SEQ ID No. 123, SEQ ID No. 68, SEQ ID No. 163 , SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: No. 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: A protein containing an amino acid sequence selected from the group consisting of Encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule having a nucleic acid sequence Protein or its mimetope; stringent high Under hybridization conditions, Column No. 9, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 17, Column No. 18, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 26, Sequence Column No. 28, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 34, SEQ ID No. 35, Sequence No. 37, Sequence No. 39, Sequence No. 40, Sequence No. 42, Sequence No. 43, Sequence No. 45, arrangement Column No. 120, SEQ ID No. 130, SEQ ID No. 154, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 1 27, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 155, SEQ ID NO: 114, SEQ ID NO: 125, Sequence Column No. 118, SEQ ID No. 128, SEQ ID No. 152, SEQ ID No. 156, SEQ ID No. 160, SEQ ID No. 1 36, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 66, Sequence No. 146, SEQ ID No. 148, SEQ ID No. 150, SEQ ID No. 80, SEQ ID No. 82, SEQ ID No. 142, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 76, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 , SEQ ID NO: 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: No. 94, which hybridizes with a gene containing a nucleic acid sequence selected from the group consisting of Under stringent hybridization conditions, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 1 29, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, Sequence No. 159, SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 67, SEQ ID No. 147, SEQ ID No. 149 , SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: No. 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 7 7, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95 A nucleic acid sequence encoding a protein containing an amino acid selected from the group consisting of Proteins encoded by nucleic acid molecules that hybridize with And selectively binds antibodies; stringent hybridization SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: No. 22, SEQ ID No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, Sequence No. 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 11 9, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, Sequence No. 79, SEQ ID No. 159, SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 67, SEQ ID No. 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 1 39, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, Sequence No. 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92and A nucleus encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 95 Tan encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule having an acid sequence A protease activity inhibitor identified by its ability to inhibit the activity of a protein; A method comprising a protected compound selected from the group consisting of these mixtures.   6. A method for producing a flea protease protein, comprising: Culturing cells capable of expressing the protein, wherein the protein comprises SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, sequence No. 15, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 23, Sequence No. 25, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 32, Sequence No. 34, SEQ ID No. 35, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 42, Sequence No. 43, SEQ ID No. 45, SEQ ID No. 120, SEQ ID No. 130, SEQ ID No. 154, SEQ ID No. 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 155, SEQ ID NO: No. 114, SEQ ID NO: 125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 66, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 80, Sequence No. 82, SEQ ID No. 142, SEQ ID No. 138, SEQ ID No. 144, SEQ ID No. 140, SEQ ID No. 122 , SEQ ID NO: 84, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 76, SEQ ID NO: 1, SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, A gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 93 and SEQ ID NO: 94 and c The method encoded by the hybridizing nucleic acid molecule.   7. A method for identifying a compound capable of inhibiting flea protease activity, comprising:       (a) SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, Column No. 24, SEQ ID No. 27, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 36, SEQ ID No. 38, arrangement Sequence No. 41, Sequence No. 44, Sequence No. 67, Sequence No. 68, Sequence No. 69, Sequence No. 70, Sequence Column No. 71, SEQ ID No. 72, SEQ ID No. 73, SEQ ID No. 96, SEQ ID No. 115, SEQ ID No. 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: No. 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, Column number 147, sequence number 149, sequence number 151, sequence number 81, sequence number 83, sequence number 143 , SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: No. 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, Column number 113, sequence number 77, sequence number 2, sequence number 5, sequence number 8, sequence number 89, sequence Isolated comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 92 and SEQ ID NO: 95 In the absence of the inhibitory compound, the protein is Contacting with a putative inhibitory compound under conditions having proteolytic activity;       (b) determining whether the putative inhibitory compound inhibits the activity A method comprising:   8. A test kit for identifying compounds that can inhibit flea protease activity. hand:       SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: No. 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: No. 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: No. 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, Sequence No. 119, SEQ ID No. 129, SEQ ID No. 153, SEQ ID No. 157, SEQ ID No. 161, SEQ ID No. 137 , SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: No. 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, Column No. 139, SEQ ID No. 145, SEQ ID No. 141, SEQ ID No. 123, SEQ ID No. 68, SEQ ID No. 16 3, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: No. 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: Amino acid sequence selected from the group consisting of 92 and SEQ ID NO: 95 An isolated flea protease protein comprising:       To determine the degree of inhibition of said activity in the presence of the putative inhibitory compound Test kit comprising:   9. An isolated flea protease protein, wherein the protein comprises In the presence of epidemic globulin, about 37 μl in about 100 μl of about 0.2 M Tris-HCl A protein that cleaves when incubated at about 18 hours.   10. Identify compounds that can inhibit flea immunoglobulin proteinase activity Method:       (A) flea immunoglobulin proteinase protein, the absence of the compound Conditions in which the protein has immunoglobulin proteinase activity in the presence Below, contacting with a putative inhibitory compound;       (b) determining whether the putative inhibitory compound inhibits the activity A method comprising:   11. 2. The isolated nucleic acid molecule of claim 1, wherein the serine protein is SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 , SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38 , SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 , SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 1 26, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67 , SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: No. 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, Selected from the group consisting of SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69 and SEQ ID NO: 85 A nucleic acid sequence encoding an amino acid sequence; Amino acid sequence selected from the group consisting of SEQ ID NO: 107, SEQ ID NO: 111 and SEQ ID NO: 113 A cysteine protease gene comprising the sequence SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 95 and SEQ ID NO: 95 A method comprising a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 77 Nucleic acid molecule.   12. 3. An isolated nucleic acid molecule according to claim 1 or 2, wherein the nucleic acid component is Offspring are larval serine protease protein, adult serine protease protein Larval aminopeptidase protein, adult aminopeptidase, larval cysteine Protein protease protein and adult cysteine protease protein. Comprising a nucleic acid sequence encoding a flea protease protein selected from the group consisting of: molecule.   13. 3. An isolated nucleic acid molecule according to claim 1 or 2, wherein the nucleic acid component is A child is a nucleic acid molecule that is a flea nucleic acid molecule.   14． 3. An isolated nucleic acid molecule according to claim 1 or 2, wherein the nucleic acid component is Offspring are Ctenocephalides, Ceratophyllus and Diamanu Snow flea (Diamanus), chicken flea (Echidnophaga), European flea (Nosopsyllus), Flea genus (Pulex), Snow flea (Tunga), Oropsilana (Or opsylla), from Orchopeus and Xenopslla A nucleic acid molecule selected from the group consisting of:   15. 3. The isolated nucleic acid according to claim 1 or 2, wherein the nucleic acid molecule is , Stenocephalidis felis (Ctenocephalides felis), Stenocephali Ctenocephalides canis, Ceratofils plisidae (Cerato phyllus pulicidae), Plex irritans, Oropsila Orchids (Oropsylla (Thrassis) bacchi), Oropsylla (Diam anus) montana), Orchopeus howardi, Xenopsila Xenopsylla cheopis and Pulex simulans An isolated nucleic acid molecule selected from the group consisting of:   16. 3. The isolated nucleic acid according to claim 1 or 2, wherein the nucleic acid molecule is , An isolated nucleic acid molecule comprising the nucleic acid molecule of Stenocepharidis felis.   17． 2. The isolated nucleic acid molecule of claim 1, wherein said isolated nucleic acid molecule is a stringent. Under hybridization conditions, nfSP18₅₃₄, NfSP18₇₇₅, NfSP18₂₂₅, N fSP24₄₁₀, NfSP24₁₀₈₉, NfSP24₇₇₄, NfSP24₇₁₁, NfSP28₇₁₁, NfSP28₉₂₃, NfSP32₉₃₃ , NfSP32₉₂₄, NfSP32₆₉₉, NfSP33₄₂₆, NfSP33₇₇₈, NfSP33₁₈₉₄, NfSP33₁₂₀₀ , NfSP33₇₂₆, NfSP40₈₄₁, NfSP40₇₁₇, NfSP5₈₀₆, NfSP11₃₀₇, NfSP8₅₁₅, NfSP8_{Four 36} , NfSP12₇₅₈, nfSP26₆₁₀, NfSP27₃₈₆, NfSP23₄₂₃, NfSP34₃₉₀, NfSP36₁₉₇, NfSP38₃₄₁, NfSP37₂₆₁ , NfSP39₂₆₇ nfSP29₆₁₂, NfSP30₆₄₁, NfSP31₆₂₆, NfSP32₄₃₃, NfSP15₈₁₅, Nf SP19₈₅₅, NfSP25₈₆₄, NfSP21₅₉₅, NfAP2₃₈₃, NfAP2₅₃₇, NfCP1₅₇₃And nfCP1_{11 09} A nucleic acid molecule that hybridizes with a nucleic acid molecule selected from the group consisting of:   18. 2. An isolated nucleic acid molecule according to claim 1 wherein nfSP18.₅₃₄, NfSP18_{7 75} , NfSP18₂₂₅, NfSP24₄₁₀, NfSP24₁₀₈₉, NfSP24₇₇₄, NfSP24₇₁₁, NfSP28₇₁₁, N fSP28₉₂₃, NfSP32₉₃₃, NfSP32₉₂₄, NfSP32₆₉₉, NfSP33₄₂₆, NfSP33₇₇₈, NfSP33_{1 894} , NfSP33₁₂₀₀, NfSP33₇₂₆, NfSP40₈₄₁, NfSP40₇₁₇, NfSP5₈₀₆, NfSP11₃₀₇, N fSP8₅₁₅, NfSP8₄₃₆.nfSP12₇₅₈, NfSP26₆₁₀, NfSP27₃₈₆, NfSP23₄₂₃, NfSP34₃₉₀ , NfSP36₁₉₇, NfSP38₃₄₁, NfSP37₂₆₁, NfSP39₂₆₇ nfSP29₆₁₂, NfSP30₆₄₁, NfSP3 1₆₂₆, NfSP32₄₃₃, NfSP15₈₁₅, NfSP19₈₅₅, NfSP25₈₆₄, NfSP21₅₉₅, NfAP2₃₈₃, N fAP2₅₃₇, NfCP1₅₇₃And nfCP1₁₁₀₉. A nucleus containing a nucleic acid molecule selected from the group consisting of Acid molecule.   19. 3. An isolated nucleic acid molecule according to claim 1 or 2, wherein , SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27 , SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44 , SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72 , SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: No. 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, Sequence No. 145, SEQ ID No. 141, SEQ ID No. 123, SEQ ID No. 68, SEQ ID No. 163, SEQ ID No. 162 , SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: No. 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and sequence Nucleus encoding a protein having an amino acid sequence selected from the group consisting of No. 95 A nucleic acid molecule containing an acid sequence; and a nucleic acid molecule encoding any of the aforementioned amino acid sequences. An isolated nucleic acid selected from the group consisting of nucleic acid sequences containing allelic variants of the offspring Child.   20. 2. The isolated nucleic acid of claim 1, wherein the nucleic acid is SEQ ID NO: 9, SEQ ID NO: 11. , SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20 , SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 45, SEQ ID NO: 120, SEQ ID NO: 1 30, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, Sequence Column No. 131, SEQ ID No. 155, SEQ ID No. 114, SEQ ID No. 125, SEQ ID No. 118, SEQ ID No. 1 28, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 78, Sequence No. 158, SEQ ID No. 132, SEQ ID No. 134, SEQ ID No. 66, SEQ ID No. 146, SEQ ID No. 148 , SEQ ID NO: 150, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 142, SEQ ID NO: 138, SEQ ID NO: No. 144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 76, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, Sequence No. 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 94 A nucleic acid molecule comprising a selected nucleic acid sequence; and a nucleic acid having any of the foregoing nucleic acid sequences A nucleic acid molecule selected from the group consisting of nucleic acid molecules containing allelic variations of the molecule.   21. 2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an oligonucleotide. An isolated nucleic acid molecule comprising a gonucleotide.   22. An isolated protein according to claim 3, wherein said protein is , When administered to animals, nomiserin protease, flea aminopeptidase And flea cysteine proteases An isolated protein that elicits an immune response against it.   23. 4. The isolated protein of claim 3, wherein said protein is administered to an animal. Nominerin protease, flea aminopeptidase, and flea An immune response to a flea protease selected from the group consisting of Induced isolated protein.   24. 3. The nucleic acid molecule of claim 1 or 2 operably linked to a transcription control sequence. A recombinant molecule comprising:   25. A recombinant virus comprising the nucleic acid molecule according to claim 1.   26. A recombinant cell comprising the nucleic acid molecule according to claim 1 or 2, wherein the recombinant cell comprises the nucleic acid molecule according to claim 1 or 2. A cell capable of expressing a nucleic acid molecule.   27. 3. The isolated nucleic acid molecule of claim 2, wherein the isolated nucleic acid molecule is a stringent Under hybridization conditions, the phase of the nucleic acid encoding the protein A nucleic acid molecule that hybridizes to a complement.   28. 4. The isolated nucleic acid molecule of claim 3, wherein the protein is: SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 11 9, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, Sequence No. 79, SEQ ID No. 159, SEQ ID No. 133, SEQ ID No. 135, SEQ ID No. 67, SEQ ID No. 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 1 39, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, Sequence No. 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 77, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and And a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 95 and SEQ ID NO: 95; Allele of a nucleic acid molecule encoding a protein that blows any of the above amino acid sequences A nucleic acid molecule selected from the group consisting of a protein encoded by a child variant.   29. 4. The isolated nucleic acid molecule of claim 3, wherein the protein is: Nomiserin protease activity inhibitors, aminopeptidase activity inhibitors, and Identify inhibitors selected from the group consisting of mycysteine protease activity inhibitors Nucleic acid molecules used for   30. 30. The isolated nucleic acid molecule of claim 29, wherein said inhibitor is an active nucleic acid. A nucleic acid molecule that can reduce flea invasion when administered to an object.   31. An isolated antibody that selectively binds to the protein of claim 3.   32. A therapeutic composition or method according to claim 4, 5 or 52, In addition, components selected from excipients, adjuvants, carriers, and mixtures thereof. A composition comprising   33. Therapeutic composition or method according to claim 4 or 5, wherein the combination. The composition is a composition comprising a sustained release formulation.   34. A therapeutic composition or method according to claim 4 or 5, further comprising: Decrease flea adhesion by methods other than reducing flea protease activity A composition comprising a compound.   35. 6. The method of claim 5, wherein the animal is an adult flea, a flea larva and A composition selected from the group consisting of animals that are susceptible to infestation by fleas.   36. 6. The method of claim 5, wherein the infestation of flea larvae comprises the therapeutic composition. A composition that is reduced by flea larvae ingesting adult flea feces that contain the material.   37. 6. The method according to claim 5, wherein the infestation of the larvae fleas reduces adult fleas feces. Reduced by ingesting larval fleas, wherein the dung is reduced by one or more of the isolated flea mites Anti-flea protein induced in a host animal in response to administration of a Rotase protein An adult flea provided by the host animal after the administration. Composition.   38. 6. The method of claim 5, wherein said animal is selected from mammals and birds. How to get out.   39. 6. The method of claim 5, wherein said animal comprises a cat and a dog. A method chosen from the group.   40. 6. The method of claim 5, wherein the fleas are of the genus Ctenoceph. alides), Cyopsillus (Cyopsyllus), Diamanus flea (Diamanus), Chick flea (Echidnophaga), European flea (Nosopsyllus), human Consists of flea (Pulex), snow flea (Tunga), and flea (Xenopsyll) A method chosen from the group.   41. 6. The method of claim 5, wherein the flea is Stenocephalidis. -Ferris (Ctenocephalides felis), Stenocepharidis canis (Ctenocep) halides canis), Pulex irritans, Plex Sim A method selected from the group consisting of lances (Pulex simulans).   42. 6. The method of claim 5, wherein the composition further comprises a flea protein. Contains compounds that reduce fleas attached by methods other than reducing activity how to.   43. An isolated flea protease protein according to claim 9, wherein A protein that cleaves immunoglobulin heavy chains.   44. An isolated flea protease protein according to claim 9, wherein A protein that cleaves the hinge region of an immunoglobulin heavy chain.   45. An isolated flea protease protein according to claim 9, wherein The mature protein has a molecular weight of about 25 kD to about 35 kD.   46. 46. The isolated flea protease protein of claim 45, The protein has a molecular weight of about 31 kD.   47. An isolated flea protease protein according to claim 9, wherein SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, A protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 73 and SEQ ID NO: 96 Quality.   48. An isolated flea protease protein according to claim 9, wherein Nominerin protease gene that cleaves immunoglobulin Encoded by nucleic acid molecules that hybridize under mild hybridization conditions A protein comprising the amino acid sequence to be made.   49. 49. The isolated flea protease protein of claim 48, A protein wherein the serine protease gene comprises the nucleic acid sequence of SEQ ID NO: 66;   50. 49. The isolated flea protease protein of claim 48, The serine protease gene is SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, Group consisting of column No. 70, SEQ ID No. 71, SEQ ID No. 72, SEQ ID No. 73 and SEQ ID No. 96 A protein comprising a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of:   51. An isolated flea protease protein according to claim 9, wherein Under stringent hybridization conditions, nfSP28₇₁₁And n fSP28₉₂₃A nucleic acid molecule that hybridizes with a nucleic acid molecule selected from the group consisting of: Encoded protein.   52. An isolated flea protease protein according to claim 9, wherein The protein has the sequence number under stringent hybridization conditions. Amino acid sequence encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of No. 66 The protein containing the column.   53. An isolated flea protease protein according to claim 9, wherein nfSP28₇₁₁And nfSP28₉₂₃Encoded by a nucleic acid molecule selected from the group consisting of Protein containing amino acid sequence   54. An isolated flea protease protein according to claim 9, wherein Nucleic acid molecule nfSP28₇₁₁Produced by a method comprising culturing cells expressing Protein.   55. An excipient and the nucleic acid molecule of claim 48 and the protein of claim 47; A therapeutic composition comprising a protective compound selected from the group consisting of:   56. 56. The therapeutic composition according to claim 55, further comprising an adjuvant, a key. And a component selected from the group consisting of carrier and mixtures thereof.   57. 56. The therapeutic composition of claim 55, further comprising a flea immunoglobulin. Reduces blood-sucking parasites by means other than reducing proteinase activity A composition comprising a compound to be added.   58. 56. The therapeutic composition according to claim 55, wherein parasites enter the animal. A composition that is administered to the animal to reduce the bleeding.