Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
2000-04-07
2003-12-02
Navarro, Mark (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S350000
Reexamination Certificate
active
06657045
ABSTRACT:
BACKGROUND
Cryptosporidium parvum
(
C. parvum
), an intestinal Apicomplexan parasite, is a significant cause of diarrheal disease worldwide (Griffiths, 1998
. Adv Parasitol
. 40:37-85). In immunocompetent individuals, the disease is usually self-limiting, but it can be chronic and life threatening in immunocompromised patients such as those with AIDS.
C. parvum
has also been associated with diarrheal disease in children in daycare centers, travelers, animal handlers and hospital personnel. Recently, the parasite has gained notoriety as the causative agent of numerous outbreaks of waterborne diarrheal disease. There is currently no effective, specific therapy approved for disease caused by this parasite.
C. parvum
infection is initiated by ingestion of oocysts which, upon exposure to favorable conditions within the host, undergo excystation. Released sporozoites attach to and invade host cells forming a parasitophorus vacuole where the parasite undergoes further intracellular development through asexual and sexual cycles eventually leading to formation of new oocysts that are capable of reinitiating the infectious cycle. The ultrastructural aspects of the processes of attachment and invasion and various factors influencing attachment have been characterized (Tzipori et al., 1998
. Adv Parasitol
. 40:5-36; Hamer etal., 1994
. Infect Immun
. 62:2208-2213). However, the molecular basis of these host-parasite interactions is not well understood (Ward et al., 1998
. Adv Parasitol
. 40:151-185).
SUMMARY
The present invention is based, in part, on the discovery of a gene, gp40gp 15 (SEQ ID NO:1). The gp40gp15 cDNA described below is a 981 nucleotide sequence which encodes a 49 KDa precursor protein (SEQ ID NO:6) of
C. parvum
. The precursor protein is proteolytically cleaved to yield two glycoproteins, gp40 (SEQ ID NO:2) and gp15 (SEQ ID NO:8). gp40 protein is a 40 KDa glycoprotein which is present in oocysts and sporozoites and is also shed from the parasite during invasion. gp40 protein mediates sporozoite attachment and invasion of host cells and is therefore useful as a target for prevention or therapy of cryptosporidiosis.
Accordingly, in one aspect, the invention features an isolated nucleic acid molecule comprising a nucleotide sequence encoding a gp40 protein or a biologically active portion thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of a gp40-encoding nucleic acid (e.g., gp40 mRNA). The gp40 nucleotide sequence, nucleotides 1-666 of SEQ ID NO:1; SEQ ID NO:3, encodes a 222 amino acid protein (SEQ ID NO:2). gp40 protein includes a signal sequence of around 30 amino acids (from amino acid 1 to amino acid 30 of SEQ ID NO:2; SEQ ID NO:4) and has a mature protein length of 192 amino acids (amino acids 31-222 of SEQ ID NO:2; SEQ ID NO:5). gp40 protein possesses a polyserine domain (at amino acids 37 to 55 of SEQ ID NO:2) with multiple predicted O-glycosylation sites. The protein also has a hydrophobic stretch of amino acids in its C-terminal region consistent with that required for GPI-anchoring.
In one embodiment, an isolated gp40 nucleic acid molecule includes the nucleotide sequence of SEQ ID NO:3, or a complement of these nucleotide sequences. In another embodiment, the isolated nucleic acid molecule of the invention includes a nucleotide sequence which hybridizes, preferably under stringent conditions, to or has at least about 60-65%, preferably at least about 70-75%, more preferably at least about 80-85%, and even more preferably at least about 90-95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence shown in SEQ ID NO:3, or a portion thereof. In yet another embodiment, the isolated nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. gp40 nucleic acid molecules can encode a protein which possesses at least one of the gp40 activities described herein, e.g., the ability to bind human intestinal epithelial cells.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5 such that the protein or portion thereof possesses a gp40 biological activity, e.g., the ability to bind intestinal epithelial cells. The protein or portion thereof encoded by the nucleic acid molecule maintains the ability to play a role in mediating the attachment and invasion of host cells by
C. parvum
. In yet another embodiment, the protein encoded by the nucleic acid molecule has at least about 60-70%, preferably at least about 80-85%, and more preferably at least about 86%, 88%, 90%, and most preferably at least about 90-95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. In one embodiment, the protein is a full length protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2.
In another embodiment, the isolated nucleic acid molecule encodes a portion of a gp40 protein, e.g., a portion which includes a sequence encoding a polyserine domain with multiple O-glycosylation sites.
In another embodiment, the isolated nucleic acid molecule encodes a gp40 protein, or portion thereof, which has at least about 55%, 65%, 75%, 85% or 95% identity to SEQ ID NO:2 or SEQ ID NO:5, and has one or more of the following activities:1) it is involved in parasite-host interactions; 2) it interacts, directly or indirectly, with a host cell, e.g., a human intestinal epithelial cell; or 3) it modulates the ability of
C. parvum
sporozoites to attach and invade a host cell.
In another embodiment, the isolated nucleic acid molecule is at least 15 (30, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:3.
Given the disclosure herein of gp40-encoding sequence (e.g., SEQ ID NO:3), antisense nucleic acid molecules (i.e., molecules which are complementary to the gp40 nucleotide sequence) are also provided by the invention.
In another embodiment, the encoded gp40 protein differs in amino acid sequence at least 1 to as many as 2, 3, 5, 10, 20 or 40 residues from a sequence in SEQ ID NO:2 or SEQ ID NO:5. In one embodiment, the differences are such that the gp40 encoded protein exhibits a gp40 biological activity, e.g., the encoded gp40 protein retains a biological activity of a naturally-occurring gp40, e.g., the gp40 protein of SEQ ID NO:2 or SEQ ID NO:5.
In another embodiment the encoded gp40 protein includes a gp40 sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
The invention also features vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce gp40 protein by culturing the host cell in a suitable medium. The gp40 protein can be then isolated from the medium or the host cell.
In yet another embodiment, the biologically active portion of the gp40 protein includes a domain or motif, preferably a domain or motif which has a gp40 activity. The motif can be e.g., a short hydrophobic region at the C-terminus, consistent with that required for addition of a GPI anchor; a polyserine domain which has multiple predicted O-glycosylation sites which may be used by the parasite to bind a host cell; a carbohydrate domain; or a N-glycosylation site.
The invention also provides an isolated preparation of a gp40 protein. In one embodiment, the gp40 protein includes the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. In another embodiment, the invention pertains to an isolated full length protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2 (encoded by SEQ ID NO:3) or the mature amino acid sequence of SEQ ID NO:5. In yet another embodiment, the protein has at least about 60-70%, p
Cevallos Ana Maria
Hamer David
Keusch Gerald
Ward Honorine
Fish & Richardson P.C.
Navarro Mark
New England Medical Center Hospitals Inc.
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