Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-03-06
2004-10-19
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023700, C435S320100, C435S069100, C435S069700, C435S243000, C435S252300, C435S325000, C435S348000, C435S006120
Reexamination Certificate
active
06806065
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to a
Rickettsia
protein, and more particularly to
Rickettsia felis
outer membrane protein and uses thereof.
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
In 1990, during a study investigating potential vectors for
Ehrlichia risticii
, rickettsia-like organisms were observed in the midgut epithelial cells of adult cat fleas,
Ctenocephalides felis
, by electron microscopy (Adams et al., 1990). The organisms were found only in a group of fleas obtained from El Labs, from which the original designation of the organism (ELB) was derived, and not in any of the other three sources of fleas. The organisms were described as having an ultrastructure and infection pattern similar to that of
R. typhi
. The organisms were 0.25-0.45 &mgr;m in diameter by 1.5 &mgr;m in length and were found not only in the midgut, but also in the tracheal matrix, muscles and reproductive tissues of the fleas. The organisms contained trilaminar cell walls that were characteristic of rickettsiae with a well-defined inner cell membrane and outer membrane. Measurements of the microcapsular layer, outer and inner leaflets of the outer membrane, and the periplasmic space strongly resembled other Rickettsia species.
The first attempts to characterize the organism involved the amplification of the 17-kDa antigen, citrate synthase (CS) and 190 kDa antigen (rompA) genes (Azad et al., 1992). The ELB agent was found to be distinguishable from
R. typhi
by restriction fragment length polymorphism (RFLP) analysis of the 17-kDa gene product digested with Aha II or Alu I. The CS gene was used to confirm that the organism found in the cat fleas was the ELB agent and not
R. typhi
, which can also be found occasionally in these fleas. The RFLP pattern of the CS gene amplified from the ELB agent in cat fleas differed from that of
R. typhi
. Attempts at that time to amplify the rompA gene from the ELB agent proved to be unsuccessful.
This study also provided evidence that the ELB agent can be transmitted transstadially and transovarially by two experimental observations. Unfed cat fleas that were negative by polymerase chain reaction (PCR) for the ELB agent tested positive for the 17-kDa protein gene of the ELB agent after feeding on infected cats (Azad et al., 1992). Also the ELB agent was present in freshly deposited eggs as determined by PCR (Azad et al., 1992).
The first serologic assays for the ELB agent were also conducted in this study. Antisera and monoclonal antibodies generated against
R. typhi
were used to examine smears of newly emerged fleas from both the El Labs and negative controls. Indirect immunofluorescent staining detected the ELB agent in the sample fleas, but not in the control fleas. In surveys of fleas in Los Angeles County, Calif. and in Texas, the 17-kDa and CS genes were used to investigate the natural occurrence of the ELB agent (Williams et al., 1992; Schriefer et al., 1994a). The results from both studies indicated that infection of the cat flea with the ELB agent is more prevalent than
R. typhi
. One study reported an infection rate of 3.8% for the ELB agent (Schriefer et al., 1994a).
The ELB agent has been identified in flea colonies from various regions of the United States through the use of RFLP analysis of PCR products of the 17-kDa and CS genes (Higgins et al., 1996). Analysis of the eight colonies showed that the colonies were infected with the ELB agent with a range of prevalence within each colony of 43 to 93%. The possible source of ELB in these colonies was subsequently traced to the El Labs, which provided fleas as starter stock or to replenish the colony. Attempts to infect mammalian cells and SCID mice with the ELB agent were not successful. Two publications are based upon the study of organisms considered to represent ELB agent propagated in cell culture (Radulovic et al., 1995a; Radulovic et al., 1995b); however, the cultured agent could not be reproducibility propagated and maintained in further culture.
In 1996, it was proposed that the ELB agent be designated as
Rickettsia felis
in recognition of its discovery and origin in the cat flea (Higgins et al., 1996). Subsequent additions to the knowledge of
R. felis
have used this name in the biomedical and scientific literature (Noden et al., 1998, Andersson & Andersson, 1999, Andersson et al., 1999, Bouyer et al., 1999).
There have been reports implicating the involvement of
R. felis
in human disease indicating its potential importance as a newly emerging pathogen. (Schriefer et al., 1994a). Given the evidence that infectious diseases are transmitted by
Ctenocephalides felis
cat fleas via
Rickettsia felis
, identification of
R. felis
proteins can lead to methods for interfering with/preventing
R. felis
infections based on knowledge of those
R. felis
proteins.
SUMMARY OF THE INVENTION
To this end, DNA from cat fleas naturally infected with
R. felis
was amplified by polymerase chain reaction utilizing primer sets specific for the 190-kDa surface antigen (rOmpA) and 17-kDa antigen genes. The entire 5513 base pair rompA gene was sequenced, characterized and found to have several unique features when compared to the rompA genes of other
Rickettsia
. Phylogenetic analysis of the partial sequence of the 17-kDa antigen gene indicates that
R. felis
is less divergent from the spotted fever group (SFG) rickettsiae than from the typhus group rickettsiae. The organism is passed transstadially and transovarially, and infection in the cat flea has been observed in the midgut, tracheal matrix, muscle, hypodermis, ovaries, and testes.
The subject invention provides an isolated nucleic acid molecule encoding a
Rickettsia felis
outer membrane protein. The invention also provides an antisense nucleic acid molecule complementary to at least a portion of the mRNA encoding the
Rickettsia felis
outer membrane protein.
The isolated nucleic acid molecules of the invention can be inserted into suitable expression vectors and/or host cells. Expression of the nucleic acid molecules encoding the
Rickettsia felis
outer membrane protein results in production of
Rickettsia felis
outer membrane protein in a host cell. Expression of the antisense nucleic acid molecules in a host cell results in decreased expression of the
Rickettsia felis
outer membrane protein.
The invention further provides a ribozyme having a recognition sequence complementary to a portion of mRNA encoding a
Rickettsia felis
outer membrane protein. The ribozyme can be introduced into a cell to also achieve decreased expression of
Rickettsia felis
outer membrane protein in the cell.
The invention further provides a method of screening a substance for the ability of the substance to modify
Rickettsia felis
outer membrane protein function, and a method of obtaining DNA encoding a
Rickettsia felis
outer membrane protein.
Further provided is an isolated nucleic acid molecule encoding a
Rickettsia felis
outer membrane protein, wherein the nucleic acid molecule encodes a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence. The second amino acid sequence is as shown in SEQ ID NO:2.
The invention further provides a DNA oligomer capable of hybridizing to a nucleic acid molecule encoding a
Rickettsia felis
outer membrane protein. The DNA oligomer can be used in a method of detecting presence of a
Rickettsia felis
outer membrane protein in a sample, which method is also provided by the subject invention.
The invention also provides an isolated
Rickettsia felis
outer membrane protein, and antibodies or antibody fragments specific for the
Rickettsia felis
outer membrane protein. The antibodies and antibody fragments can be used to detect the presence of the
R
Bouyer Donald H.
Crocquet-Valdes Patricia
Stenos John
Walker David H.
Graser Jennifer E.
Rogalsky & Weyand, LLP
The Board of Regents of the University of Texas System
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