Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
2001-03-16
2003-12-09
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C435S041000, C435S069100, C435S069300, C435S069700, C435S070100, C435S243000, C435S252100, C435S320100, C435S325000, C435S352000, C536S023100, C536S023500, C424S184100, C424S185100, C424S190100, C424S191100, C424S192100, C424S234100, C424S265100
Reexamination Certificate
active
06660269
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to molecular cloning and characterization of homologous 28-kDa protein genes in
Ehrlichia canis
, a multigene locus encoding the 28-kDa homologous proteins of
Ehrlichia canis
and uses thereof.
2. Description of the Related Art
Canine ehrlichiosis, also known as canine tropical pancytopenia, is a tick-borne rickettsial disease of dogs first described in Africa in 1935 and the United States in 1963 (Donatien and Lestoquard. 1935; Ewing, 1963). The disease became better recognized after an epizootic outbreak occurred in United States military dogs during the Vietnam War (Walker et al., 1970)
The etiologic agent of canine ehrilichiosis is
Ehrlichia canis
, a small, gram-negative, obligate intracellular bacterium which exhibits tropism for mononuclear phagocytes (Nyindo et al., 1971) and is transmitted by the brown dog tick,
Rhipicephalus sanguineus
(Groves et al., 1975). The progression of canine ehrlichiosis occurs in three phases, acute, subclincal and chronic. The acute phase is characterized by fever, anorexia, depression, lymphadenopathy and mild thrombocytopenlia (Troy and Forrester, 1990). Dogs typically recover from the acute phase, but become persistently infected carriers of the organism without clinical signs of disease for months or even years (Harrus et al., 1998). A chronic phase develops in some cases that is characterized by thrombocytopenia, hyperglobulinemia, anorexia, emaciation, and hemorrhage, particularly epistaxis, followed by death (Troy and Forrester, 1990).
Regulation of surface antigenicity may be an important mechanism for the establishment of such persistent infections in the host. Although disease pathogenesis is poorly understood, multigene families described in members of the related genera Ehrlichia, Anaplamsa, and Cowdria may be involved in variation of major surface antigen expression thereby evading immune surveillance.
Anaplasma marginale
, an organism closely related to
E. canis
, exhibits variation of major surface protein 3 (msp-3) genes resulting in antigenic polymorphism among strains (Alleman et al., 1997).
Molecular taxonomic analysis based on the 16S rRNA gene has determined that
E. canis
and
E. chaffeensis
, the etiologic agent of human nionocytic ehrlichiosis (HME), are closely related (Anderson et al., 1991; Anderson et al., 1992: Dawson et al., 1991; Chen et al., 1994). Considerable cross reactivity of the 64, 47, 40, 30, 29 and 23-kDa antigens between
E. canis
and
E. chaffeensis
has been reported (Chen et al., 1994; Chen et al., 1997; Rikihisa et al., 1994; Rikihisa et al., 1992). Analysis of immunoreactive antigens with human and canine convalescent phase sera by immunoblot has resulted in the identification of numerous immunodominant proteins of
E. canis
, including a 30-kDa protein (Chen et al., 1997). In addition, a 30-kDa protein of
E. canis
has been described as a major immunodominant antigen recognized early in the immune response that is antigenically distinct from the 30-kDa protein of
E. chaffeensis
(Rikihisa et al., 1992; Rikihisa et al., 1994). Other immunodominant proteins of
E. canis
with molecular masses ranging from 20 to 30-kDa have also been identified (Brouqui et al., 1992; Nyindo et al. 1991; Chen et al., 1994; Chen et al., 1997).
Homologous 28-32 kDa immunodominant proteins encoded by multigene families have been reported in related organisms including,
E. chaffeensis
and
Cowdria ruminantium
(Sulsona et al., 1999; Ohashi et al., 1998a; Reddy et al., 1998). Recently, characterization of a 21 member multigene family encoding proteins of 23 to 28-kDa has been described in
E. chaffeensis
(Yu et al., 2000). The
E. chaffeensis
28-kDa outer membrane proteins arc surface exposed, and contain three major hypervariable regions (Ohashi et al., 1998a). The recombinant
E. chaffeensis
P28 appeared to provide protection against homologous challenge infection in mice, and antisera produced against the recombinant protein cross reacted with a 30-kDa protein of
E. canis
(Ohashi et al., 1998a). Diversity in the p28 gene among
E. chaffeensis
isolates has been reported (Yu et al., 1999a), and studies using monoclonal antibodies have further demonstrated diversity in the expressed P28 proteins (Yu et al., 1993). Conversely, complete conservation of a p28 genes in geographically different isolates of
E. canis
has been reported and suggests that
E. canis
may be conserved in North America (McBride et al., 1999, 2000).
The prior art is deficient in the lack of cloning and characterization of new homologous 28-kDa immunoreactive protein genes of
Ehrlichia canis
and a single multigene locus containing the homologous 28-kDa protein genes. Further, The prior art is deficient in the lack of recombinant proteins of such immunoreactive genes of
Ehrlichia canis
. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
Certain embodiments of the present invention describe the molecular cloning, sequencing, characterization, and expression of homologous mature 28-kDa immunoreactive protein genes of
Ehrlichia canis
(designated p28-1, -2, -3, -5, -6, -7, -9), and the identification of a single locus (10,677-bp) containing nine 28-kDa protein genes of
Ehrlichia canis
(p28-1 to p28-9). Eight of the p28 genes were located on one DNA strand, and one p28 gene was found on the complementary strand. The nucleic acid homology among the nine p28 gene members was 37 to 75%, and the amino acid homology ranged from 28 to 72%.
In one embodiment of the present invention, there are provided DNA sequences encoding a 30-kDa immunoreactive protein of
Ehrlichia canis
. Preferably, the protein has an amino acid sequence selected from the group consisting of SEQ ID No. 2, 4, 6, 40, 42, 44, 46 and the gene has a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, 3, 5, 39, 41, 43, 45 and is a member of a polymorphic multiple gene family. Generally, the protein has an N-terminal signal sequence which may be cleaved after post-translational process resulting in the production of a mature 28-kDa protein. Furthermore, the genes encoding 28-kDa proteins are preferably contained in a single multigene locus, which has the size of 10,677 bp and encodes nine homologous 28-kDa proteins of
Ehrlichia canis.
In another embodiment of the present invention, there is provided an expression vector comprising a gene encoding a 28-kDa immunoreactive protein of
Ehrlichia canis
and capable of expressing the gene when the vector is introduced into a cell.
In still another embodiment of the present invention. there is provided a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID No. 2, 4, 6, 40, 42, 44, and 46. Preferably, the amino acid sequence is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, 3, 5, 39, 41, 43, and 45. Preferably, the recombinant protein comprises four variable regions which may be surface exposed, hydrophilic and antigenic. The recombinant protein may be useful as an antigen.
In yet another embodiment of the present invention. there is provided a method of producing the recombinant protein comprising the steps of obtaining a vector that comprises an expression region comprising a sequence encoding the amino acid sequence selected from the group consisting of SEQ ID No. 2, 4, 6, 40, 42, 44, and 46 operatively linked to a promoter; transfecting the vector into a cell; and culturing the cell under conditions effective for expression of the expression region.
The invention may also be described in certain embodiments as a method of inhibiting
Ehrlichia canis
infection in a subject comprising the steps of: identifying a subject prior to exposure or suspected of being exposed to or infected with
Ehrlichia canis
; and administering a composition comprising a 28-kDa antigen of
Ehrlichia canis
in
McBride Jere W.
Walker David H.
Yu Xue-Jie
Adler Benjamin Aaron
Baskar Padmavathi
Research Development Foundation
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