Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-04-12
1996-11-26
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 530324, 530822, G01N 33569, G01N 3353
Patent
active
055784534
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the cloning and sequencing of two Toxoplasma gondii gene fragments, the encoded polypeptides of which can be used as diagnostic antigens, for example in an enzyme-linked immunosorbent assay (ELISA), or as active components in a vaccine against Toxoplasma. The invention also extends to the production of recombinant polypeptides by expression of these gene fragments in a host cell, and to the use of the expressed recombinant polypeptides as diagnostic antigens or as vaccine components.
T.gondii is an obligate intracellular protozoan of the order Coccidia. Humans usually acquire infection by ingesting either infectious oocysts through contact with cats or cat faeces, or by eating meat that contains tissue cysts (Dubey & Beattie, 1988). Once infected the host harbours the parasite for life. In a chronically infected individual who subsequently develops an immunodeficiency, caused by either drugs or by an underlying disease, the infection can reactivate and cause morbidity and mortality. The parasite can also be pathogenic if first acquired during pregnancy and may cause abortion or congenital infections.
Diagnosis of T.gondii infections is usually based on serological demonstration of IgG or IgM antibodies. For such a test, T.gondii antigens are required, but since the parasite is obligately intracellular, these antigen preparations are contaminated with host cells. This occurs regardless of whether the parasites are grown in tissue culture or in the peritoneal cavities of mice (Abbas, 1967). In addition, parasites have to be cultured which may be hazardous for workers, and the growth of the parasite in mice or tissue culture is expensive. The production of recombinant fusion proteins in bacterial or yeast hosts overcomes these problems and allows a standardized and reproducible assay to be performed.
Experimental work leading to the present invention has included the cloning and sequencing of two gene fragments that encode polypeptides which react in the ELISA for the serological diagnosis of T.gondii antibodies in sera from naturally infected humans, sheep and cats, and in experimentally infected mice. The production of recombinant antigens in bacterial or yeast cells offers a potential for the development of highly standardized diagnostic tests and well-defined, reproducible, and inexpensive antigens.
In an attempt to clone T.gondii genes which encode polypeptides likely to be important diagnostically, two different strategies have been followed. The first was to clone genes for antigens that constituted a large part of the tachyzoites, the actively dividing stage of the parasite. To that end, a gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of T.gondii has been cloned and sequenced (Johnson et.al., 1989). However, antibodies to this antigen have since been found to be present in only a low percentage of patients with chronic toxoplasmosis (Tenter and Johnson, 1990). The second strategy was to screen a cDNA library with several different types of antibody to T.gondii, and to test the antigens commonly identified for their usefulness in a diagnostic ELISA for toxoplasmosis. Using this second strategy, the present inventors have now identified fragments of two genes termed H4 and H11, which encode polypeptides that can be used as antigens in the ELISA to measure antibodies to T.gondii in human sera and murine sera, and these gene fragments have been expressed as fusion proteins in host bacteria. Such fusion proteins have the potential to replace the requirement for T.gondii to be grown in tissue culture or the peritoneal cavities of mice (Abbas, 1967) to prepare diagnostic ELISA antigen.
According to one aspect of the present invention, there is provided a recombinant DNA molecule comprising a nucleotide sequence which codes for all or an antigenic portion of the H4 or H11 polypeptides of T.gondii. Such a recombinant DNA molecule is capable of being expressed as a polypeptide displaying the antigenicity of all or an antigenic fragment of the H4 o
REFERENCES:
patent: 5215917 (1993-06-01), De Araujo et al.
Johnson and Illana, "Cloning of Toxoplasma gondii gene fragments encoding diagnostic antigens", Gene, 99:127-132, (1991).
Tenter et al, "Recognition of recombinant Toxoplasma gondii antigens by human sera in an ELISA", Parasitol. Res., 77:197-203 (1991).
Prince et al, "Cloning of cDNAs encoding a 28 kilodalton antigen of Toxoplasma gondii", Mol. Biochem. Parasitol., 34:3-14 (1989).
Handman et al, "Detection and Characterization of Membrane Antigens of Toxoplasma gondii", J. Immunol., 124(6):2578-2583 (Jun. 1980).
Johnson et al, "Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleotide triphosphate hydrolase of Toxoplasma gondii", Gene, 85:215-220, (1989).
Johnson Alan M.
McDonald Peter J.
Scheiner Toni R.
The Flinders University of South Australia
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