Recombinant antigen of Taenia solium metacestodes

Details Recombinant antigen of Taenia solium metacestodes Recombinant antigen of Taenia solium metacestodes

2000-09-29

2003-07-08

Smith, Lynette R. F. (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S069100, C435S007100, C530S350000, C536S023100

Reexamination Certificate

active

06589752

ABSTRACT:

FIELD OF THE INVENTION
This invention is related to the field of recombinant antigen of
Taenia solium
metacestodes and a method of detecting neurocysticerocosis disease.
BACKGROUND OF THE INVENTION
Neurocysticercosis (NCC), which is caused by infection of the central nervous system with
Taenia solium
metacestodes (TSM), is a major cause of neurological diseases in Asian, African and Latin American people [1, 2, 3]. Surveillance in endemic areas showed that it is of public health concern causing considerable mortality and chronic morbidity as well as economic losses in endemic areas [3, 4]. Substantial evidence has shown that up to 50% of late-onset epilepsy is due to NCC [3-6]. In the United States, over 1,000 cases of NCC are identified each year mostly in immigrants [3, 7].
The diagnosis of NCC can be achieved with a high degree of accuracy by brain computed tomography (CT)/magnetic resonance (MR) [8-10]. These methods are, however, expensive, inaccessible in most endemic areas. Moreover, the number, size and location of the lesions and stage of infection may vary individually. The development of immunological tests, based on the detection of specific antibodies either in sera or in cerebrospinal fluid (CSF), provides a simple and reliable adjuvant for the diagnosis of NCC. Unfortunately, most of the tests employing the crude antigens lack both sensitivity and specificity; cross-reactions occur frequently with other parasitic infections, especially with cystic echinococcosis (CE) and alveolar echinococcosis (AE), which are caused by larval
Echinococcus granulosus
and
E.multilocularis,
respectively [11, 12]. Over the past two decades, many efforts have been directed toward characterizing specific antigens of TSM either from whole worm or from cyst fluid (CF) [11-16]. The low molecular weight antigenic components ranging 8-50 kDa of TSM have attracted particular attention due to their high specificity. In a study with TSM crude soluble extracts by immonoblot, two polypeptides at 8 and 26 kDa were recognized specifically by serum/CSF antibodies of NCC patients or TSM infected pig sera [11, 17] and specific as high as 98% and 100%, respectively. This assay has been widely used for immunodiagnosis of individual patients and for seroepidemiological surveys [4, 18, 19].
Our research interest has been focused on the identification and isolation of specific antigens from CF of TSM. We have previously demonstrated a 10 kDa antigen of TSM CF allowed a high reliability in detecting the specific anti-TSM antibodies in human NCC [12, 15, 16]. Biochemical studies with a monoclonal antibody (mAb) revealed the 10 IcDa antigen is a subunit of a 150 kDa thermostable protein complex [12, 20]. In an experiment to isolate the 10 kDa protein from TSM CF either by mAb-ligand immunoaffinity chromatography [12, 20] or by isoelectric focusing [16], we found that the 10 kDa protein was always linked to other two proteins (15 and 7 kDa, respectively) and could not be separated individually. Nevertheless, immunological evaluation of a fraction containing the three components by both immunoblot and enzyme-linked immunosorbent assay (ELISA) with sera/CSF from NCC and other helminthic infections including AE and CE demonstrated high sensitivity and specificity, both >90% [12, 15, 16].
In the present study, we describe the cloning and sequencing of a cDNA encoding a TSM 10 kDa protein and its expression in
E. coli
. We evaluated its diagnostic value and provided evidence that this recombinant antigen is highly useful in differentiating active NCC from chronic cases and other parasitic infections.
SUMMARY OF THE INVENTION
This invention is directed to an isolated DNA sequence encoding an antigen of
Taenia solium
metacestodes. The DNA sequence is prepared by cloning a cDNA library encoding a 10 kDa protein from
Taenia solium
metacestodes. The DNA sequence comprises the 258-base sequence of the ORF DNA sequence set out in
FIG. 2
, or its complementary strand, or other DNA sequence which hybridizes to it under stringent conditions. Alternatively, the DNA sequence comprises the 198-base sequence of the truncated fragment of the ORF DNA sequence without the N-terminal hydrophobic sequence, as set out in
FIG. 2
, or its complementary strand, or other DNA sequence which hybridizes to it under stringent conditions
This invention is also directed to a purified recombinant
Taenia solium
metacestodes protein characterized by a molecular weight about 7,000 daltons on SDS-PAGE, wherein said protein is encoded by the 198 bases of the truncated fragment of the ORF DNA sequence without the N-terminal hydrophobic sequence, set out in FIG.
2
. This recombinant
Taenia solium
metacestodes protein provides antigencity and can be used in the immunoassay to detect the presence of antibody against
Taenia solium
metacestodes and to diagnose neurocysticercosis diseases. Alternatively, a fusion protein of glutathione S-transferase and the 7,000 kDa
Taenia solium
metacestodes protein can be used as an antigen to detect the presence of antibody against of
Taenia solium
metacestodes in a mammalian subject biological fluid.


REFERENCES:
patent: 5599692 (1997-02-01), Harrison et al.
patent: 5874251 (1999-02-01), Zarlenga et al.
patent: 6156505 (2000-12-01), Steinbruch et al.
Chung,J.Y. et al. “A recombinant 10-kDa Protein ofTaenia soliumMetacestodes . . . ”, Journal of Infectious Diseases vol. 180, pp 1307-1315, Oct. 1999.*
Gencore Acc# AF076609, Sequence alignment.*
Harrison et al. “Differential diagnosis ofTaenia saginataandTaenia soliumwith DNA probes” Parasitology, vol. 100, pp. 459-461, 1990.*
Chapman et al. “Isolation and characterization of species-specific DNA probes fromTaenia soliumandTaenia saginataand their use in an egg detection assay”, Journal of Clinical Microbiology, vol. 33, No. 5, pp. 1283-1288, May 1995.*
Rishi et al. “Molecular cloning ofTaenia soliumgenomic DNA and characterization of taeniid cestodes by DNA analysis” Parasitology, vol. 97 pp. 161-176, 1988.*
Cho, SY, et al., “Biochemical properties of a purified protein in systic fluid ofTaenia Soliummetacestodes,”Korean J. Parasitol. 24:25-41 (1986).
Fernandez, V, et al. Molecular characterization of a novel 8-kDa subunit ofEchinococcus granulosisantigen B,Mol. Biochem. Parasitol. 77:247-250 (1996).
Gevorkian, G, et al. “Immunodominant synthetic peptides ofTaenia crassicepsin murine and numan cysticercosis,”Immunol. Lett. 49:185-189 (1996).
Hayunga EG, et al., “Development of a serological assay for cysticercosis, using an antigen isolated from Taenia spp cyst fluid,”Am. J. Vet. Res. 52:462-470 (1991).
Tsang VCW, et al., “An enzyme-linked immunoelectrotrasnfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Tacnia solium),”J. Infect. Dis. 159:50-59 (1989).
Zarlanga DS, et al., “ATaenia crassicepscDNA sequence encoding a putative immunodiagnostic antigen for bovine cysticercosis,”Mol. Biochem. Parasitol. 67:215-223 (1994).

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