title: High-level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izatnagar isolate) and their application in serodiagnosis of goat toxoplasmosis.
abstract: Toxoplasmosis, caused by Toxoplasma gondii, is a disease of economic importance in livestock, especially in sheep and goats, where it causes abortion. Although several serological tests are in use for diagnosis of infection, production of reliable reagents is a constraint. An 814 bp sequence coding for a truncated surface antigen surface antigen 1 (SAG1), a tachyzoite stage-specific protein, as well as a 657 bp sequence coding for granule protein 7 (GRA7), a dense granule protein were PCR amplified from the genomic DNA of T. gondii. The amplified products were ligated in pET-32b(+) and pET-32c(+) expression vectors, respectively and subsequently transformed into BL21(DE3)pLysS cells. A high-level expression of the histidine-tagged SAG1 and GRA7 fusion proteins were obtained after 7h of incubation. The recombinant proteins were purified using Ni-NTA column and were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis using reference positive sera from goat, rabbit and humans at 1:100 dilution. Subsequently, the diagnostic efficiency of the recombinant proteins, either individually or as a cocktail of the recombinant proteins, was assessed with 56 reference goat sera by enzyme-linked immunosorbent assay (ELISA). The immunoreactivity of the refolded SAG1 and GRA7 was evidenced by high OD values. The reactivity of the recombinant proteins as a cocktail preparation was more than that of individual proteins in ELISA and could detect accurately the infection in goats. This is the first report of serological detection of caprine toxoplasmosis by ELISA using a cocktail of recombinant Toxoplasma proteins.
authors.affiliation: Protozoology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
authors.forename: Gopal Viswanathan
authors.initials: GV
authors.lastname: Velmurugan
authors.forename: Anup Kumar
authors.initials: AK
authors.lastname: Tewari
authors.forename: Jammi Raghavendra
authors.initials: JR
authors.lastname: Rao
authors.forename: Surajit
authors.initials: S
authors.lastname: Baidya
authors.forename: Manchukonda Udaya
authors.initials: MU
authors.lastname: Kumar
authors.forename: Ashok Kumar
authors.initials: AK
authors.lastname: Mishra
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D015511:Goat Diseases / Q000175:diagnosis* / Q000469:parasitology; D006041:Goats; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D012698:Serologic Tests / Q000662:veterinary; D014122:Toxoplasma / Q000378:metabolism*; D014124:Toxoplasmosis, Animal / Q000175:diagnosis* / Q000469:parasitology
chemical_list: D000953:Antigens, Protozoan; C112573:GRA7 protein, Toxoplasma gondii; D015800:Protozoan Proteins; C047168:SAG1 antigen, Toxoplasma