title: Expression of Neospora caninum NcSRS2 surface protein in Pichia pastoris and its application for serodiagnosis of Neospora infection.
abstract: Neospora caninum is considerd a major cause of abortion in cattle worldwide. The antigenic domain of NcSRS2 in N. caninum is an important surface antigen present in the membrane of this parasite. In the present study, the Pichia pastoris expression system proved to be a useful tool for the production of recombinant protein. The truncated NcSRS2 gene (by removal of the N-terminal hydrophobic sequence), was cloned in the vector pPICZalphaB, and integrated on the genome of the methylotrophic yeast P. pastoris. Subsequently, the NcSRS2 protein was expressed, purified, and characterized using naturally infected cattle sera and Mab 6xhistag. The recombinant protein NcSRS2 was present in the supernatant of the culture, where later it was concentrated and purified using ammonium sulfate (∼100 mg/ml). An indirect immunoenzymatic assay (ELISA) was performed using cattle sera from endemic N. caninum area.
authors.affiliation: Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
authors.forename: Amanda Fernandes
authors.initials: AF
authors.lastname: Pinheiro
authors.forename: Sibele
authors.initials: S
authors.lastname: Borsuk
authors.forename: Maria Elisabeth Aires
authors.initials: ME
authors.lastname: Berne
authors.forename: Luciano da Silva
authors.initials: Lda S
authors.lastname: Pinto
authors.forename: Renato
authors.initials: R
authors.lastname: Andreotti
authors.forename: Talita
authors.initials: T
authors.lastname: Roos
authors.forename: Barbara Couto
authors.initials: BC
authors.lastname: Rollof
authors.forename: Fábio Pereira Leivas
authors.initials: FP
authors.lastname: Leite
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D000954:Antigens, Surface / Q000235:genetics / Q000302:isolation & purification*; D001709:Biotechnology / Q000379:methods; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis*; D005591:Chemical Fractionation; D003001:Cloning, Molecular; D003048:Coccidiosis / Q000175:diagnosis / Q000662:veterinary*; D015870:Gene Expression; D005822:Genetic Vectors; D010843:Pichia / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012698:Serologic Tests / Q000379:methods; D014730:Veterinary Medicine / Q000379:methods
chemical_list: D000953:Antigens, Protozoan; D000954:Antigens, Surface; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C115389:SRS2 protein, Neospora caninum