title: Production of Equine Infectious Anemia Virus (EIAV) antigen in Pichia pastoris.
abstract: Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropriate enzymatic digestion. P. pastoris clones transformed with the pPICZαAp26 construction were induced to produce the recombinant p26 protein (rp26) under the regulation of alcohol oxidase 1 promoter by adding methanol to the culture medium. The p26 gene expression was detected by RT-PCR and the production of rp26 was confirmed by dot blotting, Western blotting, ELISA and AGID. The P. pastoris expression system was capable of producing a functional EIAV p26 protein that can be used directly in the functionality tests without requiring laborious purification or recovery steps. This is the first reported study of EIAV p26 protein production in yeast cells.
authors.affiliation: Department of Veterinary Medicine, Federal Rural University of Pernambuco-UFRPE, Av. Dom Manoel de Medeiros, s/n, Dois Irmãos, CEP 52171-900, Recife, Pernambuco, Brazil. lcacoutinho@gmail.com
authors.forename: Luciana Cavalcanti
authors.initials: LC
authors.lastname: de Arruda Coutinho
authors.forename: André Luiz Santos
authors.initials: AL
authors.lastname: de Jesus
authors.forename: Karin Florêncio Lins
authors.initials: KF
authors.lastname: de Paiva Fontes
authors.forename: Eliane Campos
authors.initials: EC
authors.lastname: Coimbra
authors.forename: Filipe Colaço
authors.initials: FC
authors.lastname: Mariz
authors.forename: Antonio Carlos
authors.initials: AC
authors.lastname: de Freitas
authors.forename: Rita
authors.initials: R
authors.lastname: de Cássia Carvalho Maia
authors.forename: Roberto Soares
authors.initials: RS
authors.lastname: de Castro
mesh_terms: D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000956:Antigens, Viral; D011994:Recombinant Proteins