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.
Subject(s)
Antigens, Viral/metabolism , Infectious Anemia Virus, Equine/genetics , Antigens, Viral/genetics , Blotting, Western , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Gene Expression , Gene Expression Profiling , Genetic Vectors , Pichia/genetics , Plasmids , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The seroprevalence of caprine arthritis-encephalitis virus (CAEV) in the Cariri Region of Paraíba State, Brazil, was determined in 60 goat herds using the agar gel immunodiffusion test. The overall seroprevalence was 8.2%, with seropositivity in 21/60 (35%) herds and 13/15 (86.6%) municipalities. Bucks had a significantly higher frequency of infection (28.3%) than does (5.9%), and bucks that originated in other states had a significantly higher frequency of infection (76.5%) than those from Paraíba State (9.3%).