Bacterial expression of the caprine arthritis-encephalitis virus gag and env proteins and their use in enzyme-linked immunosorbent assay.
The core protein and the transmembrane protein, encoded for the structural genes gag and env, respectively, of caprine arthritis-encephalitis virus were amplified by use of polymerase chain reaction, cloned into a pGEX-2T vector, and expressed in Escherichia coli as fusion proteins with the glutathione S-transferase at their C-terminus. The recombinant proteins were purified and evaluated by use of an ELISA. Sera from 269 goats were tested, and the results were compared with those obtained by use of immunoblot analysis. When results from both recombinant ELISA (r-ELISA) were compared, it appeared that the transmembrane glycoprotein was more immunoreactive than the core protein, because it was recognized by a higher percentage of sera from infected goats. When results of the 2 ELISA (p28 r-ELISA and p40 r-ELISA) were combined in parallel, they were comparable to those of the immunoblot test, with sensitivity of 100% and specificity of 98.3%. It was also found that use of both r-ELISA makes it possible to compare the variable immunoreactivity against gag and env viral antigens, which may be correlated with the disease state. The r-ELISA, using core and transmembrane proteins, appears to be highly sensitive and specific for detection of antibodies against caprine arthritis-encephalitis virus.
Clavijo
A
A
Department of Veterinary Microbiology and Immunology, University of Guelph, Ontario, Canada.
Thorsen
J
J
D000818:Animals; D016088:Arthritis-Encephalitis Virus, Caprine / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D005189:False Positive Reactions; D015686:Gene Products, env / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D015683:Gene Products, gag / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D015511:Goat Diseases; D006041:Goats; D016180:Lentivirus Infections / Q000175:diagnosis / Q000662:veterinary*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015686:Gene Products, env; D015683:Gene Products, gag; D011993:Recombinant Fusion Proteins