Production of a recombinant major inner capsid protein for serological detection of epizootic hemorrhagic disease virus.
Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.
Luo
Lizhong
L
Canadian Food Inspection Agency, National Centre for Foreign Animal Disease, Winnipeg, Manitoba R3E 3M4, Canada. luol@inspection.gc.ca
Sabara
Marta I
MI
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D018164:Hemorrhagic Disease Virus, Epizootic / Q000235:genetics*; D007313:Insecta / Q000166:cytology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D012088:Reoviridae Infections / Q000097:blood / Q000175:diagnosis*; D012698:Serologic Tests / Q000592:standards
D036022:Capsid Proteins; D011994:Recombinant Proteins; C427245:VP7 protein, epizootic hemorrhagic disease virus-2