Expression of the non-structural protein NS1 of bluetongue virus in bacteria and yeast: identification of two antigenic sites at the amino terminus.
cDNA transcribed from bluetongue virus serotype 1 (Australia) dsRNA 5 coding for non-structural protein NS1 was amplified in a polymerase chain reaction and ligated downstream of the T7 RNA polymerase promoter in the bacterial expression plasmid pET-5b, as a fusion protein with glutathione S-transferase using the pGEX bacterial expression system or the metallothionein promoter in the yeast expression plasmid pYELC5. The linear epitopes bound by six monoclonal antibodies to NS1 were localised to two antigenic regions at the amino terminus by Western blots using a series of carboxy-terminal truncations of the NS1 protein overexpressed in Escherichia coli. Expression of truncated NS1 genes using the pGEX expression system in E. coli enabled a more detailed map of the two epitopes to be constructed. The first epitope is thought to lie between amino acid residues 40-59, while the second is defined by the peptide sequences flanking amino acid 96.
Gould
A R
AR
C.S.I.R.O., Australian Animal Health Laboratory, Geelong, Victoria.
Martyn
J C
JC
Stevenson
L
L
D001483:Base Sequence; D001820:Bluetongue virus / Q000737:chemistry* / Q000235:genetics; D002874:Chromosome Mapping; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology*
D000939:Epitopes; C078966:NS1 protein, bluetongue virus; D017361:Viral Nonstructural Proteins