Expression of the outer capsid proteins VP2 and VP5 of bluetongue virus in Saccharomyces cerevisiae.
cDNAs transcribed from bluetongue virus serotype 1 (Australia) ds RNA 2 and ds RNA 6 coding for the major neutralising antigen VP2 and the outer capsid protein VP5, respectively, were amplified in polymerase chain reactions and ligated downstream of the copper-inducible metallothionein promoter in the yeast expression plasmid pYELC5. Saccharomyces cerevisiae transformed with the recombinant plasmid pYELC5-VP2 expressed full-length VP2 only following induction with 1 mM CuSO4 and reached the maximum level after 6 h. In contrast, S. cerevisiae transformants harboring the recombinant plasmid pYELC5-VP5 expressed VP5 constitutively, although induction increased the level to a maximum after 4 h. A sheep trial was done testing the recombinant proteins, however it was shown that none of these were effective immunogens for eliciting a protective response against a subsequent challenge with bluetongue virus. An analysis of the yeast expression products for the VP2 outer coat protein using a panel of monoclonal antibodies showed that the yeast expressed VP2 was in a conformation different from native VP2 and hence probably unable to elicite an appropriate protective immune response.
Martyn
J C
JC
CSIRO, Australian Animal Health Laboratory, Geelong, Victoria.
Gould
A R
AR
Yu
M
M
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D001820:Bluetongue virus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D036022:Capsid Proteins; D002417:Cattle; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012756:Sheep; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D000914:Antibodies, Viral; D036022:Capsid Proteins; D018076:DNA, Complementary; D012367:RNA, Viral; D011994:Recombinant Proteins; C082513:VP2 protein, infectious bursal disease virus; D015678:Viral Structural Proteins