High level expression of the major core protein VP7 and the non-structural protein NS3 of bluetongue virus in yeast: use of expressed VP7 as a diagnostic, group-reactive antigen in a blocking ELISA.
The major core protein VP7 and a non-structural protein NS3 of bluetongue virus serotype 1 have been synthesized from recombinant plasmids using both an in vitro transcription/translation system and a yeast expression system. Bluetongue virus genes were transcribed under the control of the bacteriophage SP6 promoter and the regulatable yeast metallothionein promoter. An indirect ELISA showed that expression of NS3 in yeast was inducible with 1 mM CuSO4 and VP7 synthesis was constitutive but could be further induced. The preferred procedure for antigen extraction from yeast was sonication for VP7 and SDS/NaOH treatment for NS3. Yeast-expressed VP7 antigen and a monoclonal antibody were used in a blocking ELISA to distinguish sera raised against bluetongue virus serotypes from those generated to viruses of the epizootic haemorrhagic disease serogroup.
Martyn
J C
JC
C.S.I.R.O., Australian Animal Health Laboratory, Geelong, Victoria, Australia.
Gould
A R
AR
Eaton
B T
BT
D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D001820:Bluetongue virus / Q000235:genetics*; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014170:Transformation, Genetic; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D017361:Viral Nonstructural Proteins
D000956:Antigens, Viral; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins