Identification, expression in E. coli and insect cells of the non-structural protein NS2 encoded by mRNA2 of bovine coronavirus (BCV).
The coding part of mRNA 2 (ORF2) of BCV (F15 strain) was cloned and sequenced. The comparison of our sequence data with the sequence of the same ORF of BCV Quebec strain previously published revealed a major difference in the length of the C-terminal part of the NS2 protein. In vitro transcription and translation of ORF2 resulted in the synthesis of a single protein migrating with a Mr of 31 kDa. The ORF2 was fused in frame with the glutathione S transferase gene (GSH) in the pGEX vector. The fusion protein was synthesized as inclusion bodies which were concentrated and used to raise a monospecific antiserum. Alternatively the fusion protein was solubilized, purified by affinity chromatography and cleaved with Factor Xa to yield pure recombinant NS2. The ORF2 was also expressed in the baculovirus system and the recombinant proteins expressed in pro- and eukaryotic systems were compared on the basis of their size and immunoreactivity. Immunoprecipitation performed with the monospecific antiserum allowed us to identify NS2 in HRT18 infected cells, to follow its kinetic of synthesis, and to ascertain that NS2 was not incorporated in the virion as a minor structural component.
Boireau
P
P
CNEVA-Laboratoire Central de Recherches Vétérinaires, Maisons-Alfort, France.
Madelaine
M F
MF
Saulnier
D
D
Laporte
J
J
Vautherot
J F
JF
D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017938:Coronavirus, Bovine / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli; D015951:Factor Xa; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007106:Immune Sera; D009036:Moths; D017924:Nucleopolyhedroviruses; D016366:Open Reading Frames; D012367:RNA, Viral / Q000235:genetics*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D012004:Rectal Neoplasms; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000235:genetics*
D007106:Immune Sera; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; C087633:nonstructural protein, coronavirus; D015951:Factor Xa