Expression of coxsackievirus B3 capsid proteins in Escherichia coli and generation of virus-specific antisera.
Subgenomic fragments of cloned infectious coxsackievirus B3 (CVB3) cDNA up to the size of the complete coding sequence of the viral polyprotein were inserted into the prokaryotic expression vector pPLc24 and expressed in Escherichia coli. Fusion proteins, containing 54 amino acids of MS2 replicase at their amino terminus followed by different parts of the CVB3 structural proteins, were expressed from several constructs. The expression product of a plasmid encoding the capsid proteins VP4, VP2, and the amino-terminal part of VP3 was obtained in high amounts. However, primary expression products containing the complete viral capsid precursor VP4-VP1 were completely degraded, indicating the presence of domains downstream from VP3 that are accessible to E. coli proteases. This finding is consistent with the observation that the structural intact expression product of the separately subcloned VP1 gene is also extremely unstable and consequently obtained only in low amounts. Two fusion proteins of non-overlapping parts of the viral structural proteins containing VP4, VP2, and VP3 or VP1, respectively, were isolated and used for the generation of antisera in rabbits. The antisera obtained recognize distinct CVB3 structural proteins in infected cell cultures as well as from purified CVB3 preparations. In addition, significant cross-reactivity of the described antisera with the corresponding structural proteins of other enteroviruses was observed, indicating that these antisera provide a valuable tool for an improved broad spectrum diagnosis of enteroviral infections.
Werner
S
S
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Klump
W M
WM
Schönke
H
H
Hofschneider
P H
PH
Kandolf
R
R
D002213:Capsid / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D029822:Enterovirus B, Human / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D007106:Immune Sera; D010957:Plasmids; D014760:Viral Fusion Proteins / Q000235:genetics
D004279:DNA, Viral; D007106:Immune Sera; D014760:Viral Fusion Proteins