Antigenic characterisation of yeast-expressed lyssavirus nucleoproteins.
In Europe, three genotypes of the genus Lyssavirus, family Rhabdoviridae, are present, classical rabies virus (RABV, genotype 1), European bat lyssavirus type 1 (EBLV-1, genotype 5) and European bat lyssavirus type 2 (EBLV-2, genotype 6). The entire authentic nucleoprotein (N protein) encoding sequences of RABV (challenge virus standard, CVS, strain), EBLV-1 and EBLV-2 were expressed in yeast Saccharomyces cerevisiae at high level. Purification of recombinant N proteins by caesium chloride gradient centrifugation resulted in yields between 14-17, 25-29 and 18-20 mg/l of induced yeast culture for RABV-CVS, EBLV-1 and EBLV-2, respectively. The purified N proteins were evaluated by negative staining electron microscopy, which revealed the formation of nucleocapsid-like structures. The antigenic conformation of the N proteins was investigated for their reactivity with monoclonal antibodies (mAbs) directed against different lyssaviruses. The reactivity pattern of each mAb was virtually identical between immunofluorescence assay with virus-infected cells, and ELISA and dot blot assay using the corresponding recombinant N proteins. These observations lead us to conclude that yeast-expressed lyssavirus N proteins share antigenic properties with naturally expressed virus protein. These recombinant proteins have the potential for use as components of serological assays for lyssaviruses.
Kucinskaite
Indre
I
Institute of Biotechnology, V. Graiciūno 8, 02241, Vilnius, Lithuania.
Juozapaitis
Mindaugas
M
Serva
Andrius
A
Zvirbliene
Aurelija
A
Johnson
Nicholas
N
Staniulis
Juozas
J
Fooks
Anthony R
AR
Müller
Thomas
T
Sasnauskas
Kestutis
K
Ulrich
Rainer G
RG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification / Q000378:metabolism; D000914:Antibodies, Viral / Q000302:isolation & purification / Q000378:metabolism; D000956:Antigens, Viral / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D019085:Fluorescent Antibody Technique, Direct; D015870:Gene Expression; D015151:Immunoblotting; D018114:Lyssavirus / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D022701:Virosomes / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D009698:Nucleoproteins; D014764:Viral Proteins; D022701:Virosomes