Expression of two bovine rotavirus non-structural proteins (NSP2, NSP3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins.
Studies on rotavirus non-structural proteins have been hampered in the past by difficulties in obtaining monospecific reagents. To make such reagents available, we have expressed in the baculovirus system NSP2 and NSP3 (formerly called NS35 and NS34, respectively) of the bovine rotavirus RF and produced hybridomas against these proteins. Full-length DNA copies of RNA segments 7 (coding for NSP3) and 8 (coding for NSP2) of the virus strain RF were cloned and sequenced. Each cDNA was inserted in the transfer vector pVL941 and used to transfect Spodoptera frugiperda cells (Sf9). Recombinant baculoviruses encoding these proteins were obtained. Infection of Sf9 cells with these recombinant viruses resulted in a high level of expression of NSP2 and NSP3 (range of 1 microgram per 10(6) cells). Monoclonal antibodies (MAbs) were elicited by immunization of BALB/c mice with adjuvented, unpurified recombinant proteins in the rear foot pads. Fusion was performed using lymphocytes from popliteal lymph nodes with SP2/O-Ag14 myeloma line. Screening was by differential indirect immunofluorescent staining on monolayers of Sf9 cells infected with each recombinant virus. Two MAbs proved to be reactive against NSP3 and a single one against NSP2. They showed high specificity by immunofluorescence, immunoprecipitation and Western blot. The isotype of these MAbs was IgG1. Oligomeric forms of NSP3 and NSP2 proteins were detected and the existence of intra-chain disulfide bridge in NSP2 protein was suggested. The levels of synthesis and cellular localization of NSP3 and NSP2 proteins were different as shown by immunoprecipitation and immunofluorescence.
Aponte
C
C
Laboratoire de Virologie et Immunologie Moléculaires INRA, C.R.J. Domaine de Vilvert, Jouy-en-Josas, France.
Mattion
N M
NM
Estes
M K
MK
Charpilienne
A
A
Cohen
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D016367:Baculoviridae; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D006801:Humans; D006825:Hybridomas; D008969:Molecular Sequence Data; D009036:Moths; D011233:Precipitin Tests; D011994:Recombinant Proteins; D012401:Rotavirus / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins