Expression of two bovine rotavirus non-structural proteins (NSP 2, NSP 3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins

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Abstract
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 NSP 2 and NSP 3 (formerly called NS 35 and NS 34, respectively) of the bovine rotavirus RF and produced hybridomas against these proteins. Full-length DNA copies of RNA segments 7 (coding for NSP 3) and 8 (coding for NSP 2) of the virus strain RF were cloned and sequenced. Each cDNA was inserted in the transfer vector pVL 941 and used to transfectSpodoptera frugiperda cells (Sf 9). Recombinant baculoviruses encoding these proteins were obtained. Infection of Sf9 cells with these recombinant viruses resulted in a high level of expression of NSP 2 and NSP 3 (range of 1 µg per 106 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 SP 2/O-Ag 14 myeloma line. Screening was by differential indirect immunofluorescent staining on monolayers of Sf 9 cells infected with each recombinant virus. Two MAbs proved to be reactive against NSP 3 and a single one against NSP 2. They showed high specificity by immunofluorescence, immunoprecipitation and Western blot. The isotype of these MAbs was IgG 1. Oligomeric forms of NSP 3 and NSP 2 proteins were detected and the existence of intra-chain disulfide bridge in NSP 2 protein was suggested. The levels of synthesis and cellular localization of NSP 3 and NSP 2 proteins were different as shown by immunoprecipitation and immunofluorescence.