Expression and characterization of bovine rotavirus proteins in the baculovirus system

Recombinant proteins of bovine rotavirus VP2, VP4, VP6 and VP7 were expressed in insect cells using baculovirus expression vectors. Expressed recombinant proteins were similar to those of rotavirus. Recombinant viral antigens expressed in insect cells reacted with polyclonal antiserum to the VMRI strain of BRV by an indirect immunofluorescence test. Expressed recombinant proteins were characterized by radioimmunoprecipitation and their molecular mass was found to be similar to that of in wild type rotavirus. These four recombinant proteins were antigenic in guinea pigs and induced antibodies to the corresponding rotavirus proteins as detected by indirect immunofluorescence test. Recombinant viruses Bac.pVL/VP4 and Bac.pVL/VP7.D expressing VP4 and VP7, respectively, induced high levels of virus neutralizing antibodies in guinea pigs. Recombinant virus Bac.pVL/VP6 also induced low level of virus neutralizing antibodies. Recombinant proteins VP4 and VP7 inhibited receptor binding of rotavirus to MA-104 cells;To increase the expression of VP7, we have investigated the effect of removing terminal non-coding sequences, upstream and downstream of the VP7 open reading frame, on its expression in insect cells. Four constructs were generated in baculovirus transfer vector pVL1393 to be expressed as nonfusion proteins under the control of a baculovirus polyhedrin promoter. The construct pVL/VP7.A contained the full sequence of the bovine rotavirus VMRI VP7 gene including the noncoding sequences, construct pVL/VP7.B contained the VP7 gene in which the noncoding region at the 3\u27 end was deleted, construct pVL/VP7.C was composed of the VP7 gene in which the 5\u27 noncoding sequence was deleted, and construct pVL/VP7.D had the VP7 gene in which noncoding sequences upstream and downstream of the VP7 open reading frame were deleted. Recombinant DNA from these four constructs was used to generate recombinant baculovirus and VP7 expression using each recombinant baculovirus was evaluated. Insect cells infected with recombinant baculovirus Bac.pVL/VP7.D had the highest transcriptional activity. The level of protein expression by radioimmunoprecipitation was highest with recombinant baculovirus Bac.pVL/VP7.D and was at least ten fold more than that with the other three recombinants. We believe the recombinant proteins VP4 and VP7 offer a valuable tool for development of an effective vaccine for the prevention and control of rotavirus associated gastroenteritis