Construction of baculovirus transfer vector for co-expression of Rice gall dwarf virus gene S3 and S8 and identification of recombinant baculovirus.
To construct a recombinant baculovirus co-expressing the major core capsid protein gene 53 and outer capsid protein gene 58 of Rice gall dwarf virus (RGDV), the target genes (S3 and S8) were subcloned into the downstream of PH promoter and p10 promoter of the baculovirus transfer vector pFastBacDual, respectively. After transformation, pFBDS3-S8, which was identified with restriction enzyme digestion and conformed by sequence analysis, was introduced into the competent cells (Escherichia coli DH10Bac), generating the recombinant bacmid rbpFBDS3-S8. Bacmid rbpFBDS3-S8 was transfected with Sf9 (Spodoptera frugiperda) insect cells package virus by liposomal transfection method. The recombinant virus was identified by PCR. Results showed that an increased diameter, granular appearance and cells lysis, which were much different from the morphology of normal Sr9 cells were observed under fluorescence invert microscope, 72 h after infection. The gene S3 and S8 were integrated into genome of recombinant baculovirus, laying a foundation for the expression of the major structural protein gene in insect cells and the research of the function of gene being investigated.