Constructing Recombinant Baculoviruses for Protein Expression

The two recombinant plasmids used in the project were constructed into pFastBacDual cloning vector (Invitrogen, Carlsbad, CA, USA) which was described in the previous work [26 (link)] and specific plasmids construction information was provided in the supplementary material.
Following the manufacturer’s instructions for the Bac-to-Bac Baculovirus Expression System (Invitrogen), we constructed two Bacmids by the transposition of the above two plasmids with DH10BmBac^TMEscherichia coli competent cells. Bacmid DNA isolated from amplified bacterial colonies was used to transfect BmN cells to generate recombinant Bombyx mori nuclear polyhedroviruses (BmNPV), which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. Serial passages of rBmNPV/AAV2ITR-eGFP were then titrated according to the median tissue culture infectious dose (TCID₅₀), and rBmNPV/AAV2Rep-HBoV1Cap was titrated in plaque forming units (pfu) by a plaque assay as described in the manual of the Bac-to-Bac Baculovirus Expression System (Invitrogen)^® (Figure 1).