Initial transformation and screening were done in Blue-gal, and the positive clones were confirmed by dideoxy sequencing and used for transformation of Escherichia coli DH10Bac.
Cell Culture and Virus
The Bac-to-Bac baculovirus was a derivative of Autographa californica nuclear polyhedrosis virus (AcMNPV).
The recombinant virus was propagated in a monolayer culture of Spodoptera frugiperda cells, Sf9, which were grown in a monolayer culture at 27°C in Grace insect medium containing antibiotics (streptomycin, 100ig/ml; penicillin, 50 U/ml), and 10% FBS according to the manufacturer’s protocol (Invitrogen).
Generation of Recombinant Baculovirus and Transposition
Generation of recombinant baculovirus expressing rgLh in Sf9 cells was carried out using the Bac-to-Bac baculovirus expression system kit (Invitro-gen). DNA of the recombinant pFastBac transfer vector containing cDNA corresponding to the grouper Ihb and cga sequence was introduced into E. coli DH10Bac for the transposition of the sequence into baculovirus genomic DNA (bacmid) according to the manufacturer’s protocol.