The result of Southern blot indicated that orange-spotted grouper lhb and cga had been transposed into the baculovirus genomic DNA (bacmid) (Fig. 2, A and B).
Transfection of Sf9 Insect Cells
At early stages of infection, cell diameter increased and nuclei began to fill the cells (Fig. 3A, B, C). Cells stopped growing and appeared vesicular. Budded virus was released into the medium 72 h after transfection (Fig. 3D) at late stage infection, and cells released from the plate, lysed and appeared clear in the monolayer in very late stage infection (Fig. 3, E and F).
Production of rgLh in Insect Cells
To analyze the production of rgLh, the Sf9 cell monolayer was infected with recombinant virus at 10 multiplicities of infection. The medium was collected at the appropriate time (i.e., 0, 24, 48, 72, 96, and 120 h postinfection) and separated by SDS-PAGE and non-denaturing PAGE. The results in Figure 4 show that a band at 15.6 kDa and a product of 11.4 kDa could be detected by SDS-PAGE while a band in the range of about 30 kDa was detected by native PAGE under non-reducing conditions. The concentration of rgLh was about 50 mg/L.
FIG. 3. Sf9 cells infected with recombinant baculovirus. Normal Sf9 insect cells
(A);Sf9 insect cells after 24 h of transfection
(B);Sf9 insect cells after 48 h of transfection
(C);Sf9 insect cells after 72 h of transfection
(D);Sf9 insect cells after 96 h of transfection
(E); Sf9 insect cells after 120 h of transfection (F). Original magnification X200.