Steady expression of heterologous genes in insect cells based on rebuilt Bacmid vectors.

The genetic instability of genes transposed into the baculovirus genome by Bacmid technology, often caused by mini-F replicon, raises a crucial challenge for large scale production of recombinant proteins via serial passages in insect cells. To address this problem, we reconstructed Bacmid vector by decoupling the mini-F replicon from Tn7 transposition site. Two Bacmid variants, BacDC1 and BacDC2, were generated through homologous recombination. In BacDC1, the KanR-lacZα-attTn7 cassette remains in its original position while the mini-F replicon is relocated to the chitinase/v-cathepsin loci. Conversely, BacDC2 undergoes the opposite rearrangement. Using the classical Bacmid technology, two genes encoding enhanced green fluorescent protein (eGFP) and nano-luciferase (Nluc) C-terminally tagged spike 1 (S1) protein of porcine epidemic diarrhea virus (PEDV) were cloned as reporters to assess stability. Fluorescence observation analysis indicated that the genetic stability of cloned genes was markedly improved by the Bacmid variant-derived baculoviruses with sustained expression for up to 20 passages, appropriately 2-3 times longer than the wild-type control. Similarly, western blot and luciferase activity analysis demonstrated the S1-Nluc secretion from Sf9 cells infected with the dual expression variants remained stable over 10 passages, whereas the control showed significant expression decline by passage 5. These data suggest that decoupling the mini-F replicon from Tn7 transposition site significantly stabilizes cloned gene expression. The Bacmid variants thus represent promising vectors for industrial recombinant protein production and gene therapy applications.