Modification and use of fp25k gene from Autographa californica multicapsid nucleopolyhedrovirus in stably transforming insect cell line Sf9.
Aim: After serial passages in some permissive insect cells, phenotype of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) has the potential to change from many polyhedra (MP) to few polyhedra (FP). This phenomenon is related to mutations occurred in a gene named fp25k which codes a 25 kDa protein. The AcMNPV fp25k mutants produce less occlusion-derived viruses (ODVs) and more budded viruses (BVs) than the wild type, usually resulting in reduction of foreign protein expression in the baculovirus expression vector system. To overcome the deficiency resulted from the mutations of fp25k in baculovirus expression vector system, we modified fp25k and constructed a transgenic insect cell line expressing FP25K protein. Methods:The AcMNPV fp25k gene was modified in a site which is easy to be mutated during passage, and a modified fp25k, named mfp25k, was produced in this study. mfp25k was combined to pIZT/V5-His vector, the recombinant vector was further used to transfect the Spodoptera frugiperda cells (Sf9), and Sf9 cells which were not successfully transformed were eliminated gradually by Zeocin selection. Results: We successfully modified AcMNPV fp25k in TTAA site and got recombinant vector pIZT/V5-mfp25k. After transforming Sf9 cells and selecting with Zeocin, we finally got stable transgenic Sf9 cell line, Sf9-mfp25k, which contains mfp25k gene in the genome. Sf9-mfp25k transgenic cells and normal Sf9 cells were both infected with AcP2, the AcMNPV fp25k mutant. The results indicated that the transgenic Sf9-mfp25k cells expressing FP25K protein could rescue the phenotype of AcMNPV with fp25k mutations. Conclusion: The transgenic Sf9-mfp25k cells expressing FP25K protein could make up for the shortage of baculovirus caused by fp25k mutations. Our results provide a novel way to build stable baculovirus-insect cell expression system.