Rapid titration of recombinant baculoviruses based on NanoLuc secretion in early infection

The baculovirus expression vector system has become a powerful tool for recombinant protein production and gene delivery. However, existing titration methods for baculovirus are not economical in terms of test time and cost. A titration method based on NanoLuc secretion that allows for titration of recombinant baculoviruses at 4 h post infection (hpi) is described. In the assay, the envelop protein GP64 signal peptide-guided NanoLuc was secreted into the culture medium in proportion to the virus amount during early infection under combined control by the homologous region 5 (hr5) enhancer and the promoter of immediate early gene 1 (ie1) plus L21. Two timepoint standard curves of luciferase activity to virus titers of 5-9 logs were established with excellent linearity and correlation coefficients (slope = 1.050, R² ≥ 0.9969) using a secretory Nanoluc (secrNluc) - inserted standard baculovirus. Through the assay, the titers of three recombinant viruses prepared independently were calculated by directly measuring luciferase activity in the supernatant at 4 and 6 hpi, with greater accuracy compared to the endpoint dilution assay. These results show the efficacy of this proposed method as a streamlined assay for rapidly titrating recombinant baculoviruses.