Production of influenza virus-like particles from stably transfected Trichoplusia ni BT1 TN-5B1-4 cells

Abstract

We investigated the production of influenza A/ Korea/01-2-9/2009 (H1N1) virus-like particles (VLPs) containing four structural proteins, matrix protein 1 (M1), matrix protein 2 (M2), neuraminidase (NA) and hemagglutinin (HA), using stably transfected Trichoplusia ni BT1 TN-5B1-4 (TN-5B1-4) cells. Recombinant M1, M2, NA and HA were expressed as bands with molecular weights of 28, 17, 60, and 70 kDa, respectively, in stably transfected TN-5B1-4 (TN-5B1-4/M1-M2-NA-HA) cells. VLPs were purified from the culture medium of the TN-5B1-4/M1-M2-NA-HA cells by pelleting on a sucrose cushion, followed by ultracentrifugation in a sucrose density gradient. The four structural proteins were released together from the TN-5B1-4/M1-M2-NA-HA cells, and were co-purified from the same fractions of the sucrose density gradient, indicating that recombinant M1, M2, NA, and HA self-assembled into VLPs in the TN-5B1-4/M1-M2-NA-HA cells. Recombinant baculoviruses (rBac/NA and rBac/HA) expressing recombinant NA and HA were generated and used to co-infect stably transfected TN-5B1-4 (TN-5B1-4/M1-M2) cells expressing recombinant M1 and M2, resulting in the production of high-molecularweight VLPs and the release of self-assembled VLPs with diameters of 80 ~ 120 nm. The production of VLPs was greatly enhanced in the TN-5B1-4/M1-M2 cells co-infected with rBac/NA and rBac/HA, compared with the TN-5B1-4/M1-M2-NA-HA cells. Our results suggest that a stably transfected-insect cell expression system might be useful for producing VLPs for the development of recombinant vaccines against influenza.

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

“…The High Five cell line is conventionally accepted as a higher protein producer host compared to Sf9 cells (14,46). Therefore, several works have focused on using this cell line for VLP production (15,(47)(48)(49). However, quality assessment of VLPs and the associated contaminants has not been addressed in detail.…”