TRANS-COMPLEMENTATION OF Ac109 KNOCK OUT BACULOVIRUSES BY TRANGENIC INSECT CELL LINES FOR THE PRODUCTION OF VIRION-FREE RECOMBINANT PROTEINS

AMALFI SABRINA; MATTERA RAMIRO EZEQUIEL; PLASTINE MARÍA DEL PILAR; TABOGA OSCAR; ALFONSO VICTORIA

Santa Fe

Simposio; IX Simposio Latinoamericano de Tecnología de Cultivos Celulares; 2022

Universidad Nacional del Litoral

The baculovirus expression system is widely used for the production of recombinant proteins, which in many cases co-purify with viral particles. We previously described ac109 of Autographa californica multiple nucleopolyhedrovirus as an essential gene for the production of infectious viral progeny. This work aimed to use an ac109 knockout virus (Ac109KO) trans-complemented from transgenic insect cells to produce recombinant proteins with minimal levels of co-produced virions. First, different promoters and ac109 nucleotide sequences were evaluated in their ability to trans-complement Ac109KO. We also determined the strategies that produced lower levels of regenerated wild type (wt) viruses by recombination. Then, we selected a modified codon sequence of ac109 driven by its own promoter zone (pac109) or by a synthetic polyhedrin-based promoter as the best genetic constructs and obtained two polyclonal Sf9-derived cell lines that rendered high levels of complemented viruses and less than 1% of wt viral particles. Finally, the production of a reporter protein was evaluated by fluorometry in Sf9 cells infected with complemented or control baculoviruses. Interestingly, Ac109KO viruses obtained by trans-complementation with both developed Sf9 cell lines expressed higher levels of the reporter gene than wt viruses. In conclusion, the use of defective baculoviruses complemented with transgenic Sf9 lines is a promissory tool to obtain protein products with very low levels of budded virion contaminations.