Příprava bakulovirových transferových vektorů pro in vitro produkci izoenzymůP4501A1 a P4503A4

The Construction of a Baculovirus Expression System for in vitro Production ofIsoenzymes P4501A1 and P4503A4

The aim of this work is the construction of an expression system for in vitro synthesis of microsomalmonooxygenases P4501A1 and P4503A4, which catalyze oxidative transformations of most xenobioticsin both animal and human organisms. cDNAs encoding both proteins were obtained followingthe UBMTA protocol by the courtesy of holders, and amplified by established methods. Baculovirustransfer vectors were used to clone these cDNAs. These vectors contain a strong polyhedrinepromoter surrounded by sequences homologous to that of baculovirus DNA, allowing the recombinationof the vector with the viral DNA, and hence the production of a protein. Established methodsand PCR were used to insert cDNA into the vectors, and the insertion was verified by the PCRmethod with specific primers and using restriction endonucleases.

Key words:
P4501A1 – P4503A4 – baculovirus transfer vector