NR-17606 牛痘病毒

The entire vaccinia virus (VACV) Western Reserve (WR) genome (~195 Kb) (NCBI Accession AY243312) with a green fluorescent protein (GFP) sequence, two loxP sites, and a zeomycin resistance gene (zeo^R) was cloned as a bacterial artificial chromosome (BAC) and grown in Escherichia coli (E. coli) DH10? cells harboring a mini-lambda prophage. The integrated mini-lambda encodes the red recombination system, comprising a 5' to 3' exonuclease (Exo), a single-strand DNA binding protein (Bet), and a nuclease inhibitor (Gam), under the control of the temperature-sensitive λ cI857 repressor. E. coli DH10β/VAC-BAC/λ provides a system to achieve recombination between the VAC-BAC artificial chromosome and electroporated linear DNA with homologous sequences to facilitate production of recombinant VACV expression vectors or mutagenesis of the VACV genome. The chloramphenicol acetyl transferase (cat) gene promoter in the VAC-BAC is inactivated by replacement with the zeo^R gene, making NR-17606 chloramphenicol sensitive and zeomycin resistant. Subsequent reactivation by inclusion of a cat promoter in the recombination cassette allows selection of recombinant VAC-BAC based on chloramphenicol resistance.

Each vial contains approximately 200 ?L of NR-17606 in Luria-Bertani (LB) broth supplemented with 10% glycerol.

The presence of authentic VACV WR and GFP sequences in NR-17606 has been confirmed by PCR amplification and partial nucleotide sequencing. The sequence of the entire VACV WR genome has not been confirmed due to the large size of the plasmid insert. The presence of the integrated mini-lambda prophage also has not been confirmed at BEI Resources.