2.2∣Preparation of pVAX-For t 2 plasmid for vaccination
For t 2-encoding fragment (GenBank accession EU678971) was amplified by
PCR and the PCR products were subcloned into pVAX1 (Life Technologies,
Carlsbad, CA) . pVAX1 is a 3.0 kb plasmid vector designed to
conform with the Food and Drug Administration (FDA) document published
in 1996 (Points to consider on Plasmid DNA Vaccines for Preventive
Infectious Disease Indications. PTC document Docket No 96-N-0400). The
eukaryotic expression plasmid pVAX-For t 2 was then propagated inE. coli DH5α and large-scale purification was performed with the
EasyPrep EndoFree Maxi Plasmid Extraction kit according to the
manufacturer’s instructions (Tools, New Taipei City, Taiwan).