Bacterial manipulations, PCR and sequencing

AH02LA BAC with TK, gE and gI deletion (BAC^{PRVΔTK/gE/gI}) constructed previously in our lab [4], was used to generate PRV-S BACs. Electroporation was carried out as described earlier [19]. Plasmid and PRV-S BAC DNA were performed with commercial kits (QIAGEN) according to manufacturer’s instructions. PRV-S BAC was confirmed by RFLP with BamH I.

Primers KAN ins S F/KAN ins S R (Table 1) with two EcoR I restriction sites in both terminals for cutting and ligation were used to insert a kanamycin resistance gene into plasmid S-T. Primers (PRV ins S cas UL11-10 F/R, PRV ins S cas UL35-36 F/R, PRV ins S cas UL46-27 F/R or PRV ins S cas US2-1 F/R; Table 1) were used to insert the S^cas-KAN into the UL11-10, UL35-36, UL46-27 or US2-1 of BAC^{PRVΔTK/gE/gI} through the En Passant protocol [20]. Specific primers (S cas check F/R, Table 1) were used to verify the sequence of the inserted S gene expression cassette. A pair of primers (PRV BAC H1 F and PRV BAC H2 R) were used to amplify a DNA fragment (H1-H2-gI-ΔgE) including the gI gene, part of gE with the deletion of the 1286 bp fragment (position 13 to 1298), and upstream and downstream homologous sequences using PRV LA-A^B strain [17] DNA as template. The primers PRV ΔgE check F and PRV ΔgE check R were used for sequencing gI and ΔgE gene. S expression cassette were confirmed by sequencing (S cas check F and S cas check R).

Primers for PCR, sequencing or RT-PCR