Modification of PCV-2 virulence by substitution of the genogroup motif of the capsid protein

Allemandou, Aude; Grasland, Béatrice; Hernandez-Nignol, Anne-Cécile; Kéranflec'h, André; Cariolet, Roland; Jestin, André

The PCV-2b DNA clone was constructed from the tandem infectious DNA clone already constructed and characterized [21] . A primer pair, PCV-2b forward and reverse primers (Table 1) , was designed in the rep gene to amplify by PCR a 1.5 PCV-2b DNA clone containing only one copy of the cap gene. The PCR reaction mix included 0.2 mM of dNTP mix, 0.2 μM of each primer, 2 mM of MgSO 4 , 1 unit of Platinum Taq Polymerase High Fidelity (Invitrogen) and its buffer to 1X in the final concentration. The PCR reaction was composed of a first denaturing step of 2 min at 94°C, then 30 amplification cycles with one denaturing step of 30 s at 94°C, an annealing step of 30 s at 56.3°C and an elongation step of 2 min at 72°C; and finally an elongation step of 10 min at 72°C. The fragment of 1.5 genome copy was purified on 1% agarose gel with the Ultrafree DA kit (Millipore, Bedford, MA, USA) and then cloned in the pCR4 vector, which was transformed in XL1 Blue MRF' cells.