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 construction of PCV-2a clone was carried out in two stages. A long fragment corresponding to almost all the PCV-2a was amplified by PCR from DNA extracted from PCV-2a infected cells (see Table 1 , PCV-2a long forward and reverse primers). A second fragment corresponding to the rep gene of the PCV-2a genome was amplified by PCR with the PCV-2a short forward and reverse primers (Table 1) . For both fragment amplification, the PCR 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, Carlsbad, CA, USA) and its buffer to 1X in final concentration. The PCR program for both reactions 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 and 54.5°C (respectively for the long and short fragments) and an elongation step of 1.5 min at 72°C; and finally an elongation step of 10 min at 72°C. Both fragments were cloned separately in pCR4 vector with the TOPO TA kit (Invitrogen) and transformed in XL1 Blue MRF' electro-competent cells (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Then, the two plasmids were digested by NdeI and NotI enzymes (New England Biolabs GmbH, Frankfurt am Main, Germany). The rep gene enclosed between the NdeI and NotI sites was ligated in the plasmid containing the PCV-2a genome and digested by the same enzymes. The ligation mix was subsequently transformed in XL1 Blue MRF' electro-competent cells.