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 primers used to mutate the PCV-2a DNA clone were PCV-2a/(SNPISI) forward and reverse first and then the PCV-2a/motif 2b forward and reverse. To mutate the genogroup motif in the PCV-2b DNA clone, the primers PCV-2b/(TNKRSV) forward and reverse and PCV-2b/ motif 2a forward and reverse were used. All the mutagenesis reactions were carried out with the QuickChange II XL mutagenesis kit (Stratagene), with some modifications. Pfu Ultra HF DNA polymerase was replaced in the PCR mix by Pfu Turbo DNA polymerase. The PCR protocol was also optimized: the first denaturing step was extended to 2 min at 95°C; then the 18 amplification cycles with one denaturing step of 1 min at 95°C, an annealing step of 50 s at 60°C and an elongation step of 6.5 min at 68°C; and finally an elongation step of 10 min at 68°C. The PCR product was digested with DpnI enzyme for one hour, and 4 μL of the digested PCR product was transformed in XL-10 Gold chemically competent cells according to the manufacturer instructions. Each mutant DNA clone was checked by sequencing the 1.5 copies of the insert.