The capture ELISA was performed in Selleck Olaparib triplicate. A P (virus strain)/N (negative control) value > 2.1 was considered positive. Analysis of ORF2 from different strains Multiple alignments
of amino acid sequences in the capsid protein of six strains of PCV2 (PCV2a/LG, PCV2a/CL, PCV2a/JF2, PCV2b/SH, PCV2b/YJ and PCV2b/JF) were performed using Clustal W within the DNASTAR software (version 7.0). Construction of PCV2-ORF2-CL/YJ chimeras and mutants Plasmids pMD18/PCV2a-CL, pMD18/PCV2b-YJ and pMD18/PCV2a-LG, containing the complete genomic sequences of the PCV2a/CL, PCV2b/YJ and PCV2a/LG strains, were constructed as described previously [20, 21]. Plasmid pMD18/PCV2a-JF2 containing entire genomic sequences of PCV2a/JF2 strain was constructed as described by Guo et al. [20] with primers Q-R and Q-F (Table 2). A series of chimeric pMD/PCV2- ORF2-CL/YJ (Figure mTOR inhibitor 1a) containing regions deletion of pMD/PCV2-CL-ORF2 fused with the corresponding ORF2 regions of YJ-ORF2 were constructed by fusion PCR or mutation PCR. Briefly, the pMD18/PCV2a-CL templates were respectively
PCR-amplified using primers A-F and A-R, C-F and C-R, E-F and E-R, or G-F and G-R (Table 2) according to the instructions that accompany the KOD-plus kit (Toyobo, Japan). Those PCR products that did not contain regions (aa 47-72, 80-94, 110-154 or 190-210) of PCV2a/CL capsid protein were respectively gel purified, and subsequently
served as the templates for fusion PCR using primers B-F and B-R, D-F and D-R, F-F and F-R, or H-F and H-R (Table 2), which inserted the corresponding regions for of PCV2b/YJ capsid protein. The fusion PCR products were then used to transform Escherichia coli strain Top10 according to the manufacturer’s recommendations (Takara, Dalian, China). The resulting chimeric plasmids were verified by sequence analyses (BGI, Beijing, China) and were respectively designated as rCL-YJ-1, rCL-YJ-2, rCL-YJ-3 and rCL-YJ-4 (Figure 1a). Mutations were introduced into the pMD/PCV2a-CL-ORF2, pMD/PCV2a-LG-ORF2, pMD/PCV2a-JF2-ORF2 and pMD/PCV2b-YJ-ORF2 by PCR using a set of primers (Table 2) by QuickChange Lightning Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s recommendations. The resulting plasmids were verified by sequence analyses (BGI) and were designated as rCL-YJ-5, rCL-YJ-1-51, rCL-YJ-1-57, rCL-YJ-1-59, rCL-YJ-1-63, rLG-YJ-1-59, rJF2-YJ-1-59 and rYJ-CL-1-59 (Figure 1a-c).