2. Materials and Methods
The plasmid containing full-length PCV2b was mutated into PCV2b-6M with primers b-d-F and b-d-R, and the PCV2d plasmid was mutated into PCV2d-6M with primers d-b-F and d-b-R. The sequence of the primers was shown in Table 1. Methods and primers for construction of double copy infectious cloning were referred with articles published in our laboratory (Zhang et al., 2018). The four dual-copy plasmids constructed were named as 2PCV2b, 2PCV2b-6M, 2PCV2d and 2PCV2d-6M. Genome sequence was used to determine the success of site-specific mutations.
The four different recombinant plasmids were transfected into porcine kidney-15 (PK-15) cells free of PCV using Lipofectamine 3000 (Invitrogen). The four viruses were harvested 72 hour (72h) post transfection and store at -80°C. The empty pEASY-Blunt vector was included as negative controls. The indirect immunofluorescence assay (IFA) and mRNA amplification were performed to determine the success of virus rescued (Cheung et al., 2003). Viral load of 10th passage was calculated using a real-time quantitative PCR. The viral titer and one-step growth curve of the 10th generation virus were calculated at different harvest time points. All assays were repeated at least three times, with each experiment performed in triplicate. One-way ANOVA and Student’st tests were used to compare results between different groups. All statistical analysis was performed using GraphPad Prism. P< 0.05 was considered to be statistically significant.
Table 1 Site-directed mutagenesis primer name and sequence