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