When the genomic DNA of SEZ strain ΔhasB was used as template, a 2265-bp band encompassed the length of its homologous arms and the deleted region of the szp gene. However, when the genomic DNA of SEZ-Cap was used as template, a 2160-bp fragment could be amplified, indicating that the length of the partial szp gene was subtracted and the cap gene was incorporated (Fig. 1c). The PCR products were further cloned and sequenced. The result showed that
part of the szp gene had been successfully replaced by the recombinant szp-cap gene, coding for the fusion protein with partial Cap protein sequence (see Supporting Information, Data S1). In addition, using RT-PCR with primers located in the cap
gene frame of the szp-cap gene also confirmed a 276-bp fragment yield from the SEZ-Cap selleck compound strain but no transcription from the parental SEZ ΔhasB strain (Fig. 1c). The nearly identical growth curves of SEZ-Cap and SEZ ΔhasB indicated that incorporation of cap into the szp gene did not have a significant influence on the growth of SEZ strain ΔhasB. A 276-bp PCR fragment was consistently amplified using primers PCV-S-1 and PCV-S-2 this website from SEZ-Cap from each of 25 serial passages, implying that the cap gene was stably inserted into the genome (data not shown). To study attenuation of the SEZ-Cap strain, virulence of the two strains was assessed in BALB/c mice. Results showed that SEZ-Cap was nearly fourfold less virulent than the parental strain (Table 2). To test whether the transcription level of cap was reduced when incorporated into the szp gene, we compared that of the recombinant szp-cap gene in the SEZ-Cap strain and the original szp gene in the parental SEZ ΔhasB strain by quantitative RT-PCR. The comparison was carried out using the strains either cultured in TSB broth (in vitro) or recovered from infected mice (in vivo). Analysis of the dissociation curves from infected samples and bacteria
cultured Levetiracetam in vitro revealed a single melting peak, and no specific fluorescence signal was detected from negative control samples. The result showed that transcription levels of cap in the recombinant strain were not statistically different from that of szp in the parental strain both in vitro and in vivo. Immunofluorescence labeling of the cells was performed using mouse anti-PCV2 antibody as the primary antibody and FITC-conjugated goat anti-mouse IgG as the secondary antibody. The green fluorescence of the immunostained capsid fusion protein was observed on SEZ-Cap cells, whereas control cells of SEZ strain ΔhasB were not immunostained (Fig. 2). Flow cytometry was used to quantitatively analyze the cell-surface display of the cap-anchor. As shown in Fig. 3, the recombinant strain showed significantly more intense fluorescence signals than the parental strain SEZ ΔhasB.