(1999) SEZ-Cap and SEZ ΔhasB strains were applied in duplicate t

(1999). SEZ-Cap and SEZ ΔhasB strains were applied in duplicate to multiwell slides. The slides were air dried and then fixed in 100% methanol for 10 min at −20 °C.

The slides were incubated with the mouse sera against the PCV2 (1 : 20), and preimmune mice serum was used as negative control. After washing, the slides were incubated with fluorescence isothiocyanate (FITC)-labeled affinity-purified antibody to mouse IgG (H + L) (Santa Cruz, CA). After a final wash, the slides were examined selleckchem with a fluorescence microscope (Zeiss, Germany). Surface expression of the capsid protein by SEZ was determined as previously described (Rubinsztein-Dunlop et al., 2005), with some modifications. About 5 × 106 bacteria were incubated with PCV2-positive serum or normal mice serum, which was diluted 10-fold in phosphate-buffered saline/bovine serum albumin (PBS-BSA) and incubated at room temperature with bacteria in a total volume of 500 μL for 45 min. The bacteria were harvested by centrifugation at 6000 g for 5 min and washed

with PBS-BSA. Goat antimouse IgG-FITC (10 μg) (Santa Cruz) was added, and the bacteria were incubated for 45 min at room temperature, washed and analyzed with a FACSCalibur check details flow cytometer (Becton Dickinson, San Jose, CA). Forward and side scatter were used to exclude debris and aggregates, and 10 000 gated events were recorded. The mean fluorescence intensity and percentage of fluorescent PFKL bacteria (brighter than 10 fluorescence intensity units on the FL1 axis) were calculated for each sample. To evaluate the efficacy of recombinant live vaccine against PCV2, 6-week-old female BALB/c mice were randomly divided into three groups (10 mice per group). The mice in group 1 were immunized twice at 2-week intervals by intraperitoneal injection with 1 × 106 CFU SEZ-Cap (0.5 mL). Group 2, serving as a positive control, were vaccinated with commercially available PCV2-inactive vaccine (Nannong Hi-tech Co. Ltd, Nanjing, China)

and group 3, serving as a negative control, were vaccinated with SEZ ΔhasB strain at an equal dose and using the same protocol. Fourteen days after the second vaccination, sera were obtained from each group by tail vein bleeding and the antibodies were measured using the commercial PCV2 ELISA IgG kit (Ingezim Circovirus IgG, Ingenasa). Data are presented as mean ± SD and were analyzed using a t-test. Values of P < 0.05 were considered significant. To gain the recombinant strain expressing the capsid protein of PCV2, a fragment of the ORF2 gene lacking the nuclear localization signal sequence which possesses rare codons encoding arginine and proline and suppressing high-level expression (Liu et al., 2001) was cloned. The truncated cap gene was incorporated into the szp gene of SEZ strain ΔhasB designated as SEZ-Cap through homologous replacement.

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