The results show that this xylose promoter from can be used to efficiently induce the expression of PPV heterologous protein. Open in a separate window Figure 2 Expression of VP2 protein in rLc393:pPG611.1-VP2. targeting. The immunogenicity of the two recombinant strains was analysed after intragastric administration of live bacteria to mice. Our data have indicated that intragastric intubation of two recombinant strains could induce the specific mucosal and systemic immune response against PPV. The higher anti-VP2 specific immune responses were obtained with the recombinant strain producing the VP2 in the extracellular milieu. Materials and methods Bacterial strain and growth conditions ATCC 393 kindly supplied by Jos Seegers (NIZO, Holland), was produced in Mann Rogosa Sharpe (MRS) medium (Sigma, St Louis, MO), at 37 anaerobically, without shaking. For the analysis of expression of VP2 protein, recombinant strains were produced in basal MRS medium supplemented with 2% xylose. Antibiotic concentration used for the selection of transformants was 10 g/ml of chloromycetin, Cm (Sigma). Plasmids, DNA manipulation and transformation The expression plasmid pPG612.1, a type of secretion expression vector containing ssUsp secretion signal peptide sequence and the expression plasmid pPG611.1, a type of cell-surface expression plasmid containing the structures of ssUsp secretion signal sequence and cell wall anchor domain name, were kindly supplied by Jos Seegers (NIZO, Holland). Nucleic acid manipulation and cloning procedures were performed according to standard procedures.31 A gene fragment of about 174 kb encoding the VP2 structural polypeptide of PPV was obtained from the genome of PPV strain LJL12 by polymerase chain reaction (PCR) amplification with the primers 5-CGAGGATCCTATGGTTCACTGGTTCGACGACCGCGAG-3 (forward) made up of a HI site (underlined) and 5-AGCTTCTCGAGCCATGCTACCTGATTAACCGAGTAACTG-3 (reverse) made up of an I Nodakenin site (underlined). PCR amplification conditions were as follows: 95, 5 min; 30 cycles of 94, 1 min; 55, 1 min; 72, 12 min; 72, 10 min for the final extension. The PCR product of VP2 gene was cleaved with HI and I restriction endonuclease (MBI) and inserted into the corresponding sites of pPG611.1 and pPG612.1 digested by HI and I respectively, giving rise to pPG611.1-VP2 and pPG612.1-VP2 (Fig. 1). Open in a separate window Physique Nodakenin 1 The construct of recombinant vectors expressing VP2 protein. The gene fragment encoding VP2 structural polypeptide of PPV was amplified by PCR with the primers. The PCR product was cleaved with HI and I restriction endonuclease and inserted into the corresponding sites of pPG611.1 and pPG612.1, respectively, giving rise to pPG611.1-VP2 and pPG612.1-VP2. Electroporation of was carried out as previously described32 with some modifications. In brief, PTGIS recombinant plasmid DNA (10 l) was added to 150 l of 393, gently mixed at 4 for 5 min and subjected to a single electric pulse (25 F of 25 kV/cm). The mixer was incubated in MRS medium without Cm at 37 anaerobically for 2 hr. Recombinant strains were selected on MRS-agar medium made up of 10 g/ml of Cm. The presence and integrity of the constructions carried by the 393 transformants were checked by extraction of recombinant plasmid DNA following by restriction analysis and sequencing. Protein expression and Western blot analysis To analyse the expression of the VP2 fusion protein by xylose-induced rLc393:pPG611.1-VP2, overnight cultures grown in basal MRS medium supplemented with xylose were collected by centrifugation at 12 000 for 10 min. The pellets were washed twice with sterile 50 mm Tris-Cl, pH 80 and lysed in a Bead-Beater (Biospec, Bartlesville, OK) by vigorous shaking. The lysates were centrifuged at 15 000 for 10 min and the supernate were Nodakenin examined using 10% sodium dodecyl sulphateCpolyacrylamide gel electrophoresis (SDSCPAGE). Western blot analysis: Proteins were electrotransferred onto a nitrocellulose membrane and the immunoblots were developed using mouse anti-VP2 serum at a dilution of 1 1 : 1000 with phosphate-buffered saline (PBS). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Nodakenin (Sigma) diluted at 1 : 2000 was used and visualization of immunolabelled bands were then carried out using the Chemiluminescent Substrate reagent (Pierce, Rockford, IL) according to the manufacturer’s training. To assay.