[23]. The purity of His-cSipC and flagellin (FliC) was verified by 10% SDS-PAGE followed by CBB staining, and the concentration of proteins was quantified by Bradford’s method (Bio-Rad). In order to prepare anti-cSipC serum, 8–10-week-old female BALB/c mice were immunized intraperitoneally (i.p.) with the purified protein. Ten micrograms of protein with Freund’s complete adjuvant (FCA) was injected into a mouse 3–4 times at 3-week intervals between each administration. The care and use of experimental animals complied

with local Animal Welfare Laws and Guidelines. Total blood was collected two weeks after the last booster and INCB28060 supplier serum was prepared by centrifugation. The antibody’s specificity was checked by western blotting analysis. The anti-flagellin antibody used in this study was the same as that prepared previously [24]. As the expression vector for cell-surface anchoring of the heterologous antigens, the plasmids pLP401::cSipC, pLP401::cSipC = FliC, and pLP401::FliC = cSipC were constructed from pLP401 by the same technique as described previously [5]. In brief, DNA fragments encoding these antigens were amplified AZD5363 concentration from SE #40 chromosomal DNA by PCR with primers IGM389 and IGM390 for cSipC. In order to construct the fusion protein, FliC = cSipC,

overlap PCR was performed. As a first step, DNA fragments encoding FliC and cSipC were synthesized using chimeric primers that included both sequences of fliC and truncated sipC; IGM200 (gaa aag gat ccg

gca caa gtc att aat aca aac agc ct) and IGM423 (ttt aag cgc gcc tct ttc att acg cag taa aga gag gac gt) for the front segment (FliC-) and IGM422 (acg tcc tct ctt tac tgc gta atg aaa gag gcg cgc tta aa) and IGM390 for the rear segment (-cSipC). As a second step, the two segments were connected and amplified by PCR using primers IGM200 and IGM390. Another chimeric gene encoding cSipC = FliC was prepared by the same technique but using different primers, IGM389 and IGM421 (gta tta atg act tgt gcc ata gcg cga ata ttg cct gcg a) for the front segment (cSipC-), IGM420 (tcg cag gca ata ttc gcg cta tgg cac aag tca tta ata c) and IGM201 (tcg ccg tcg aca cgc agt aaa gag agg acg tt) for the rear segment (-FliC), and IGM389 and much IGM201 for the connection. These PCR products were digested with BamHI and XhoI, and inserted into the same restriction sites of pLP401. The ligated plasmid was then introduced into E. coli JM109 for cloning. In order to convert it into a mature plasmid, the constructed plasmid was treated with NotI followed by self-ligation. The preparation of competent cells and electroporation of L. casei were carried out in accordance with the method of Pouwels et al. [25]. The procedure to confirm the expression and surface presentation of heterologous proteins was described previously [5]. Briefly, transformed bacteria were grown, collected, and disrupted in SDS-PAGE sample buffer.