iniae vaccine component. Conclusions In summary, this study MAPK inhibitor presents MtsA as a novel solute-binding protein that can contribute to iron transport. This is the first ABC transporter member to be identified from S. iniae. We have shown that MtsA is a lipoprotein which can bind to heme, and is expressed in vivo during Kunming mice infection by S. iniae HD-1. More

importantly, this is the first report on the cloning of ABC transporter lipoprotein from S. iniae genomic DNA, and its immunogenicity is indicative of its possible use as an S. iniae subunit vaccine. Methods Bacterial strains and growth conditions Streptococcus iniae HD-1 was isolated from Threeband sweetlips (Plectorhynchus cinctus) from Guangdong province, PRC. The microorganism was stored in our lab and cultured according to the methods described by Zhou et al [45]. Briefly, S. iniae isolate HD-1 cells were grown in brain heart infusion broth (BHI, Oxoid Ltd.), and BHI broth with 1.5% agar (Guangdong Huankai Microbial Sci. & Tech, Co., Ltd.) was used as Selleckchem BI2536 the solid medium. Escherichia coli DH5α and BL21 (DE3) strains (Beijing Newprobe Biotechnology Co., Ltd.) were used for gene

cloning and protein expression, respectively. Cloning and reverse transcription analysis of mtsABC Genomic DNA was extracted from the S. iniae HD-1 strain using the Wizard genomic DNA purification kit (Promega Co., Ltd.), as recommended by the manufacturer, Megestrol Acetate and the material was quantified by measuring the absorbance at 260 nm. PCR was carried out with 1 μg of DNA using the primers listed in Additional file 1, Table S6. The primers were designed based on the conserved regions of the published amino acid sequence of metal ABC transporter (Additional file 1, Table S6-1), and the full-length product was obtained by SiteFinding-PCR (Additional file 1, Table S6-2, 6-3), as described by Tai et al [46]. The PCR products were sequenced

to rule out spurious mutations (Invitrogen Co., Ltd.). S. iniae HD-1 cells grow to the logarithmic phase were harvested by centrifugation, and total RNA was extracted by the Pure Yield™ RNA midiprep system (Promega, USA, Co., Ltd.). Total RNA was then incubated with RNase I at 37°C for 30 min to remove the contaminating genomic DNA. The material was quantified spectrophotometrically by ultraviolet absorption spectrometry (CE2302, Gene Quest), and its integrity was verified on a 0.8% agarose gel. First-strand cDNA was synthesized from 1 μg total RNA using the first-strand cDNA synthesis kit with ReverTra Ace-α-reverse transcriptase (Toyobo Co., Ltd.). The cDNA synthesized above was used as the template to amplify genes using the ORF-specific primers listed in Additional file 1, Table S7, and the PCR products were sequenced at Invitrogen Corporation to confirm their specificity. Expression of recombinant MtsA The genomic DNA of S.