Additional file 1: Figure S1. of Improvement of alkalophilicity of an alkaline xylanase Xyn11A-LC from Bacillus sp. SN5 by random mutation and Glu135 saturation mutagenesis

Schematic diagram of a high-throughput screening of the positive mutant. Table S1. Primers used for random mutagenesis, site-directed mutagenesis and site saturation mutagenesis. (DOC 325 kb

Kinetics of specific anti-SjGALE IgG induced in rSjGALE-immunized mice.

<p>Sera of 10 immunized mice per group were collected on days 15, 30, 45, 60, 75 and 90 after the first immunization and analyzed by an ELISA. Results are presented as the mean absorbance of one trial measured at 450 nm for each group of one experiment. Statistically significant differences of vaccinated mice compared to the PBS + adjuvant control group is denoted by one asterisk (<i>p</i><0.05).</p

Western blot analysis of the SjGALE protein level at different stages of the <i>S. japonicum</i> life cycle.

<p>Total protein lysates from different stages of <i>S. japonicum</i> as indicated were separated on 12% polyacrylamide gels and electrophoretically transferred to nitrocellulose filters. Blots were probed with mouse anti-SjGALE and anti-α-tubulin as a loading control. (A) Tubulin was used as a control to determine the sample preparation. (B) Extracts were prepared from various stages of the <i>S. japonicum</i> life cycle and subjected to immunoblotting with anti-rSjGALE serum. (C) The western blot bands were converted into a histogram by measuring the optic density of the autoradiogram band.</p

Differential gene expression of SjGALE.

<p>Samples (A) throughout six stages of <i>S. japonicum</i> and (B) between the male and female adult worms were analyzed by real-time PCR. Data were normalized against amplification of an internal housekeeping control gene (β-tubulin). The data are the means ± SD of one representative of three independent experiments.</p

IgG1 and IgG2a immune profile induced by vaccination with rSjGALE or PBS.

a<p>Days after the first immunization.</p>b<p>Statistically significant compared to group of animals immunized with PBS.</p

Cytokine profile of rSjGALE-immunized mice.

<p>Ten days after the last immunization, splenocytes were isolated and assayed for IL-4, IL-10, IFN-γ and TNF-α production in response to rSjGALE (25 µg/ml) or medium alone, as a control. The results are presented as mean ± SD for each group. Statistically significant differences between cytokines produced after rSjGALE stimulation compared with unstimulated splenocytes are denoted by an asterisk (<i>p</i><0.01).</p

The complete protein sequence of SjGALE in relation to <i>S. mansoni</i>, mouse and human GALE.

<p>Clustal X alignment of the derived amino acid sequences of SmGALE (XP 002578272.1), MmGALE (NP_848476.1), and HsGALE (NP 001008217). The regions with high identity and similarity between GALE sequences are shown in color. The epimerase domain (continuous box), active sites (#) and NAD binding sites (*) are also indicated.</p

Protection level induces by BALB/c mice immunization with rSjGALE plus Freund’s adjuvant and infected with 30 cercariae.

*<p>Statistically significant compared to the control group (p<0.05),</p>**<p>(p<0.01).</p

Immunolocalization of SjGALE from adult male and female <i>S. japonicum</i>.

<p>Polyclonal anti-rSjGALE antibody was used as primary antibody and serum from naive mice was used as a negative control. Alexa Flour 488-conjugated (green) anti-mouse IgG was used as a second antibody. DAPI was used to stain nuclei (blue). (A): male adult worm. (C): male and female paring worms. (E): negative control. (B, D and F): DIC images of A, C, E, separately. (G): Detail of the image from panel (C) for detection of SjGALE (60× magnification).</p