Fig 1.
The predicted three-dimensional model of T. spiralis TsSP protein.
A: The predicted three-dimensional structure of TsSP protein, there are 4 α-helixes (in red), 14 β-strand (in yellow), and 19 irregular coils (in green); B: Catalytic residues Ser-His-Asp form a pocket-shaped functional domain. The active site of TsSP was highlighted with red color.
Fig 2.
Sequence alignment of serine protease from Trichinella spiralis (ABY60762) with other species of the genus Trichinella and other organisms.
The serine proteases was from Trichinella spp.: T. britovi (KRY59723.1), T. nativa (KRZ48330.1), T. zimbabwensis (KRZ02345.1), T. pseudospirali (KRY01512.1), T. nelsoni (KRX27556.1), Homo sapiens (CAB91984.1), Mus musculus (EDL11329.1), Anopheles gambiae (CAB90819.1). The multiple sequences alignment was performed in the Clustal X and displayed using BOXSHADE. Black shade indicated that residues identical to TsSP, and conservative substitutions were shaded grey. The numbers at the end of each sequence represent the score and percent identity to TsSP.
Fig 3.
Cladogram of analysis of TsSP.
The maximum parsimony tree of serine protease from 19 organisms calculated using MEGA. The GenBank accession numbers of each serine protease are as follows: Trichinella spiralis (ABY60762.1), Trichinella nativa (KRZ48330.1), Trichinella nelsoni (KRX27556.1), Trichinella britovi (KRY59723.1), Trichinella pseudospiralis (KRY01512.1), Trichinella zimbabwensis (KRZ02345.1), Homo sapiens (CAB91984.1), Mus musculus (EDL11329.1), Caenorhabditis elegans (CCD65324.1), Drosophila melanogaster (NP_001262565.1), Trichuris trichiura (CDW53654.1), Ascaris suum (ERG81033.1), Necator americanus (XP_013308358.1), Brugia malayi (XP_001900088.1), Wuchereria bancrofti (EJW79555.1), Haemonchus contortus (CDJ82043.1), Echinococcus granulosus (EUB62466.1), Anopheles gambiae (CAB90819.1), Schistosoma haematobium (KGB34910.1). Bootstrap values which are higher than 80 are indicated on branches.
Fig 4.
SDS-PAGE analysis of the rTsSP.
M: protein molecular weight marker; 1: protein lysates from recombinant E. coli before being induced, 2: protein lysates of recombinant E. coli induced by IPTG; 3: rTsSP purified by Ni-NTA-Sefinose Column. Arrow indicates rTsSP.
Fig 5.
Serum anti-rTsSP IgG titers determined by ELISA.
Microtiter plates were coated with 2 μg rTsSP /ml and incubated overnight at 4°C. After being blocked with 5% nonfat milk, the plates were incubated at 37°C for 1 h with different dilutions of immune sera. Normal mouse sera (n = 20) diluted at 1:100 were assayed as negative controls. HRP-conjugated IgG was utilized as the second antibodies. The coloration was developed by incubation with the substrate OPD. The absorbance at 490 nm was assayed following adding 2 M H2SO4. The cut-off values (0.202) are expressed with a dotted line.
Fig 6.
Western blot analysis of rTsSP antigenicity.
(A) SDS-PAGE analysis of ML ES antigens from T. spiralis (lane 1), ML crude antigen (lane 2), and rTsSP (lane 3). (B) Western blotting of the rTsSP. T. spiralis ML ES antigens (lane 1) and ML crude antigens (lane 2) and rTsSP (lane 3) were probed with mouse infection sera. The natural TsSP protein in ML ES (lane 4), ML crude antigens (lane 5) and rTsSP (lane 6) were identified by using anti-rTsSP serum. Normal mouse sera did not probe the ES (lane7) and crude antigens (lane 8), and rTsSP (lane 9). (C) Western blotting of ML ES (lane 1 and 4) and crude antigens (lane 2 and 5), and rTsSP (lane 3 and 6) probed with immune sera from mice vaccinated with ES and crude antigens.
Fig 7.
RT-PCR assay of TsSP transcription at different T. spiralis phases.
RT-PCR assay of the mRNA transcription of TsSP (A) and GAPDH (B) at various T. spiralis stages. M: DNA marker; Lane 1: ML; Lane 2: IIL; Lane 3: AW at 3 dpi; Lane 4: AW at 6 dpi; Lane 4: NBL.
Fig 8.
Anti-rTsSP IgG level of immunized mice determined by ELISA using antigens from various stages.
OD values shown for each group (n = 13) were the arithmetic mean ± standard deviation (SD) of serum anti-rTsSP IgG levels.
Fig 9.
Western blot analyses of the TsSP expression levels at diverse T. spiralis phases.
Expression levels of TsSP protein with 45.2kDa in crude antigens of diverse T. spiralis phase (AW, NBL, ML and IIL) were determined by Western blot with 1:100 dilutions of anti-rTsSP serum. The graph reveals the relative protein expression assayed with densitometry in 3 independent experiments. Asterisk (*) shows statistical differences (P < 0.05) relative to the ML. Pound sign (#) indicates remarkably statistical differences (P <0.01) relative the ML.
Fig 10.
Immunolocalization of TsSP at different T. spiralis phases.
A-I: IFT with intact worm of different T. spiralis phases probed by anti-rTsSP serum. The apparent immunostaining is observed on cuticles of ML (A), 6 hpi IIL (B), 24 hpi IIL (C), 3 dpi female adult (D), 6 dpi female adult (E), and NBL (F). The ML probed by infection serum (G) was utilized as a positive serum control; the ML incubated with normal mouse serum (H) and PBS (I) were applied to negative controls. When worm sections were incubated by anti-rTsSP serum, positive staining is seen in cuticles and stichosomes of ML (J), IIL (K) and female adult at 3 dpi (L). The ML probed by infection serum (M) was employed as a positive serum control; ML reveals no staining with normal mouse serum (N) and PBS (O) as a negative control. Scale-bars: 100 μm.
Fig 11.
Scatter plot of optical density values of rTsSP-ELISA (A) and ES-ELISA (B) for detection of anti-Trichinella IgG in serum samples of patients with trichinellosis and other parasitic diseases. The cut-off value is represented by the dotted line.
Table 1.
Detection of anti-Trichinella IgG antibodies in serum samples of patients with trichinellosis and other parasitic diseases by rTsSP-ELISA.
Fig 12.
Kinetics of serum anti-Trichinella IgG in mice experimentally infected with 300 muscle larvae.
Anti-Trichinella IgG was detected by rTsSP-ELISA (A) and ES-ELISA (B). The cut-off value is represented by the dotted line.