Table 1.
Specific oligonucleotide primers used to obtain full length cDNA sequence.
Figure 1.
Purification of native Ts-PCHTP with anion exchange chromatography followed by size exclusion chromatography.
(A) and Ni-NTA agarose purification (B). 1) markers; 2) soluble fraction after ultracentrifugation; 3) supernatant after 70% saturation with ammonium sulfate; 4) unbound fraction of anion exchange chromatography on DEAE-cellulose; 5) Ts-PCHTP after size exclusion chromatography on Superdex 75; 6) Ts-PCHTP after Ni-NTA chromatography. Proteins were visualized by Coomassie Blue staining.
Figure 2.
Organization of ts-pchtp gene and protein chain.
A) Comparison between Ts-PCHTP cDNA and whole genome shotgun sequence, ABIR01001777 (start 65194–end 67089) from Trichinella spiralis showed 6 exons and 5 introns of the 1896 bp gene. B) Open reading frame of translated protein sequence contains signal peptide (exon 1), two homology poly-cysteine domains (exon 2–4 and exon 5–6) and histidine tail on C-term end (exon 6). The 2 potential glycosylation sites are shown. C) Comparison between poly-cystein domain 1 and 2. Data showed 38.5% identity at 174 amino acid. Conservative cysteine residues are in bold.
Figure 3.
Comparison of the Ts-PCHTP pcd domains with Caenorhabditis elegans proteins and other nematode ESTs sequences.
Bordered groups pcd-1_T.sp and pcd-2_T.sp contains pcd domains of the Ts-PCHTP and related ESTs (Table S1); bordered group R01H10.4 contains ESTs pt39d02.y1 (Trichinella spiralis) and HTAB-aae58a04.b1 (Heterorhabditis bacteriophora); and bordered group T19C3.2 (YSV2_CAEEL) contains EST ph72c07.y1 (Ostertagia ostertagi) and hypothetical protein CBG15264 (C. briggsae). Homologous amino acids residues among groups are in grey boxes. Amino acids residues homologous for all proteins are shown with white letters.
Figure 4.
Deglycosylation native Ts-PCHTP with N-Glycosidase F.
1) eluted protein fraction after Ni-NTA chromatography; 2) deglycosylation with N-Glycosidase F; 5) marker. Proteins were visualized by Coomassie Blue staining.
Figure 5.
Total Reflection X-ray Fluorescence (TXRF) of the native Ts-PCHTP.
1 M of the native Ts-PCHTP bind about 1.5 M Fe; 2 M Zn and 2 M Ni.
Figure 6.
Immunofluorescent localization of Ts-PCHTP in T. spiralis larvae.
(A) The intense staining of the encapsuled larva (L) after reaction with polyclonal serum. Nc - nurse cell, M - host muscle. (B) Labeling of the Ts-PCHTP in encapsuled larva (L) with anti-histidine antibody. Scale bar: A, B - 60 µm.
Figure 7.
Immunogold electron microscopic localization of Ts-PCHTP in T. spiralis.
(A) Strong accumulation of gold particles in the cuticle (C), hypodermis (H) and muscle (M) layers, revealed by polyclonal serum. (B) Control sample. Scale bar: A, B - 3 µm.