关键词:
 日本血吸虫；抗原表位；重组表达；淋巴细胞增殖试验

Abstract:

 Objective  To synthesize and fusion express the predicted T-cell epitopes of Schistoso-
ma japonicum., and analyze their immunogenicities. Methods   The plus and minus oligo-nucleic acid
strands of epitopes P7, P17, P18 were synthesized following their DNA sequence, respectively.
The Nco I restriction enzyme sites were added to the 5' end of epitope gene and the Xho I restriction
enzyme sites were added to the 3' end of epitope gene. The plus and minus strand of each epitope
gene was annealed to form double strand DNA fragments. Then the double strand DNA fragments
encoding epitope peptide were cloned into the site between Nco I and Xho I of plasmid pET32c(+)
to construct recombinant plasmid which was transformed into E.coli DH5a. The recombinant plas-
mid containing P7, P17, P18 genes respectively was identified by PCR, restriction digestion and
DNA sequencing, and then transformed into E.coli BL21 (DE3) for expressing the fusion protein.
The fusion protein of peptide-thioredoxin(Trx) was expressed by inducing with IPTG and analyzed
with SDS-PAGE. The fusion proteins were purified with N12+ column affinity chromatography.
Meanwhile, the peptides P7, P17, P18 were synthesized artificially following their amino acid se-

quence. By using the purified epitope peptide fusion proteins and synthesized epitope peptides, the
splenic cells of C57BL/6J mice immunized with ultraviolet-attenuated cercaria of Schistosoma japon-
icum were stimulated respectively. The stimulation activity of fusion proteins and synthesized pep-
tides were assayed by detecting the incorporation rate of 3H-thymidine. Results  The double strand
DNA fragments of epitopes P7, P17, P18 were successfully cloned to form recombinant plasmids,
all of which could express a 20 kDa fusion protein. Both the fusion protein and synthesized epitope
peptides of P7 and P17 were able to stimulate the lymphocyte cells to proliferation effectively. Con-
clusion  The peptide P7 and peptide P17 are testified as T-cell epitopes of Schistosoma jaPon,icum.
 

Key words:  Schistosoma japonicum, Antigen epitope, Recombinant expression, Lymphocyte proliferation assay