Aim: To clone and sequence the gene encoding paramyosin of S. japonicum (Chinese strain) and to study the expression of the DNA-based vaccine encoding the full-length paramyosin of S. japonicum in vivo.
Methods: Total RNA was isolated from adult S. japonicum using TRIzol reagent. The full-length cDNA encoding paramyosin of S. japonicum was amplified by RT-PCR and cloned into pGEM-T vector and sequenced by the method of dideoxy-mediated chain-termination. The cDNA encoding paramyosin of S. japonicum was subcloned into the expressive plasmid vector pCDNA/AMP(pCMV-Sjc97), and the recombinants were identified by restriction enzyme digestion and sequencing. The immunofluorescence assay was used to study the expression of Sjc97 in vivo in mice.
Results and conclusion: The 2.6 kb cDNA encoding the full-length paramyosin of Chinese S. japonicum has been successfully cloned and sequenced for the first time. The full-length sequence of paramyosin of S. japonicum was determined. Comparison of the nucleotide sequence and the deduced amino acid sequence of Sjc97 with that of S. japonicum paramyosin (Philippine strain) (Sjp97), S. japonicum paramyosin(Japanese strain) (Sjj97), S. mansoni (Sm97), B6 and Y6 clone (the partial cDNA encoding paramyosin of Chinese strain) showed that Sjc97 differed from Sjp97 by 16/2,601 nucleotide and 3/866 amino acid substitutions (99.4% on nt-level and 99.7% on aa-level in homology); from Sjj97 by 20/2,601 nucleotide and 2/866 amino acid substitutions (99.2% on nt-level and 99.8% on aa-level in homology); and from B6 by 11/1,329 nucleotide and 1/443 amino acid substitutions (99.0% on nt-level and 99.8% on aa-level in homology); from Y6 by 13/1,329 nucleotide and 1/443 amino acid substitutions (98.9% on nt-level and 99.8% on aa-level in homology); Sjc97 differed from the Sm97 by 2,235/2,601 nucleotide and 34/866 amino acid (91.0% on nt-level and 96.0% on aa-level in homology). The plasmid expression vector encoding the full-length paramyosin of Chinese S. japonicum has been successfully constructed. The pCMV-Sjc97 vaccine could express Sjc97 protein in vivo in mice after intramuscular immunization.