OBJECTIVE: To obtain the expression of Schistosoma japonicum 8 kDa calcium-binding protein gene subcloned into the pET32a(+) soluble expression plasmid and carry out purification and immunoreactivity assessment of the expressed protein. METHODS: The recombinant plasmid was transferred to BL21 E.coli. strain, and induced with IPTG before bacterial lysis ultrasonically. The supernatant was analysed by SDS-PAGE after centrifugation. The target protein was purified with Ni-NTA agarose and transferred to the PVDF film for immunoreactivity test by Western blotting with anti-6-His antibody, the serum of rabbits 6 weeks after cercariae infection and the serum from UV-attenuated cercariae-immunized rabbits. The purified protein was used as the coating antigen for enzyme-linked immunosorbent assay (ELISA) to examine the sera from normal and cercariae-infected rabbits. RESULTS: The molecular weight of the target protein was 29,000 after IPTG induction. The fusion protein presented a single band immunoreactive to anti-6-His antibody, which was not reactive to the serum from cercariae-infected or UV-attenuated cercariae-immunized rabbit. CONCLUSIONS: The calcium-binding protein is expressed as thioredoxinfusion protein with a relative molecular weight of about 29,000. The fusion protein possesses no immunoreactivity to cercariae-infected or UV-attenuated cercariae-immunized rabbit sera, possibly because of the specificity of the protein at the cercariae stage.
OBJECTIVE: To obtain the expression of Schistosoma japonicum 8 kDa calcium-binding protein gene subcloned into the pET32a(+) soluble expression plasmid and carry out purification and immunoreactivity assessment of the expressed protein. METHODS: The recombinant plasmid was transferred to BL21E.coli. strain, and induced with IPTG before bacterial lysis ultrasonically. The supernatant was analysed by SDS-PAGE after centrifugation. The target protein was purified with Ni-NTAagarose and transferred to the PVDF film for immunoreactivity test by Western blotting with anti-6-His antibody, the serum of rabbits 6 weeks after cercariae infection and the serum from UV-attenuated cercariae-immunized rabbits. The purified protein was used as the coating antigen for enzyme-linked immunosorbent assay (ELISA) to examine the sera from normal and cercariae-infected rabbits. RESULTS: The molecular weight of the target protein was 29,000 after IPTG induction. The fusion protein presented a single band immunoreactive to anti-6-His antibody, which was not reactive to the serum from cercariae-infected or UV-attenuated cercariae-immunized rabbit. CONCLUSIONS: The calcium-binding protein is expressed as thioredoxinfusion protein with a relative molecular weight of about 29,000. The fusion protein possesses no immunoreactivity to cercariae-infected or UV-attenuated cercariae-immunized rabbit sera, possibly because of the specificity of the protein at the cercariae stage.