Expression of Schistosoma japonicum 8 kDa calcium-binding protein gene and evaluation of the immunoreactivity of its fusion protein.
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.
Xu
Xing
X
Department of Parasitology, First Military Medical University, Guangzhou 510515, China.
Peng
Hong-Juan
HJ
Chen
Xiao-Guang
XG
D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015801:Helminth Proteins / Q000276:immunology*; D051379:Mice; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D012549:Schistosoma japonicum / Q000276:immunology*
D002135:Calcium-Binding Proteins; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins