Résumé
Objective To obtain genes encoding the novel molecules for diagnosis of schistosomiasis.Methods Juvenile S.japonicum cDNA library was immunoscreened to obtain positive clones.By DNA sequencing and sequence analysis,the target gene was amplified by PCR and subcloned into prokaryotic plasmid pET28a.The recombinant plasmid was transformed into E.coli BL21 followed by expression of the protein induced by IPTG.The protein was identified by Western blotting.Results 34 positive clones were obtained,24 of which were chosen to be sequenced,13 of which were Sj22600 gene.The protein were recognized with sera of infected rabbits and patients with acute and chronic schistosomiasis by Western blotting.Conclusion The gene coding for Sj22600 membrane protein was screened with high frequency in the cDNA library.The E.coli BL21 transformed with the recombinant plasmid can express the fusion protein,which shows immunoactivity.
Résumé
Objective To clone and express a membrane protein(Tetraspanin 2) gene of Schistosoma japonicum (SjTsp2). Methods A pair of primers was designed to amplify the SjTsp2 gene which was subcloned into prokaryotic plasmid pET28a(+). The recombinant plasmid was transformed into E. coli BL21(D3) and followed by expression of the protein induced by IPTG. The protein was purified by affinity chromatography and used to immunize BALB/c mice. Dilution of antibody against SjTsp2 was determined by ELISA. The protein was also identified by Western blotting. Results Big loop of SjTsp2-A, 228 bp, was amplified in vitro by PCR. Its deduced amino sequence shared 52% similarity with SmTsp2. The soluble recombinant SjTsp2-A was expressed in the experiment and high dilution antibody against the recombinant (1 ∶ 32 000 in maximum) was produced in immunized mice. SjTsp2-A reacted positively with sera of acute and chronic schistosomiasis patients but not with sera from healthy persons by Western blotting. Conclusion SjTsp2 has been expressed and shows certain antigenicity.