OBJECTIVE: To prepare soluble recombinant signal transduction protein 14-3-3 of Schistosoma japonicum (rSj14-3-3) and investigate its immunologic features. METHODS: The cDNA fragment of signal transduction protein 14-3-3 gene was prepared from Schistosoma japonicum adult worm mRNA, and was subcloned to the downstream of glutathione S transferase gene of expression vector pGEX-4T-3 to construct a recombinant expression plasmid 14-3-3/pGEX-4T-3. The recombinant plasmids were transferred into E. coli BL21, the transforments containing recombinant plasmid were induced by IPTG, and the expression situation of fusion protein GST-14-3-3 was observed by SDS-PAGE. The pure recombinant 14-3-3 protein was prepared by digesting the fusion protein GST-rSj14-3-3 with thrombin and affinity chromatography. The specific antibody against rSj14-3-3 protein was prepared by an immunized rabbit with rSj14-3-3 protein. The immunogenicity and immune reactivity of rSj14-3-3 protein were analyzed by Western blotting. RESULTS: The gene encoding signal transduction protein 14-3-3 of Schistosoma japonicum Jiangsu strain was cloned successfully, the homology of open read frame sequence to the registrated sequence of Sj14-3-3 protein in genbank was 99.08%. A 55 kilo Dalton fusion protein GST-rSj14-3-3 was expressed by transferring the recombinant plasmid 14-3-3/pGEX-4T-3 into E. coli BL21 and induced with IPTG. The high titer antibody against rSj14-3-3 was produced by the immunized rabbit with rSj14-3-3 ,and could recognize the nature and recombinant rSj14-3-3 proteins. CONCLUSIONS: The soluble rSj14-3-3 protein has been prepared successfully in this study, and it has good immunogenicity and reactivity.
OBJECTIVE: To prepare soluble recombinant signal transduction protein 14-3-3 of Schistosoma japonicum (rSj14-3-3) and investigate its immunologic features. METHODS: The cDNA fragment of signal transduction protein 14-3-3 gene was prepared from Schistosoma japonicum adult worm mRNA, and was subcloned to the downstream of glutathione S transferase gene of expression vector pGEX-4T-3 to construct a recombinant expression plasmid 14-3-3/pGEX-4T-3. The recombinant plasmids were transferred into E. coli BL21, the transforments containing recombinant plasmid were induced by IPTG, and the expression situation of fusion protein GST-14-3-3 was observed by SDS-PAGE. The pure recombinant 14-3-3 protein was prepared by digesting the fusion protein GST-rSj14-3-3 with thrombin and affinity chromatography. The specific antibody against rSj14-3-3 protein was prepared by an immunized rabbit with rSj14-3-3 protein. The immunogenicity and immune reactivity of rSj14-3-3 protein were analyzed by Western blotting. RESULTS: The gene encoding signal transduction protein 14-3-3 of Schistosoma japonicum Jiangsu strain was cloned successfully, the homology of open read frame sequence to the registrated sequence of Sj14-3-3 protein in genbank was 99.08%. A 55 kilo Dalton fusion protein GST-rSj14-3-3 was expressed by transferring the recombinant plasmid 14-3-3/pGEX-4T-3 into E. coli BL21 and induced with IPTG. The high titer antibody against rSj14-3-3 was produced by the immunized rabbit with rSj14-3-3 ,and could recognize the nature and recombinant rSj14-3-3 proteins. CONCLUSIONS: The soluble rSj14-3-3 protein has been prepared successfully in this study, and it has good immunogenicity and reactivity.