[Secreted expression of signaling protein 14-3-3 of Schistosoma japonicum in Pichia pastoris system with primary evaluation on its antigenicity].

OBJECTIVE

To express signaling protein Sj14-3-3 in Pichia pastoris and compare its antigenicity with prokaryotic expression one.

METHODS

Sj14-3-3 gene was amplified from pET28a-Sj14-3-3 recombinant plasmid, cloned into vector pMD18-T followed by sequencing. The Sj14-3-3 gene was subcloned into the expression vector pPICZalpha-B and transformed into Pichia pastoris X-33 by electroporation. The transformants were identified by sequencing. Three transformants with high copies were obtained when selected under zeocin, and expression was induced with methanol. The culture supernatant was collected and tested by SDS-PAGE and Western blotting. The specificity and sensitivity of eukaryotic expression rSj14-3-3 in Pichia pastoris were compared with that from prokaryotic expression by detecting sera of patients with schistosomiasis by indirect ELISA.

RESULTS

The Sj14-3-3 gene was integrated into Pichia pastoris, and the gene of interest detected by PCR was with 1 300 bp. After induction by methanol, the Sj14-3-3 gene was expressed and secreted into the medium. The molecular weight of the recombinant protein was determined as about Mr 35 000 by SDS-PAGE. Western blotting showed that the protein has a high specificity against mouse-anti-Sjl4-3-3 monoclonal antibody. The recombinant protein had a promising immune reactivity. Indirect ELISA showed that by using eukaryotic expression rSj14-3-3 in Pichia pastoris, the positive rate in 36 cases of acute schistosomiasis was 81%, with no cross-reactivity in 12 cases of Clonorchis sinensis, 9.3% cross-reactivity in 32 cases of normal sera. While using prokaryotic expression rSj14-3-3 in E.coli, the positive rate in 36 cases of acute schistosomiasis was 88.9%, with 16.7% cross-reactivity in 12 cases of Clonorchis sinensis, 12.5% cross-reactivity in 32 cases of normal sera. There was no statistically significant difference of the results (P>0.05).

CONCLUSION

The recombinant protein Sj14-3-3 of eukaryotic expression in Pichia pastoris has been successfully harvested and shows a promising immunological potential.