[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).


CONCLUSIONS
The recombinant protein Sj14-3-3 of eukaryotic expression in Pichia pastoris has been successfully harvested and shows a promising immunological potential.
Zheng
Mei-Juan
MJ
Department of Biochemistry and Molecular Biology, Bengbu Medical College, Bengbu 233003, China.
Li
Min
M
Li
Cong-Lei
CL
Chu
De-Yong
DY
Wang
Zhi-Cheng
ZC
Luo
Fei
F
Luo
Qing-Li
QL
Shen
Ji-Long
JL
D048948:14-3-3 Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction; D012549:Schistosoma japonicum / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012554:Schistosomiasis japonica / Q000097:blood
D048948:14-3-3 Proteins; D000909:Antibodies, Helminth; D015801:Helminth Proteins; D007074:Immunoglobulin G