[Purification, enzyme activity and immunology study of recombinant protein glyceraldehyde-3-phosphate dehydrogenase of Clonorchis sinensis].
OBJECTIVE
To produce prokaryotic recombinant protein glyceraldehyde-3 phosphate dehydrogenase of Clonorchis sinensis (CsGAPDH), analyze its enzyme activity and immunological function. Methods The recombinant CsGAPDH was purified according to the protocol of GST-Bind kit and was digested with thrombin proteinase and eluted with wash buffer. The BALB/c mice were inoculated with the purified protein. The antisera collected from the mice were used to detect the titres of IgG antibodies by ELISA, and Western blotting was used to identify the specificity of the antisera with the purified CsGAPDH. S-P immunohistochemistry method was used to confirm the expression and distribution of CsGAPDH in adult Clonorchis sinensis with the polyclonal antibodies from immunized BALB/c mice. The CsGAPDH catalytic activity was evaluated employing the conventional substrate glyceraldehyde-3-phosphate (3-GAP).


RESULTS
SDS-PAGE showed a single purified protein band. Gel scanning analysis revealed that the protein purity of CsGAPDH was 90%. ELISA analysis showed an increased IgG value. S-P immunohistochemistry analysis demonstrated that the recombinant plasmid pGEX-4T-1 GAPDH expressed and distributed in muscle cell membrane of immune mice. Western blotting result suggested that CsGAPDH protein contained essential epitopes with high antigenic activities. This protein CsGAPDH could catalyzed 3-GAP with enzymatic active unit of 2872 U min(-1) ml(-1).


CONCLUSIONS
The recombinant protein CsGAPDH shows a proper enzymatic activity and immunogenicity.
Zhang
Yong-li
YL
Department of Medical biology, Guangdong Pharmaceutical University, Guangzhou 510224, China.
Wu
De
D
Yu
Xin-bing
XB
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood / Q000276:immunology; D015153:Blotting, Western; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000276:immunology*; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology*; D007106:Immune Sera / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology* / Q000302:isolation & purification
D000909:Antibodies, Helminth; D015801:Helminth Proteins; D007106:Immune Sera; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases