Immunodiagnosis And Immunoprotection Of Recombinant SjGST And Sj14-3-3 In Schistosomiasis Japonica

Objective To express and purify rSjGST from pET28a/rSjGST/E.coli BL21 as recombinant antigens for the diagnosis of schistosomiasis and to generate monoclonal antibodies against rSjGST for evaluation of its poteintial in enzyme-linked immunosorbent assay(ELISA). Method: BALB/c mice were immunized by rSjGST. HGPRT - SP2/0 cells were fused with mice spleen B lymphocytes. The hybridoma cells were cultured in the HAT selective medium. The positive clone was screened by ELISA. The McAb to rSjGST was purified from the peritoneal fluids with Protein G Sepharose 4B Fast Flow and subjected to the detection for the circulating SjGST in the sera of patient with acute schistosomiasis. Result: A strong band of protein with a molecular weight of 26ku was observed in SDS-PAGE gel stained by Comassie blue. The hybridoma cells were produced and monoclonal antibodies against rSjGST were harvested from the cell culture supernatant after comfirmation by ELISA and Western-blotting. The result of ELISA showed that the positive rates were 90.6% in detecting specific antibodies against SjGST and 59.4% in detecting circulating SjGST. Conclusion: The present study indicated that the 26ku rSjGST, combined with its McAb in paralell, could be used as a pair of probes for the diagnosis of schistosomiasis. To evaluate the role of PLGA( polylactic-co-glycolic acid)delayed release microsphere administration system in molecular vaccine for schistosomiasis and to observe the synergism of rSj14-3-3 , rSjGST and mIL-12 ,and the effect of CTL and Th activated by mIL-12 against Schistosoma japonicum. Expression of rSj14-3-3 and rSjGST were purified though His-Bind Purification Kit. To construct eukaryotic expression plasmid pcDNA3.1(+)mIL-12, BALB/c mice immunized with rSj14-3-3, rSjGST,and plasmid mIL-12/PLGA delayed release microsphere were challenged by cercaria infection. Six weeks after challenging infection, the mice were sacrificed and the worm and egg reduction rates were calculated. Worm reduction rate was found to be 27.6% in single rSj14-3-3 group, 34.9% in rSj14-3-3+PcDNA3.1(+)mIL-12 group, 37.5% in rSj14-3-3+PcDNA3.1(+)mIL-12 /PLGA delayed release microsphere group, 38.8% in rSj14-3-3+rSjGST+PcDNA3.1(+)mIL-12/PLGA delayed release microsphere group,respectively. The number of eggs in liver tissue was reduced by 35.3%,49.1%,50.5%,43.1%, respectively. PLGA delayed release microsphere administration system might induce humoral and cell-mediated immunity and the effect of vaccines was enhanced but no synergistic effect was observed between rSj14-3-3 and rSjGST..