Objective:To construct trivalent-epitope DNA vaccine encoding SjFABP,SjGAPDH and Sj26,and evaluate the protective immunity efficacy against Schistosoma japanicum by pharmacological trials.Methods:The monovalent antigen gene SjFABP,SjGAPDH and Sj26 were obtained by RT-PCR.Sj26 and SjGAPDH are spliced into covalent fusion gene of Sj26.SjGAPDH by recombinant PCR.pVIVO2-SjFABP/Sj26.SjGAPDH is constructed by subcloning the SjFABP and Sj26.SjGAPDH into mcs1 and mcs2 of pVIVO2-mcs respectively.The expression of fusion antigen protein in MCF-7 cell line transfected with pVIVO2-SjFABP/Sj26.GAPDH was determined by indirect immunofluorescence assay(IIF)and reverse transcriptase-polymerase chain reaction(RT-PCR).The evaluation of vaccine's immuno-protection efficacy is tested by pharmacological trials.Results:The trivalent-epitope DNA vaccine pVIVO2-SjFABP/Sj26.SjGAPDH was successfully constructed.In the trivalent-epitope DNA vaccine group,the worm reduction rate and the egg reduction rate were 58.6% and 59.8% compared with control group.Conclusion:The trivalent-epitope DNA vaccine pVIVO2-SjFABP/Sj26.SjGAPDH could induce higher protection efficiency than both monovalent and bivalent vaccine,and paves the way for development of DNA vaccine against Schistosoma japanicum.

Objective To screen the host bacterial strain of bivalent DNA vaccine against Schistosoma japonicum and optimize the condition for fermentation of the strain. Methods The host bacterial strain as well as the carbon source, nitrogen source and phosphate of fermentation medium were optimized by synthetic evaluation of dry cell weight, plasmid DNA yield and stability of plasmids in subcultured recombinant strain. Fermentation medium was further optimized by central combination design response surface test, then verified. Results E. coli DH5α was screened as the host bacterial strain of DNA vaccine against Schistosoma japonicum. The optimal fermentation medium consisted of(m / v)1. 0% sodium chloride, 1. 0% yeast Extract, 0. 3% phosphate in which the molar ratio of dipotassium hydrogen phosphate to potsssium dihydrogen phosphate was 1 ∶ 4, 1. 68% glycerol, 2. 28% beef juice and 4. 21% bean juice. Under the optimized fermentation condition, the dry cell weight increased from 1. 57 mg / ml to 5. 68 mg / ml, while the plasmid DNA yield from 3. 94 μg / ml to 36. 52 μg / ml. Conclusion The host bacterial strain of DNA vaccine against Schistosoma japonicum was screened, and the fermentation condition was optimized, by which the plasmid DNA yield increased significantly, indicating that the screened host bacterial strain and the optimized fermentation condition were suitable for large scale production of DNA vaccine against Schistosoma japonicum.

DNA vaccine encoding a multivalent antigen is a novel approach of protective immunization. Four Schistosoma japonicum candidate antigen genes, glyceraldehyde-3-phosphate dehydrogenase (SjGAPDH), 23 kDa transmembrane protein (Sj23), 14 kDa fatty-acid binding protein (SjFABP) and 26 kDa glutathione-S-transferase (Sj26), are recombined into two pieces of fusion genes SjFABP.Sj23 and Sj26.SjGAPDH, respectively. Tetravalent DNA vaccine pVIVO2-SjFABP.Sj23/Sj26.SjGAPDH is constructed by co-expressing these two fusion genes. The super-coiled DNA vaccines for large-scale clinic application were purified by sequential chromatographies including group separation chromatography and affinitive chromatographies. The purified DNA vaccines were evaluated for in vivo and in vitro transfection assay. The immunoprotective properties of the different kinds of constructed DNA vaccines were appraised by pharmacological trials. The pharmacological trials results showed that tetravalent DNA vaccine has higher protective efficiency than other tested DNA vaccines.

Objective:To test the expression of DNA vaccine of Schistosoma japonicum using prokaryotic expressed proteins SjFABP and SjGST of S.japonicum.Methods:Specific primers were designed,SjFABP and SjGST genes were amplified by RT-PCR and were cloned to pGEM-T Easy vector and sequenced,then were subcloned to build prokaryotic expression vectors pET30a-SjFABP,pET30a-SjGST and eukaryotic expression vector pVIVO2-SjFABP-SjGST.The E.Coli BL21(DE3) cells were transformed with the recombinant plasmids pET30a-SjFABP and pET30a-SjGST respectively,then induced by IPTG to express the recombinant proteins which were purified through His-Bind Ni-Agarose.The purified recombinant proteins were used to recognize sera from mice induced by pVIVO2-SjFABP-SjGST.Results:The SjFABP(399 bp) and SjGST(657 bp) genes PCR products were obtained,and the recombinant plasmids pET30a-SjFABP,pET30a-SjGST and pVIVO2-SjFABP-SjGST were constructed,the prokaryotic expressed proteins were detected by SDS-PAGE.The rSjFABP and rSjGST can be recognized by the sera from mice induced by pVIVO2-SjFABP-SjGST using Western blotting.Conclusion:The SjFABP and SjGST genes were effectively expressed in prokaryotic cells,and can be recognized by antibody induced by DNA vaccine pVIVO2-SjFABP-SjGST.