Studies On Schistosoma Japonicum 18kDa Molecular Antigen And Candidate Gene DNA Vaccine

Objective To develop the effective vaccine candidate molecular against Schistosma japonicum (Sj), Sj adult worm and immature egg soluble antigens (SjAWA and SIEA) were separated and purified by column choromatography, SDS-PAGE and electroelution technique. At the same time, a new gene discovered was subcloned into the eukaryotic expression vector pcDNA3. Finally the immunoprotective effect as an anti-Schistosomiasis vaccine was observed in mice immuned by the plasmid pcDNA3/SJCWL01 and SjAWA18kDa, respectively.Methods (1) SjAWA and SIEA were separated and purified by column choromatography, SDS-PAGE and electroelution technique. The fractions were analysed and identified using the silver staining and coomassie brilliant blue staining after SDS-PAGE. (2)The monospecific antibody against the SjAWA18kDa antigen was produced with various immune ways. Immunity analysis was detected with above antibody as a probe. (3) S. japonicum cercaria cDNA library was screened with sera from rabbits vaccinated with irradiated cercariae. The novel gene (named SJCWL01, GenBank accession number are AY855919) was subcloneed into the eukaryotic expression vector pcDNA3.The positive recombinant was identified by PCR and restriction enzyme digestion. The plasmid pcDNA3/SJCWL01 was transformed into E.coli DH5 a for preparing DNA vaccine. The experimental mice were immunized with purified pcDNA3/SJCWL01 DNA vaccine and SjAWA18kDa purified by the column choromatography. Then, they were percutaneously challenged with 40±1 S. japonicum cercaria at the 2nd week after the last immuniztion. The worm and egg reduction rates were counted to evaluate immunoprotective effect. In addition, the expression of pcDNA3/SJCWL01 in the muscle tissue was observed by immunohistochemistry (IHC) method.Results (1)Five different protein antigens,i.e. SjAWA18kDa, SjAWA26/28kDa, SjAWA14kDa, SIEA42kDa, SIEA130kDa were obtained after the isolation and purificaion through column choromatography and/or SDS-PAGE and electroelution technique. (2)The monospecific anti-sera against SjAWA18kDa was produced successfully. Western blotting results showed there were common antigenic epitope between SjAWA 18kDa and recombinant fusion protein encoded novel gene SJCWL01. The results of immunohistochemistry revealed that specific reaction against SjAWA18kDa antigen were mainly seen in the gastrodermis and parenchyma of adult worms by optical microscopy. (3)The recombinant pcDNA3/SJCWL01 DNA vaccine was purified after SJCWL01 gene subcloned into pcDNA3 and transformed E.coli DH5α. Then mice were immunized with purified SjAWA18kDa and pcDNA3/SJCWL01 DNA vaccine. Compared with control group,the worm reduction rates of DNA vaccine group and SjAWA18kDa group is 27.6%,30.7%, and 39.5%,50.7% in the egg reduction rates, respectively.Conclusion (1) The results showed the column choromatography or SDS-PAGE and electroelution technique is a simple and effective method for the isolation and purificaion of molecular antigens of Schistosoma japonicum. (2)The monospecific anti-sera against SjAWA18kDa antigen was successfully produced. (3) The recombinant plasmids on pcDNA3/SJCWL01 were successfully constructed .Both of the SjAWA18kDa and pcDNA3/SJCWL01 could induce partial protective immunity against S. japonicum infection in mice. They showed the potential value as a DNA vaccine and a native subunit molecular vaccine.