Isolation, Identification And DNA Vaccine Of Pairing-Associated Proteins Of Schistosoma Japonicum

ObjectiveSchistosomiasis is a severe world public-health problem, with morethan 600 million people exposed to infection and nearly 200 millionpeople infected. Currently, schistosomiasis control strategy is mainlybased on the treatment of infected individuals by chemotherapy with safeand effective drugs, while it can't prevent schistosome re-infection. Manyscientists have been trying to make anti-schistosomiasis vaccines torelieve the pathologic lesions. A unique trait of schistosomes is theirsexually dimorphic character. Female schistosomes from single-sexinfection are stunted in size and sexually immature. The pairing betweenmale and female is necessary for the development, maturing and eggproduction of schistosome, and the eggs laid by the mature femaleparasite is the main pathogenic source for the disease. Vaccination can betargeted towards the prevention of infection or to the reduction of parasitefecundity. The national schistosome vaccine unitive test carried out in2000 showed the natural molecular vaccine (Vaccine No.1) constructedby Wang SP et al. was the best vaccine of 16 vaccines. In order to revealdifferentially expressed proteins of Schistosoma japonicum in pairing.Two-dimensional gel electrophoresis and mass spectrometry wereperformed between male worms of single-sex infection and male wormsof bisexual infection in schistosoma japonicum. 9 proteins were identified.The gene encoding 22.7 kDa Pairing-Associated Proteins SJCHGC ofSchistosoma japonicum male worm was chosen as a vaccine antigenmolecular. Recombinant plasmid pcDNA3/SJCHGC was constructed. The protective ability of pcDNA3/SJCHGC immunized in mice andpossible immune mechanism were performed to value the potentiality ofbeing the effective component of vaccine against schistosomiasis, and tolay a solid foundation for the construction and field-test of the vaccine inthe future.Methods1) Two-dimensional gel electrophoresis and mass spectrometry wereperformed between male worms of single-sex infection and male wormsof bisexual infection in schistosoma japonicum, and further validated bysemi-quantitative RT-PCR analysis.2) By using bioinformatics, the cDNA sequence encoding SJCHGCwas analyzed, including searching the ORF, translating the nucleotide toprotein sequence, similarity searches, secondary and tertiary structurepredication.3) The recombinant plasmid pEGFP-C1/SJCHGC was transfectedinto COS-7 cells by using cationic lipids (Lipofectamine 2000). Theexpression and subcellular localization of the fusion protein in transfectedCOS-7 cells were analyzed by fluorescence microscopy. The expressedproducts were identifed bv RT-PCR, SDS-PAGE and Western blotting.4) The SJCHGC gene was amplified by PCR, and cloned into theeukaryotic expression vector pcDNA3. The recombinant plasmidpeDNA3/SJCHGC was identified by PCR, restriction enzyme digestionand sequence analysis. The mice were immunized withpcDNA3/SJCHGC. Two weeks after the third vaccination, the antigengenes were present in muscular tissue of mice by PCR, and a challengeinfection was carded out with S.japonicum cercariae. The worms andeggs were collected and counted at the 42th day after the challengeinfection.5) The mice were immunized with pcDNA3/SJCHGC, and challenged with S.japonicum cercariae at the 6wk after theimmunization. Sera obtained from mice at 0 week (pre-immunization), 2weeks, 4 weeks and 6 weeks after immunization were analysedquantitatively for the presence of total IgG by ELISA. The specificanti-SJCHGC IgG was detected by Western blot. The proliferationactivity of spleen T lymphocytes was tested using MTT assay. By theculture of spleen cells 6 weeks after challenge infection, IFN-γand IL-4after stimulation with AWA were quantified by ELISA kits.Results1. Isolation and Identification of Differentially-Expressed Proteins ofthe male worm of Schistosoma japonicum in PairingThe good 2-DE pattern with high resolution and reproducibility wasobtained. 11 protein spots were incised from sliver staining gel anddigested in gel by trypisin. 11 maps of peptide mass fingerprints (PMF)were obtained and 9 proteins were identified.2. Sequence Analysis of Schistosoma japonicum SJCHGC cDNA byBioinformatics MethodThe knowledge encoding 198 amino acids, mol. wt 22.76kDa,isoelectric point 4.84; which had antigenicity and containingphosphorylating and alkylating sites.3. Construction of eukaryotic expression vector of pEGFP/SJCHGCand its expression and localization in COS-7 cellsThis fusion protein was localized in the cytoplasm of COS-7 cells asdetected by fluorescence microscopy. The results of RT-PCRamplification showed that there was one band with 597 bp in COS-7 cellstransfected with pEGFP-C1/SJCHGC and no any bands in COS-7 cellstransfected with the empty plasmid pEGFP-C1. Western blotting confirmed that pEGFP-C1/SJCHGC expressed a fusion protein.4. Construction and immunoprotection of DNA vaccine pcDNA3/SJCHGC of Schistosoma japonicum in miceThe SJCHGC gene was amplified. SJCHGC cDNA was successfullycloned into a eukaryotic expression vector pcDNA3. The antigen genecould be stably existing in the muscular tissue of mice and could beexpressed in the muscular tissue cells of mice. Immunization experimentshowed the naked pcDNA3/SJCHGC provided 29.70% worm reductionrates, 47.25% liver egg reduction rates, 51.77% Intestine egg reductionrates and 25.90% liver eggs reduction rates per female in micerespectively.5. Construction and immunoprotection of DNA vaccine pcDNA3/SJCHGC of Schistosoma japonicum in miceThe SJCHGC gene was amplified by PCR, and cloned into theeukaryotic expression vector pcDNA3. The recombinant plasmidpcDNA3/SJCHGC was identified by PCR, restriction enzyme digestionand sequence analysis. The mice were immunized with pcDNA3/SJCHGC. Two weeks after the third vaccination, the antigen genes werepresent in muscular tissue of mice by PCR, and a challenge infection wascarded out with S.japonicum cercariae. The worms and eggs werecollected and counted at the 42th day after the challenge infection.Conclusion1) The experiments show that male worm proteins would be affectedby pairing of S.japonicum. Comparison of the schistosomes proteomebetween male worms of single-sex infection and male worms of bisexualinfection is important in theorical and practical significance forelucidating the schistosome mechanism of development and reproduction, and may permit the identification of protein candidates for thedevelopment of novel vaccine or new targets for drug developmentagainst schistosomiasis.2) Bioinformatics is very helpful in structural and functional studiesof the Schistosoma japonium. The bioinformatic analysis of the epitopesand specific functional sites suggests that the proteins encoded can beregarded as potential vaccine molecules against schistosomiasis.3) The COS-7 cells were transfected by the recombinant plasmidpEGFP-C1/SJCHGC, and the SJCHGC gene was expressed in thetransfected COS-7 cells. The expression and subcellular localization ofthe fusion protein were analyzed by fluorescence microscopy. Theproteins expressed show the antigenicity of S.japonicum. The use of GFPfor cell marking in novel gene SJCHGC demonstrated a new technologyfor functional study of novel gene SJCHGC.4) The recombinant plasmid pcDNA3/SJCHGC has beenconstructed and expressed successfully in vitro and in vivo, and it caninduce partial protection immunity against S.japonicum infection inmice.5) DNA vaccine could induce significant cellular and humoralimmune response, and it could induce predominant Th1 type immuneresponses which contribute to the protective immunity against challengeinfection in mice.