Screening And Identification Of The Specific IgE Antibody Related Antigen And Mimic Epitopes Of Schistosoma Japonicum (Chinese Mainland Strain)

At present, schistosomiasis is still a main parasitic disease in the world, which threatens severely the health and life of human beings. On the control strategies, the comprehensive measure based on the chemotherapy of praziquantel, and combined with mollusiciding and environment modification as well as health education,has made great achivements. But in the marshlands and lake regions, it seems impossiable to control schistosomiasis and its transmission in these areas because of the complicated environments , unfavourable in doing the mollusiciding and the frequent movement of human beings and livestockes. The reinfection takes place frequently in the epidemic areas. Facing this condition, the current control measures have too many difficulties to cope with. Therefore, the new strategy of control measure by vaccination and the researches on the related fields have been paid more attention.Under the guidance of the progressive theories and-8-understandings on the immunology and immunopathology of schistosome infection,seeking for the new vaccine-candidate molecules is also the important aspects of the vaccine research work. So far,a lot of data from field sero-epidemiological investigations and laboratory studies have demonstrated that the schistosome specific IgE antibody may play an important role in defense against the infection. Thus, the new approch of inducing the specific IgE responses by vaccination is feasible. In this study,we at first aimed at obtaining the gene encoding the specific IgE antibody related proteins by immunoscreening the Schistosoma Juponi CUM adult worm cDNA library with the pooled high-titer IgE antisera from the individuals living in the schistosome epidemic regions. The target gene was then subcloned into plasmid vector and induced by IPTG for its expression. After that, the recombinant protein was purified and used for the identification and characterization of its immunogenicity. The effect of the induced specific IgE antibody on the hepatic granuloma formation and fibrosis after challenge infection with schistosome cercariae was evaluated. The mechanism of ki 11 ing the schistosomulae in vitro depending on the specific IgE antibody were observed. The distribution of the protein encoded by the cloned gene was determined by immuno-electromicroscopy with protein A- colloidal gold. In order to further analyse the epitope features of the protein relevant to the specific IgE antibody, the phage display library of random peptides was screened by pooled sera with the specific IgE antibody. The immunogenicity and the protection effects on the challenge infection of the cloned-9-phages were also assessed.1.Cloning of the Gene Encoding the Specific IgE Antibody Related Antigen of Schistosoma japonicumThe serum samples from the inhabitants living in the epidemic regions of Schistosomaisis japonica were collected and detected by ABC-ELISA method for their SWAP specific IgE antibody. The sera with high titer of the specific IgE levels were chosen and pooled. After absorbed with Protein G-Sepharose beads to remove the IgG antibody, the pooled sera were then used for immunoscreening of the cDNA library of Schistosoma japonicum adult worms. Goat anti-human IgE antibody were used as second antibody to make sure that the positive clones were IgE related. Through three cycles of screening, the inserted cDNA fragments of the positive clones were amplified by PCR and sequenced. The results showed that the inserted cDNA fragment from one clone was 1200 bp in length, with a ORF of 507 bp which encoded 169 amino acids. The deduced molecular weight of the protein was 19. 3 kDa. According to the sequence of the ORF,a pair of new probes, in which EcoR land Not I sites were incorporated respectively,were designed and used to amplify the target gene. Then, the gene was cloned into vector pGEM-T and subcloncd into expression vector pGEX-6p-l and induced by IPTG. Western blot analysis showed that the fusion protein expressed could be recognized by the specific IgE antibody in t...