Studies On The Genes Of Schistosoma Japonicum Related To IgG3 Antibody In Microtus Fortis

Schistosomiasis, caused by Schistosomes, is a serious parasitic zoonosis afflicting both human and animals in the world. In recent years, a great attention was paid in the study of vaccines against Schistosomiasis by genetic engineering techniques. Now several candidate antigens with potential immune protection of Schistosomiasis have been obtained.Although a significant progress has been made in the screening of candidate antigen genes of Schistosomes, protective effects of all available antigens obtained by conventional gene engineering techniques are not so satisfied. Therefore, establishment of new techniques and strategies to screen effectively protective antigen genes would be an alternative to resolution of the above problem.Recent studies have shown that Microtus fortis is naturally genetous resistance to Schistosomiasis and humoral immunity, especially the specific IgG3 antibody in sera of Microtus fortis could play a relatively important role for Microtus fortis against Schistosomiasis japonicum. To screen the antigen genes of Schistosotna japonicum that related to the specific IgG3 antibody and provide valuable information for searching Schistosomiasis vaccine candidates, the Schistosoma Japonicum adult worm cDNA library was immunoscreened with the sera of uninfected Microtus fortis and Goat anti-mouse IgG3-HRP.Two positive clones were obtained and then were amplified using RACE technique (rapid amplification of cDNA ends), which resulted in acquiring two of novel genes of Schistosoma japonicum. One gene is 780 base pairs in open reading frame, encoding 259 amino acids and the other is 1169 bp.By cloning the N-l gene into pET~28a(+) expression vector, a recombinant of pET-28a(+)/N-l was constructed and expressed in E.coli. The expression product was analyzed by SDS-PAGE and Western-blotting. The result revealed that the molecular weight of recombinant protein was approximately 35KD and can be specifically recognized by mouse serumagainst Schistosoma japonicum adult worm antigen preparation (SWAP), suggesting that the recombinant protein possesses good antigenicity. Furthermore, the N-l gene was successfully cloned into plasmid pBacPAK-His, an insect virus transfer vector, which may pave the way for further study on feasibility of application on Schistosomiasis using eukaryotic expression product of N-l gene.In order to evaluate the feasibility of N-l gene as DNA vaccine candidate in the controlling Schistosomiasis, the gene was cloned into eukaryotic expression vector pcDNAS and then animal protection experiment was carried out. 28.64% worm burden reduction and 21.73% eggs number reduction were obtained in Kunming mice immunized with recombinant plasmid, indicating the prospect for N-l gene DNA vaccine as a potential vaccine against Schistosomiasis.In conclusion, two novel genes of Schistosoma japonicum related to the IgG3 antibody of Microtus fortis were firstly cloned by immunoscreening the Schistosoma Japonicum adult worm cDNA library and RACE technique. Furthermore, the N-l gene was expressed in E. coli successfully. The DNA vaccine contain N-l sequence was constructed and used to vaccinate the mice, significant protection was obtained when comparing with control group. Therefore, this study has provided the theoretical and methodological basis for the development of an effective vaccine against Schistosomiasis.