Schistosoma Japonicum: Cloning Of Novel Genes And Study On The Construction And Protection Of DNA Vaccines

Schistosomiasis is one of the worldwide parasitic diseases that threaten the health of human and livestock. Though great achievement on controlling Schistosomiasis had acquired through mass chemotherapy to human and livestock synchronously and elimination of Oncomelania snails in transmission focuses, control of this disease has been hampered by frequent re-infection and high cost of chemotherapy. One complementary approach for controlling schistosomiasis is iiornunoprophylaxis of the disease. DNA vaccines have been termed The Third Generation of Vaccine. The advantages of DNA vaccine over conventional vaccine are that they are ease of construction, testing, production and they are able to induce protective cytotoxic T-cell responses as well as helper T-cell and humoral immunity. Our present studies specifically reports immunoscreening new antigen molecules with sera from infected Bubahis bubalis(Bb), constructting DNA vaccine; and evaluating their immune protection against Schistosoma japonicum(Sj).A, Ctudies of New DNA Vaccines against Schistosoma. japoniemn1. Immunoscreening of Schistosoma japonicum Adult Worm cDNA library by Sera from Infected Bubahis bubalisIt is reported that Biibalus bubalis(Bb) is a low-permissive host for Schistosoma japomcum(Sj). In order to find out more antigen genes for developing anti-schistosorniasis vaccine, a Sj adult worm cDNA library was screened using sera from infected Bb with Sj. Seven positive clones were obtained. After automatical excision with helper phage , positive clones were further identified by PCR. The size of Sj cDNA fragments in positive clones range from 0.8kb to 2kb. After DNA sequencing, it was discovered that there were two new Sj genes of these clones. These new Sj genes were designated Sj-IB1 and Sj-Rho GTPase and registered in GeneBank. It was worthwhile to further study of immune protection of these cloned molecules.2. Construction and Protective Immunity Studies of Sj-Rho GTPase DNA Vaccine and Recombinant Vaccine against Schistosoma japonicum.One novel gene, Sj-Rho GTPase (GeneBank accession number:AF521619), coding for 192 amino acids, was obtained from immunoscreening Sj adult worm cDNA library using sera taken from Bb infected with Sj. In order to evaluate the immunoprotective effect of the gene, an eukaryotic expression plasmid (Sj-Rho GTPase/pcDNA3.1) and a prokaryotic expression plasmid (Sj-Rho GTPase/pGEX-5x-3) was constructed. Sj-Rho GTPase/pcDNA3.1 was identified by sequencing and Sj-Rho GTPase/pGEX-5x-3 was identified by restriction enzyme digestion, SDS-PAGE and Western blot. In order to test the protective immunity induced by the DNA vaccine and rccombinant vaccine of Sj-Rho GTPase in mice, fifty mice were immunized three times with an interval of 3 weeks, and challenged each with 40 cercariae of Sj 3 weeks after final immunization. At day 42 after challenge, all mice were sacrificed, the numbers of worms and hepatic eggs were counted. Compared with the control group, the amount of worm reduces 18.71%, 22.16% and 39.20% in the group of mice immunized by DNA vaccine or by recombinant protein or by DNA plus recombinant protein. It is concluded that egg reduction was 20.07% , 34.55% and 49.55% respectively. The Sj-Rho GTPase gene could induce partial protective immunity against Sj in mice. The DNA priming-protein boosting vaccination regimen was better than the single vaccination.3. The Study Mechanism of Protective Immunity of Sj-Rho GTPase DNA Vaccine and Recombinant VaccineThe aim is to explore the mechanism of the protective immunity of the Schistosoma japonicum(Sj) Rho GTPase DNA vaccine and recombinant vaccine. Kun Ming mice were immunized with Sj-Rho GTPase DNA vaccine, or with recombinant vaccine or with DNA-priming then protein-boosting. The sera before each immunization and before challenge were collected. The antibody IgG, IgGl and IgG2a against Sj-Rho GTPase was detected by ELISA. The mice immunizedwith DNA vaccine and DNA-priming then protein-boosting produced predominanlly IgG2a; whcrcas...