Identification And Characteristion Of Octopamine Receptor And Elongation Factors In Schitosoma Japonicum

Schistosomiasis japonica is a widely distributed zoonotic parasitic disease andseriously harm to people’s health in southern China. The main prevention measuresis to control the source of infection and the praziquantel treatment for insect hosts isthe inprotant link. But practice shows that the chemotherapy drugs cannot stop theepidemics of chistosomiasis, and drug treatment may cause the risk of drugresistance for a long time repeatedly in schistosomiasis. thus enhancing the researchand development of new drugs and vaccines is an important direction in the currentschistosomiasis prevention and control research project. Based on the results ofprevious proteome research, the Schistosoma japonicum octopamine receptor (OAR)and protein synthesis elongation factor (EF) gene were selected as the object in thisthesis.Their expression profile and functions in the Schistosoma japonicum wasinvestigated.1. Cloning and Characteristion of Schistosoma japonicum OARThe cDNA fragment of Schistosoma japonicum OAR was amplifed by PCR,the sequence contains1422bp and encoding474amino acids. Real-time quantitativePCR was employed to analysis the transcription profile of SjOAR in7d,14d,21d,28d Schistosomula,35d,42d adult worm and42d female and male, the results showthat the adjusted gene transcription copy number was similar in schistosomula andadult worm, but the number of female worm is higher than that of the male. on thebasis of the antigencity and conservation, A261bp sequence was selected andinserted into the vector to successfully construct the recombinant plasmidSjOAR-pET-32a. The plasmid was transformed into E.coli BL21(DE3), and inducedto express a recombinant protein with a molecular weight of32kD. Therecombinant protein can be probed by anti-SWAP sera, and the specific antibodydeveloped from it can identify native SjOAR in western-blotting, respectively. Specific staining was primarily present in muscle tissue under tegument andreproductive organs of worms probed with SjOAR-specific serum.In the vaccine trial, the BALB/c mice were immunized three times withISA206-formulated recombitant protein, and then challenged with schistosomecercaria. The purified SjOAR-pET-32a with adjuvant induced38.20%(p<0.01)liveregg reduction and28.12%(p<0.01)worm burden reduction, compared with those ofthe control mice.Recombitant protein SjOAR-pET-32a have high levels of specificIgG in ELISA result and the recombitant protein may play an important role for theprotective effect on the vaccinated mice, and this protein induces a Th1and Th2mixed immune response in mouse. The test results in mice administrated withoctopamine receptor inhibiting drugs, cis(Z)-flupenxio and Mianserin, show thatcompared with the PBS control group, the5d tail vein injection of cis (Z)-flupenxiogroup received reduction rate of30.90%of worm burdern and47.31%of the liveregg with the extremely significant difference (P<0.01),25d group obtainedreduction rate15.49%of worm burdern and49.95%of the liver egg significantly (P<0.01), the Mianserin group mice obtained reduction rate of20.27%of wormburdern and28.87%of the liver egg with no significant difference (P>0.05).Theresults suggested that the Schistosoma japonicum octopamine receptor may possesspotential as an anti-schistosome drug target.2. Investigation of Schistosoma japonicum elongation factorPCR was empolyed to clone the Schistosoma japonicum elongation factor,which is the size of642bp, contains the entire ORF. Sequence alignment anddomain analysis suggested that the gene may be the EF-beta type. A recombinantplasmid SjEF-pET-28a containing SjEF was successfully constructed, therecombinant protein was purifed and applied to preparation of polyclonal antibodies.Western blotting analysis showed that the recombinant protein had good antigenicityand immunogenicity. Immunohistochemical analysis showed SjEF are mainlydistributed in the surface membrane and a substantial part of Schistosoma japonicum,evenly distributed in the muscle tissue. After the mice immunized with therecombinant protein, and induced high levels of specific IgG antibodies. Further thelevel of IgG2a and IgG1antibodies specific to SjEF were determined three timesafter immunization, and the ratio of IgG1/IgG2a were calculated, the results showedthat the SjEF induced Th2type immuno-response tend. The levels of IL-2, IL-10,IL-12but IL-4, INF-gamma in sera of the immunized mice were increased comparing to206adjuvant group. These findings initially clear characteristics ofSjEF, also provides the basis for further study.