| Our study has aimed at the identification of Sj male worm derived new vaccine candidates and the immunoregulation of cytokines on the nucleic acid vaccine.In order to investigate gender-specific antigen and its immunoprotection against Sj, profile of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was studied to compare the protein components among two antigen origins: male adult worm soluble antigen and female adult worm antigen. The antigenicity and gender specificity of the protein fraction were determined by Western blot, and the immunoprotection was assessed by the worm and egg reduction rates. Subsequently SDS-PAGE disclosed a male specific rich band at first time with molecular weight of 44.6kDa that could not be seen in female adult worm soluble antigen. This band was excised from SDS-PAGE and purified followed by mouse immunization. We found that 44.6kDa antigen recognized with immunized sera in Western blot was male worm specific. And the test group vaccinated with 44.6kDa antigenplus FCA revealed significant worm and egg reduction rates as 39.31 % and 41.98 % respectively (p<0. 001) , compared with control groups. Further investigation on its immunoprotection against Sj male worm is of importance.To select epitope mimics of Sj male adult worm soluble antigen and explore the immunoprotection against Sj in mice, phage random peptide library was screened with purified IgG from anti-5/ male worm antigen sera. Positive phage clones collected through three rounds of biopanning were detected by Dot-ELISA. As a result the specific phages binding to IgG were enriched after three rounds of biopanning. Positive clones detected by Dot-ELISA were reacted with the specific IgG. Sera from mice immunized with mixed positive phage clones gave a rise to a specific antibody and the immunoprotection against Sj was induced by immunization of mixed positive phage clones with worm reduction rate of 31.72% and egg reduction rate of 51.54%, indicating the anti-reproduction action to Sj. Therefore the antigenic epitope mimics of Sj male adult worm antigens are obtained by phage peptide-display library and reveal the immunoprotection against Sj, providing new foundation on the molecular basis for further search of the specific .male worm antigen epitope composition and for artificial synthesis of peptidevaccine.In order to obtain male-specific gene and its encoded protein thatcould be a target for anti-male worm development, Sj adult worm cDNA library was immunoscreened with anti-male adult worm antigen sera. Positive clones were identified firstly by PCR and further by sequencing and data analysis through Internet. Consequently eleven positive clones were obtained and finally three novel genes designated as 5/-MA, Cp8 and S/-p8 respectively were testified by GenBank (GenBank accession number are AF519808, AF524896 and AF517843). The deduced proteins of the three novel genes predictably encoded three possibly important nucleic and cytoplasmic signal transduction molecules. Further investigation on whether they are male worm gender-specific genes is of great attraction. Moreover the novel gene Sj-MA cDNA was firstly subcloned into a prokaryotic expression vector to construct recombinant plasmid, which was secondly transformed into E.coli. The recombinant fusion protein was highly expressed as insoluble inclusion bodies in the E.coli and was applied to mice for assessment of protective immunity against Sj. Our study showed that the subcloned novel gene was effectively expressed in the transformed E.coli. A 54.8kDa fusion protein was revealed by SDS-PAGE and its antigenicity was confirmed with the recognition by both anti-male adult worm sera and anti-GST sera in Western blot. Mice vaccinated with the fusion protein revealed significant worm reduction rate ( 34.29% p<0. 001 ) and egg reduction rate ( 48.76% p<0. 001 ), compared with the control groups. Taken together, the novelgene Sj-MA can be expressed in E.coli as a fusion protein that can elicit immunity against Sj, indicatin... |
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