[Boost effect of recombinant IL-4 on protection of Schistosoma japonicum cathepsin B DNA vaccine in mice against the parasite].

OBJECTIVE: To investigate the enhancement effect of IL-4 expression plasmid on cathepsin B DNA vaccine of Schistosoma japonicum (Sj) in mice. METHODS: The recombinant IL-4 plasmid constructed by cloning PCR amplified product of murine IL-4 gene into eukaryotic expression vector pcDNA3.1 was co-injected intramuscularly with Sj cathepsin B expression plasmid DNA to mice as the test group. The other three groups of mice were set up as control including IL-4 expression plasmid, Sj cathepsin B expression plasmid and two vacant vector plasmids. The expression of IL-4 and cathepsin B was visualized by immunohistochemistry. Challenge infection in mice was carried out 3 weeks after the last vaccination and immune protection was assessed by worm and egg reduction rates. RESULTS: The recombinant mIL-4 plasmid and cathepsin B DNA vaccine were expressed in muscular cells of the vaccinated mice. Immunization with cathepsin B DNA plus recombinant mIL-4 plasmid yielded a 43.2 % of worm reduction rate and a 76.6% of egg reduction rate, showing a significant difference (P<0.01, P<0.05) compared with that of cathepsin B DNA vaccine alone. CONCLUSION: As an adjuvant, IL-4 DNA can improve the protective effect of cathepsin B DNA vaccine in mice against S. japonicum infection.

[Cloning and characterization of new genes of Schistosoma japonicum].

OBJECTIVE: To clone and characterize new genes of Schistosoma japonicum, Sj, and to provide efficient vaccine candidates. METHODS: Sj adult cDNA library was screened with rabbit sera raised against male worm soluble antigen. The inserted cDNA fragments from the positively selected clones were amplified with PCR and further sequenced, as well as characterized through internet NCBI GenBank software. RESULTS: Eleven positive clones were obtained and two were verified by GenBank as new, including a novel gene designated as Sj-P8 (GenBank accession No. AF517843) and a new partial cDNA of Sj myosin (GenBank accession No. AY770506). The two new genes encoded a transmembrane protein of 75 amino acids and a myosin protein fragment of 212 amino acids respectively. CONCLUSION: The newly obtained genes may provide useful information for the research on Sj vaccine.

[Immunoscreening of Schistosoma japonicum adult worm cDNA library with sera vaccinated with cercaria antigen and analysis of novel genes].

OBJECTIVE: To explore antigens possessing common immunogenicity with Schistosoma japonicum (Sj) cercariae antigens, and to find out new candidate antigens for schistosomiasis diagnosis and vaccine. METHODS: Sj adult cDNA library was screened using sera from rabbits vaccinated with Sj cercariae antigen, the inserts of positive clones were amplified by PCR, all positive clones were sequenced and the data were analysed using Nucleotide BLAST software of NCBI and Expert Protein Analysis System of Swiss Institute of Bioinformatics. RESULTS: Thirteen positive clones were obtained after three rounds of immunoscreening, and all amplified by PCR. Among four novel genes, SjCAI, SjCA, SjCAI-2 and SjCAI-3 (GenBank accession number: AF495883, AF515834, AY118086 and AY129303, respectively) encoded proteins with 353, 161, 137 and 72 animo acids respectively. Sj CAI protein contained six DNA-binding zinc fingers and showed some homology to gastrula zinc finger protein XLCGF48.2; proteins encoded by SjCA, SjCAI-2 and SjCAI-3 respectively contained N-glycosylation sites and phosphorylation sites. CONCLUSION: Novel genes were obtained by immunoscreening Sj adult cDNA library.

[Study on the subcloning, expression and immunoprotection with Schistosoma japonicum CAI gene].

OBJECTIVE: To subclone and express the new gene of Schistosoma japonicum (Sj) CAI and evaluate the immunoprotective effect of the recombinant molecule. METHODS: The cDNA of SjCAI gene was subcloned into expression vector pGEX-5X-3 to form recombinants which were then used to transform to E. coli strain ER 2566. Expression was induced by IPTG. The mice were vaccinated with the expressed protein and the immunoprotective effect was tested. RESULTS: Fusion protein of SjGST-CAI was highly expressed in E. coli as inclusion bodies. The worm reduction rate and the liver egg reduction rate in vaccination group of SjGST-CAI were 29.87% and 63.71%, respectively. CONCLUSION: SjCAI gene can be highly expressed in E. coli after subcloning into pGEX-5X-3 vector and the expressed fusion protein can induce immunoprotective effect against Sj in mice.

Schistosoma japonicum: isolation and identification of peptides mimicking ferritin epitopes from phage display library.

In an attempt to isolate and identify the antigenic epitopes on ferritin of Schistosoma japonicum (SjFer) and to test their protective potentiality against Schistosoma japonicum (S.j), polyclonal antisera against SjFer was prepared to screen a 12-mer random peptide library. Three rounds of biopanning were performed and resulted in an enrichment. Six peptides selected randomly from the third elute were all found to be positive by evaluating the binding to anti-SjFer sera by ELISA and Western blotting. Three amino acid sequences were deduced from the six phage clones by sequencing. When they were used to vaccinate mice, the three peptides could induce significant reduction in adult worms (26.7%, 20.4%, and 25.9%) as well as in liver eggs per gram (LEPG) (40.0%, 38.2%, and 40.8%). This result showed that three mimotopes on SjFer were obtained and they could induce significant protective efficacy against S.j.