ABSTRACT
The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.
Subject(s)
Animals , Mice , Antigens, Helminth , Allergy and Immunology , Cloning, Molecular , Cyclophilins , Genetics , Allergy and Immunology , DNA, Complementary , Genetics , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Immunization , Mice, Inbred BALB C , Recombinant Proteins , Genetics , Allergy and Immunology , Schistosoma japonicum , Genetics , Allergy and Immunology , Schistosomiasis japonica , Vaccines, Synthetic , Allergy and ImmunologyABSTRACT
The gene fragment encoding the egg-shell precursor protein of Schistosoma japonicum was amplified with RT-PCR by using PCR primer designed according to the 423 bp cDNA fragment of the Philippine strain of S.japonicum, the corresponding mDNA fragment of Chinese strain as template and then the 5' and 3' ends of this gene cDNA were amplified with 5' RACE and 3' RACE by using a series of primers designed according to the result of sequencing. Result of sequence analysis showed that this fragment, named as Sj423, contained a complete open reading frame (ORF) of gene encoding the egg-shell precursor protein of S.japonicum.(Chinese strain). As demonstrated by sequencing analysis. No intron could be detected in this gene fragment. This gene was subsequently expressed in E.coli after cloning into the expression vector pET28c(+). The molecular mass of the expressed product of this gene was 20.9 kDa as revealed by SDS-PAGE analysis, and Western blot analysis showed that the recombinant protein expressed could react well with the rabbit antiserum against the worm antigen of S.japonicum;indicating the good antigenicity of this expressed product.