A Preliminary Study On SjAIf Of Schistosoma Japonicum

Schistosomiasis was one of the most prevalent parasitic zoonosis in China, which restricting the economy development of breeding industey and endangering people’s health. Praziquantel synthesis successfully in 1977, since then it became the only kind of massive use of drugs for the treatment of schistosomiasis. The reports about the appearing of praziquantel-resistant worms in recent years make it urgent to develop new drug to treat schistosomiasis. Some studies suggest that growth and development of Schistosoma japonicum in different hosts has some relevance to somatic cell apoptosis, which provide a new thinking for development of new drug to treatment and prevention of schistosomiasis. S.japonicum AIF is located in the inner mitochondrial membrance, performing oxidoreductase and pro-apoptosis dual function. The base sequence of Sj AIF was obtained in our previous experimennts.And in order to research its biological features and functions, the paper launched the following research.1 Cloning and expression analysis of a functional fragment of apoptosis-inducing factor(AIF) gene in Schistosoma japonicumThe functional fragment of Schistosoma japonicum apoptosis-inducing factor(Sj AIF) was obtained by PCR, which is 828 bp, encoding 276 amino acids. Sj AIF contains no signal peptide by bioinformatics analysis. The gene contain a 4Lii(human apoptosis-inducing factor) domain, a pyridine nucleotide-disulfide oxidoreductase domain and a FAD/NAD(P)-binding domain superfamily. Sj AIF is 68% homologous with Schistosoma haematobium apoptosis-inducing factor(Sh AIF) by multiple alignment. PCR technique was employed to amplify the functional fragment of Sj AIF by employing a c DNA of 14 d schistosomula as template. The fragment of AIF was subcloned into a p ET28a(+) vector and the recombinant plasmid was transformed into competent E.coil/BL21 for producing recombinant protein. The recombinant proteinmolecular weights about 35 k Da and is expressed in the pellet. The expression level of Sj AIF was determined at several different development stages of schistosomula and adult worms by using real-time RT-PCR. Real-time PCR analysis revealed that the expression of Sj AIF was lower in 7 d, 14 d and 21 d than that in other stages. The expression differences of Sj AIF from rats and BALB/c mice showed that Sj AIF expressed in 14 d, 23 d, 32 d and 42 d four stages in Wistar rats and BALB/c mice and the relative expression of Sj AIF from rats was higher than that from BALB/c mice in the four stages. Western-blotting showed that the recombinant protein had good immunogenicity. The vaccinated group showed a good ability to induce Ig G as examined by ELISA. The distribution of the protein in Schistosoma japonicum was analyzed by immunolocalization. Immunolocalization analysis revealed that the Sj AIF was mainly distributed in tegyment and parenchyma.2 The expression analysis of recombinant eukaryotic plasmid pc DNA3.1(+)-Sj AIFThis experiment applies PCR technique to amplify the functional fragment of Sj AIF. The fragment of Sj AIF was subcloned into a pc DNA3.1(+) vector and the recombinant plasmid was transfected into 293 T cell by liposome transfection method for producing recombinant protein. Western blotting showed that the recombinant plasmid was successfully expressed in 293 T cells, and the recombinant protein molecular weights about 31 k Da. FITC-Annexin-V and PI were used to stain unicellular sample of different apoptosis stages. Flow cytometry results revealed that the proportion of early apoptotic cells of experimental group was 35.35%, the proportion of late apoptotic cells was 4.53%, and the proportion of early apoptotic cells of negative control group was 6.7%, the proportion of late apoptotic cells was0.7%. The proportion of apoptotic cells of experimental group was higher than that of negative control group. Presumably, Schistosoma japonnicum apoptosis-inducing factor(Sj AIF) has a certain effect on apoptosis-promoting.