Studies On The Differential Expressed Protein Phosphoglycerate Mutas Gene Of Schistosomula Of Schistosoma Japonnicum

Schistosomiasis caused by schistosome, is a wide spread parasitic zoonosis that causes serious healthy problem to both human and animals. Schistosome presents differential gene expression pattern at each developmental stage of its life cycle, which results in dramatic changes in its biology,morphology,special metabolism and development. Phosphoglycerate mutas proteins together with their downstream effectors form a set of key glycolytic pathways. The PGAM glycolytic pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis. Based on the studies on the stage and gender differential proteome on Schistosoma japonicum, one new gene encoding glycolytic conduct proteins PGAM have been cloned.One EST (GenBank accession NO.AAL30898.2)was searched in the schistosoma EST bank by using a peptide sequence obtained from two-dimentional electrophoresis conbimed with peptide mass fingerprinting and sequencing as query. With PCR technique, one signal conduct protein encoding genes SjPGAM (GenBank accession EU374631)were cloned . Bioinformatic analysis showed that the gene had typical characteristics of PGAM family proteins: there were more than 100 conserved sites, containing a Histidine sites followed by a highly conserved distribution of cycteines and three glycosylation sites. Sequence analysis showed that SjPGAM shared 79% similarity to Clonorchis sinensis and 58% to human PGAM. The ORF of SjPGAM contained 1 003 nucleotides, encoding 250 amino acid with a molecular weight of 28.26 kD. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of SjPGAM was highest in the 19 days schistosomula, followed by 14 days schistosomula, 32 days adult worms, 42 days male, 27 days adult worms, 42 days male, 7 days schistosomula, 42 days adult and 42 days female, suggesting that the gene is aschistosome stage differential expression gene. The SjPGAM cDNA fragment was subcloned into a modified expression vector pET-28a(+) and transformed into E.coli BL21(DH5α) cells, and the production of recombinant SjPGAM protein was analysed. In the presence of IPTG, the 31kD fusion protein was expressed in included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum specific to Schistosoma japonicum adult worm antigen preparation. To analyse the function of SjPGAM gene, the purified rSjPGAM protein induced 16.48% worm reduction and 28.42% liver egg reduction.We obtained one glycolytic enzyme protein PGAM encoding genes for the first time, which is stage differentially expressed and may play an important role in the development of schistosomula. Further studies like the catalysis mechanism will assist us to understand more about the new knowledge on the regulation mechanism of the PGAM during the development of Schistosoma japonicum.