Studies On A Novel Serine/threonine Specific Protein Phosphatase SjPP Gene Of Schistosoma Japonicum

Schistosoma japonicum is the agent of a zoonotic infection which can result in serious disease both humans and in the domestic animal reservoir hosts. Schistosomiasis remains a major public health problem in China. Microtus fortis is the only one mammalian animals with resistance to schistosomiasis found in the epidemic area in China. It has been found that IgG₃ antibody in the sera from M. fortis is one of the effective factors associated with the native resistance. The blood fluke adult worm cDNA library was immunologically screened with the sera from M. fortis and five EST sequences were obtained and one of them was demonstrated as a serine/threonine specific protein phosphatase (PP) gene in our previous studies . PPs play an equally important role with protein kinases in the control of protein phosphorylation controlling a large variety of cellular events, such as cell division and growth, signal transduction and metabolism. There was little reported about PPs of S. japonicum. Here, we reported the further studies on the SjPP.1. With 5'-RACE technique based on the sequence of EST received in our laboratory, a novel gene including a complete open reading frame was obtained and named as SjPP. The ORF of SjPP contains 906 nucleotides, endoding 302 amino acid protein with 34.7kD molecular weight and pI 5.51. Sequence analysis showed that the deduced amino acid had the serine/threonine specific protein phosphatases signatures, for example GDXHGQ, GDXVDRG, LRGNHE and okadaic acid combinig site SAPNYC, revealing high homology to other protein phosphatases including human PP6 (72%), PP4C (59%), rat PPV (71%), fruit fly PPV (70%), fission yeast PPE1 (63%), budding yeast SIT4p (61%). Real-time PCR analysis of SjPP from the various stages of the parasite's life cycle demonstrated that the mRNA levels were highest in the 31d adult worms (100%), followed by 44d female worms (89.4%), 44d male worms (68.5%), 14d (49.2%) and 7d (47.5%) schistosomulum, suggesting a stage-and-sex different regulation.2. The immunogenicity and protective efficacy of a recombinant protein rSjPP and a DNA vaccine encoding SjPP was evaluated in BALB/c and Kun-ming mice.2.1 The SjPP cDNA fragment was subcloned into a modified expression vector pET28a(+) and transformed E. coli BL21(DE3) cells were analysed for the production of recombinant SjPP protein fused to an N-ternimal His₆ tag. In the presence of IPTG, the 39kD fusion protein was expressed in included bodies. Western-blotting revealed that rSjPP is a native antigen and the result of indirect ELISA indicated that the rSjPPprotein could be recognized by IgG, especially IgG₃ antibody in the sera from M. fortis.2.2 Purified, refolded through dialysis rSjPP showed serine/threonine specific protein phosphatase activity. The optimal pH value for the activity was pH8.0 and the optimal temperature was 60°C. lmM⁴mM Ni2+ enhanced the PPase activity, whereas Mn2+, Mg2+ and Ca2+ couldn't cause enhancement in activity. Surprisingly, we didn't find rSjPP activity was inhibited by Calyculin A, OA or PHI-1.2.3 The purified rSjPP from E. coli was injected to mice and induced significant anti-rSjPP antibody responses. The recombinant protein induced 18.18% worm reduction, as well as 15.44% liver egg reduction and 32.23% faecal egg reduction in BALB/c mice;While 17.8% worm reduction, 40.8% liver egg reduction in Kun-ming mice. The PPase activity of soluble proteins of adult worms from mice immunized with rSjPP was significantly inhibited when compared with those from the mice in blank control group(P<0.001). This implied the reduction of SjPP activity induced by the vaccination with rSjPP might play an inportant role on the decrease of egg production.2.4 SjPP fragment was inserted into the pCMV-Script vector to generate a DNA vaccine pCMV-SjPP. The DNA vaccine induced 26.8% worm reduction, 23.9% liver egg reduction and 31.9% faecal egg reduction in BALB/c mice;While 10.2% worm reduction, 21.5% liver egg reduction in Kun-ming mice.3. The f-SjPP protein expressed in enkaryotic cells and an siRNA molecular S312 were successfully obtained.3.1 Trough Bac-to-Bac baculovirus expression vector system, we construced the recombinant Bacmid-SjPP and obtained infective virus containing SjPP gene. A soluble fusion protein, f-SjPP, was expressed in TN5B1-4 cells infected with recombinant virus and the expressed protein reach a peak at 72h post infection and could be recognized by rabbit anti-rSjPP sera in western blotting tests.3.2 we designed and screened a siRNA molecular S312, which deduced 64.1% reduction of SjPP mRNA expression in schistosomula japonicum interfered for 8 days with a concentration 150nM.