| Schistosomiasis is a severe threat, widespread parasitic zoonosis. The current strategy for control of schistosomiasis aims at the reduction of morbidity and mainly involves the treatment of patient or animals with praziquantel. However, chemotherapy can not prevent re-infection, and some isolates of Schistosoma mansoni(S. mansoni) that are resistant to high doses of praziquantel have been found. Therefore, it is necessary to develop an efficient and safe vaccine for control of schistosomiasis. Glycolytic pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis, and3-phosphoglycerate kinase (PGK) is a key enzyme in glycolysis, it was found in all creatures. Without it, metabolism function of organism will be disturbed. On the basis of the differentially expressed genes between mice and rats, as well as the study of tegument surface proteins of the S. japonicum, SjPGK was selected to carry out the following studies.1、Coloning and Expression of PGK of Schistosoma japonicumSjPGK was cloned in this study. Bioinformatics analysis, revealed that the full length cDNA of SjPGK were1398bp with an Open Reading Frame of1251bp, encoding416amino acid, it’s theoretical molecular weight is44382.21Daltons, theoretical isoelectric point is6.57. The sequence of SjPGK has94%similarity with that of SmPGK. Real-time PCR revealed that mRNA of SjPGK had transcribed in various stages of S. japonicum with the highest expression level in21d worms, and the male had the higher expression level than that of the female. SjPGK were highly expressed in the worms from susceptible host, mice compared to those from the unsusceptible host, rats. We successfully constructed pET28a (+)-SjPGK recombinant prokaryotic expression plasmid, recombinant protein was in soluble expression in E.coli (DE3) with molecular weight of44kD. Western blotting analysis showed that the recombinant protein had good antigenicity and immunogenicity.2、The enzymatic characteristics analysis of the recombinant protein pET28a (+)-SjPGK The fusion protein activities was assayed in the reverse reaction from3-phosphoglycerate to1,3-diphosphoglycerate,using glyceraldehydes3-phosphate-dehydrogenase.The reaction was measured by the absorbance changes at340nm.The activity of the recombinant protein SjPGK was125U/mg. Kinetic analysis with respect to3-PGA as substrate gave a Km of2.69mmol/l and ATP as substrate gave a Km of1.51mmol/l.The optimal pH and temperature for the reaction were8.0-9.0and30℃-35℃respectively.3、The immune protection experiment of mice and the distribution of SjPGKCompared with the control group, BALB/c mice vaccinated with purified rSjPGK three times showed a34.5%worm reduction (P<0.05) and a32.2%egg count reduction (P>0.05).The change of the specific IgG and subtypes IgG1, IgG2a antibodies were measured by ELISA.The result showed that the induced specific IgG and subtypes IgG1, IgG2a were maintained in a high level after the first immune, which is probably relate to the immune protection induced by recomibant protein in mices.The expression of rSjPGK in different stages of S. japonicum was detected by immunofluorescence.We found that SjPGK had expression in various stages and mainly expressed in schistosome tegument surface membrane.The tegument surface proteins of42day’s worms of S. japonicum was prepared,western blotting analysis showed that the protein of SjPGK abound in schistosome tegument surface membrane.The study provided a basis for the further study of the biological function of SjPGK, and new thought to screen new candidate molecules of vaccine against schistosomiasis. |
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