| Schistosomiasis is one of the most serious parasitic diseases in morbidity and mortality, which infects at least 207 million people and a large number of animals in 76 countries, with an estimated 700 million people at risk. Schistosomiasis japonica, which is caused by the parasitization of adult male and female worms of Schistosoma japonicum within mesenteric or vesicular veins of the host, is the only zoonotic schistosomiasis that has proved to be the most difficult to be controlled among the 5 schistosome species that infect humans.Development and application of subunit vaccine is considered as an effective method of controlling the spread of schistosomiasis. Several vaccine candidates have been tested in both laboratory and clinical settings but none of these is succeed. Screening protective antigen is the key to the preparation of vaccines.Membrane protein is generally viewed as the most susceptible target for vaccines and drugs. Sjc23, a member of tetraspanin family, expressed in all infective parasite stages, is antigen of some interest in terms of both antiparasite vaccination and immunodiagnosis.In our investigations, the gene fragment encoding Sjc23-LED was amplified by PCR and cloned into the pET-22b expression vector. The His-tagged recombinant Sjc23-LED protein was expressed and purified by a His GraviTrap column.It was found that antibody responses in mice to Sjc23 were more rapid than other antigens,but intriguingly, the antibodies induced by Sjc23 were dominantly IgG2a type which has been proved to be inefficient in immunoprotection. Thus, Sjc23 antigen is likely a pro-Th2 antigen utilized by S. japonicum to modulate host immune system and facilitate parasite invasion and immune evasion.Owing to Sjc23 had affinity to normal human serum, we performed a classical ELISA assay to test the possible binding property of the serum component. Sjc23-LED was incubated respectively with purified human IgA,IgE,IgG,IgM and albumin. Only non-immune human IgG was found to bind Sjc23-LED, while the others did not show any binding activity. In order to confirm the binding between Sjc23-LED and human IgG, a pull-down assay was performed. We used Sjc23-LED as a bait protein immobilized on the nickel-Sepharose beads to capture the immunoglobulins that would interact with it. Similarly, only IgG was precipitated by Sjc23-LED immobilized Sepharose, but not IgA, IgE or IgM. Compared with ELISA assay, pull-down assays require higher affinity between the ligand and receptor due to the high stringency of particle precipitation and washing steps. Thus the binding between Sjc23-LED and IgG was specific.Porcine and bovine are the most common susceptible animals in epidemic area. They play an important role in transmission of human schistosomiasis. Here, we also performed ELISA and pull-down assays to test the affinity of Sjc23-LED to porcine and bovine IgG. However, very weak signal was observed with porcine IgG, but none was detected with bovine IgG was seen.I t is likely that the parasite is less adapted in animals than in humans.Further, Fab and Fc fragments of human IgG were respectively incubated with Sjc23-LED immobilized Sepharose beads to identify which region in human IgG that binds to Sjc23-LED, the result was that only the Fc fragment was precipitated, but not the Fab fragment, and the binding between Fc fragment and Sjc23-LED was also confirmed in ELISA assay.The relatively small molecular mass of the Sjc23-LED made it possible to perform inhibition/competition assay with synthetic overlapping peptides against the binding of Sjc23-LED with IgG. Seven peptides were chemically synthesized with 9-10 amino acids overlapped between adjacent peptides. In the competition assays, peptide number 3 and 4 completely blocked the binding between Sjc23-LED and human IgG, but not the other peptides. There is a region of amino acids (-KIQTSFHCC-) that overlapped between the two peptides, thus it is likely the structure formed by the 9 amino acids mediated the binding of Sjc23-LED with the Fc fragment of human IgG. Structural analysis of the Sjc23-LED shows that the IgG-binding motif is located on the second helix of the molecule. And the C-terminal side of the motif is conserved in all TSP family proteins, but the acids before it are very different from the others.This is the first report that an epitope of schistosomal ligand and its immunoglobulin receptor are defined, which provides further evidence of immune evasion strategy adopted by S. japonicum. Our data indicated that only low cytophilic antibodies to this antigen were generated after parasite invasion or immunization by its affinity to non-immune IgG molecule, it is may be beneficial to the process of schistosomal parasites evade host immune expulsion. With the discovery of this study, it may be necessary to reconsider the vaccine development approach based on this antigen. |
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