Study On The Adhesion Of Some TSP1-domain Containing Proteins Of Cryptosporidium Parvum To Epithelial Cells

Cryptosporidium is recognized as an enteropathogen of great worldwide medicaland veterinary importance,which causes cryptosporidiosis in humans and animals.Theinfection of Cryptosporidium parvum is considered to be the most serious for thehealth of human and animals.Cryptosporidiosis is a self-limited diarrheal disease inimmunocompetent persons,while in immunocompromised persons,it’s severe andeven life threatening.Efforts to develop novel antibodies or vaccines have beenhampered by the lack of knowledge on Cryptosporidium parvum pathogenesis and apaucity of organelle specific markers throughout the parasite’life cycle.Host-parasite interactions mediate the attachment of Cryptosporidium parvum tohost cells and invasion of the cell membrane.These are complex processes thatinvolve multiple parasite and host molecules. As for designing strategies to combatthis disease,the knowledge of the molecular basis in these processes is crucial forresearching the pathogenic mechanisms underlying infection.The proteins of parasitewhich contain TSP1domain play important role in adhesion and invasion of hostcells by apicocomplex parasites. Analysis of the Cryptosporidium genomes revealedthe presence of at least12other genes possessing one or more TSP-1domains,whichconstitute a CpTSPs family.All of them are predicted to have signal peptides andsome domains involved in cell-cell interaction, such as EGF, Apple, Notch or Kringledomain.Previous study suggested that TRAP-C1related to adhesion closely. Althoughthe function of CpTSPs have yet to be elucidated, they may also be involved ingliding motility and invasion as their sequence homology and structural similaritieswith TRAPs of other apicomplexans.In this study, CpTSP6、CpTSP7、CpTSP8、CpTSP10were expressed in E.coil.BL21. Then three methods,Western blotting、affinity ELISA and Cell blot,wereapplied to detect whether the proteins above could adhere to intestinal epithelialcells.Primer pairs of CpTSP6、CpTSP7、CpTSP8、CpTSP10genes were designed according to the sequeces in GenBank. The size obtained of CpTSP6、CpTSP7、CpTSP8、CpTSP10genes after PCR amplification were420bp、587bp、603bpand897bp respectively.The target fragments were cloned into pMD18-T vector.Afteridentified by PCR,enzyme digestion and sequencing correctly,the inserts with targetfragments were subcloned into pGEX-4T-1expression vectors. The recombinantplasmid pGEX-4T-1-TSP6、 pGEX-4T-1-TSP7、 pGEX-4T-1-TSP8andpGEX-4T-1-TSP10were transformed into E.coli BL21(DE3). Expression vectorswere idedtified by enzyme digestion and sequencing comparison.The expressionconditions were optimized.0.1mmol/L IPTG were used to induce the expression ofrecombinant proteins. The recombinant proteins were soluble in supernatant aftersonication and further purified by Gluthathione-Sepharose4B. rGST-TSP6、rGST-TSP7、rGST-TSP8and rGST-TSP10were identified by SDS-PAGE and therelative molecular mass were41KDa、47KDa、45KDa and58KDa respectively.The purified proteins were confirmed by Western blot.BALB/c mice were immunized with purified proteins rGST-TSP6、rGST-TSP7、rGST-TSP8and rGST-TSP10,as well as Freund’s adjuvant，to prepare the polyclonalantibodies repectively.After interacting with Caco-2、 HCT-8and MDBK cellsrepectively,the four proteins were detected by Western blotting,it was shown thatrGST-TSP6and rGST-TSP8could bind to these cells,while GST proteincouldn’t.Analysis of affinity ELISA showed that the binding of rGST-TSP6andrGST-TSP8to three cells were significantly different(P＜0.05) compared with thecontrol group.Moreover,the binding was dose-dependent and in Saturable manner asmeasured by affinity ELISA.The four recombinant proteins were transferred to NCmembranes after native gel electrophoresis,then the proteins on NC membranes wereincubated with three kinds of suspension cells, as verified by Cell blot, it was shownthat rGST-TSP6and rGST-TSP8were capable of adhesion to those three hostcells.While the adhesion could be blocked by the polyclonal antibodies ofrGST-TSP6and rGST-TSP8,which suggested that the binding were specific.Taken together, these findings suggest that CpTSP6and CpTSP8involved in the adhesion process between Cryptosporidium parvum and host cells.