Localization Arid Adhesive Propetry Of TRAP-C2and TSP9of Cryptosporidium Parvum

Cryptosporidium parvum is an apicomplexan parasite of great veterinaryimportance that has been recently recognized as one of the most commonenteropathogens causing diarrheal illness in humans (eg. AIDS patients). Indeveloping countries C.parvum infection accounts for a significant fraction of severeand life threatening diarrhoea episodes that afflict malnourished children. Therefore,the aim was to demonstrate the antibody against surface antigens on the infectivesporozoite stage of C.parvum neutralizes infectivity. The identification ofparasite-encoded molecules implicated in the interaction with host cells may providenovel targets for immunological and pharmacological therapy against C.parvuminfection.To date, many C.parvum antigens have been characterized. Furthermore, theseantigens have common domains such as TSP1-like domain. So we chose two genesnamed TRAP-C2and TSP9which have the common domains to study theirfunctions.In this study, CpTRAP-C2-1, CpTRAP-C2-2and CpTSP9were prokaryoticexpressed in E.coil. BL21. First we constructed recombinant plasmids withgenetic engineering techniques. Primers were designed based on the genesequence of TRAP-C2-1, TRAP-C2-2and TSP9fragments, and then amplifiedthe target gene fragment by PCR. The sizes of respective fragments were933bp,909bp and1314bp. The fragments were connected to cloning vector pMD18-T,and then identified by enzyme digestion and sequencing. The correct insertswere subcloned into the expression vector pET-28a and pGEX-4T-1. Expressionvectors were idedtified by enzyme digestion and sequencing comparison. We purifiedthe expected recombinant antigens TRAP-C2-1-His, TRAP-C2-1-GST,TRAP-C2-2-His, TRAP-C2-2-GST, TSP9-His and TSP9-GST by affinitychromatography and identified by SDS-PAGE and Western blot detection. The results indicated that all of the recombinant proteins can be used for the later study.The rabbits were immunized with the purified His-tagged recombinant proteinsemulsified with Freund’s adjuvant. When the polyclonal antibodies were prepared,CpTRAP-C2-1, CpTRAP-C2-2and CpTSP9were co-localized wih gp900by indirectimmunofluorescence assay. Then three methods, Western blotting, affinity ELISAand Immunofluorescence, were applied to detect whether the GST-taggedrecombinant proteins above could adhere to HCT-8cells. The results indicatedthat all of the recombinant proteins CpTRAP-C2-1, CpTRAP-C2-2, CpTSP9expressed in C.parvum sporozoites could bound to HCT-8cells. This result wasconfirmed by C.parvum in vitro infection assay. Therefore, we could speculatethat the three proteins in this study were important for attachment and invasionof host cells by Cryptosporidium parvum.