Screening And Identifition Ofadherence-related Protein Gene Of Cryptosporidium Parvum And Its Immunoprotection

Cryptosporidium parvum ribosome display library was screened with intestinal epithelial cells(IECs)according to established method in our laboratory. Subsequently, Protein was selectively and specifically bound to IECs using a multi-step panning procedure.Amino acid sequence was analysised by ScanProsite and InterProScan. The target gene was cloned into Pet-28a and expessed in BL21. The expressive levels were determined by SDS-PAGE and Western -blot analysis of the cell lysates.The localization of the protein(the expressive product of the target gene)was determined by immunofluorescent-antibody technique using specific antibodies. The BALB/c mice were immunized with the recombiant protein to detect the responses of specific humoral and cell immunity. The efficacy of recombiant protein was investigated in BALB/c mice.At the same time, the target gene was inserted into eukaryotic expression vector pVAX1 and transfected into Hela cell. The expression cell strain was selected with G418. The specific reombiant proteins were detected in Hela cells by indirect Immune fluorescence and Westen blot assay. The BALB/c mice and the adult pregnat goats were immunized with the reombiant eukaryotic expression vector to detect the responses of specific humoral and cell immunity. And then its efficacy was investigated in BALB/c mice and the adult pregnat goats.The results were as follows:Constrution of the ribosome display library of C.parvum.There were 1.3×10 ¹³DNA in this library and translated on ribsome using the kit (Promega) to generateternary PRM complexes. After five rounds of selection,six genes of C. parvum were selected. The gene which has an N-terminal signal peptide and transmembrane regions was named CP585.Construction and expression of prokaryotic expression vector.The recombinant plasmids pET-28a-CP585 was constructed and expressed in E.coli host cells BL21.The recombinant proteins were purified and detected its specificity by Western-blot. The mice were immunized with CP585 protein that indicated the responses of specific antibodies and cell immunity.The result of immune protect experiment showed that oocysts number of immunized mice was decreased significantly compared with control groups. The decreased worn rate of the experiment was 47.87%. Immunofluorenscence staining showed that CP585 protein was present on the surface of sporozoites and oocysts.Construction and expression of the Eukaryotic expression vector. The recombinant plasmids pVAX1-CP585 was constructed by cloning the CP585 gene into eukaryotie expression vecto pVAX1 and expressed in Hela cells.After transfected intoHela cells,the expression cell strains were selected with G418. The specific recombiant protein was detected in Hela cell strains by indirect immunofluorescence assay and Western-blot. The BALB/c mice were immunized with pVAX1-CP585 plasmid.The responses of pecific humoral and cell immunity was significant ( P<0.05). The experiment that the BALB/c mice were inculated with C.parvum oocysts showed pVAX1-CP585 had immuno-protective effect on C.parvum infection, the decreased worn rate of the experiment was 60.52%. The adult pregnat goats were inoculated intranasally with pVAX1-CP585 pasmid. The results showed that pVAX1-CP585 could induce immune response of goats and the vaccinated goats can tranfer the immunity to offspring conferring protection against C.parvum infection.Oocyst number shedding by the kids of experiment were shorter than those of control groups.The decreased worn rate of the experiment was 49.29%.The study laid a good foundation of the further research about prevention and cure of cryptosporidiosis.