Screening And Immunologic Characterization Of A Surface Adherence-Related Protein Gene Of Cryptosporidium Parvum

Cryptosporidium is an enteric protozoan parasite that causes diarrheal disease in humans and domesticated animals worldwide. Cryptosporidium parvum is generally considered to be the most serious parasite species in all of Cryptosporidium species. Although a large number of anti-parasitic drugs have been tested against Cryptosporidium, control of Cryptosporidiosis remains problematic due to the absence of approved preventive measures and lack of consistently effective parasite-specific pHarmaceuticals. Antibody and vaccine would be the effective measures for treatment and prevention of Cryptosporidiosis. The screening of vaccine candidate antigen against Cryptosporidiosis was critical for controlling Cryptosporidiosis. The attachment to and invasion to host epithelial cells of C.parvum sporozoites were the critical life cycle stages for causing Cryptosporidiosis. Sporozoites attach to intestinal epithelial cells with surface protein and then invade the cells. So the surface proteins of sporozoites closely related to attach to host cells would become the vaccine candidate antigen, and the screening of new vaccine candidate antigen gene of C.parvum was important for prevention and cure of Cryptosporidiosis. In this study, in order to gain a new suface adherence-ralated protein gene of Cryptosporidium parvum, we screened Cryptosporidium parvum T7 phage display library with intestinal epithelial cells(IECs)according to established method in our laboratory. Subsequently, the target gene was cloned and expressioned with Molecular Biology experimental methods. The localization of the protein(the expressive product of the target gene) in C. parvum was determined by immunofluorescent -antibody technique. And consequently, the influence of the anti-recombinated-protein immunoserum agaist the development of Cryptosporidium parvum was observationed by cell culture in vitro methods. In vivo, the responses of specific humoral and cell immunity were detected by indirect ELISA and flow cytometry after the ICR mice were immunized with the recombined protein by intraperitoneal injection. Meanwhile, animal protective experiment was taken to observe the Immunoprotective effect of the recombined protein. This study has established foundations for prevention of cryptosporidiosis by screening and studying on immune characteristic of a new suface adherence-ralated protein gene of Cryptosporidium parvum.Screening of adherence-related protein gene of C.parvum When the wells of 96-well plates were covered with monolayer intestinal epithelial cells, the T7 bacteriophage display library was added to wells. After five rounds of panning, the final enriched specific clones that display C.parvum adhesion protein gene products on the surface of T7 bacteriophage were plated. The phage DNA were extracted and sequenced by TaKaRa. Nucleotide sequences were analyzed by BLAST searches with the GenBank database. Amino acid sequence analysis were performed with ScanProsite and InterProScan. About 100 clones were then randomly picked from individual plaques, and their DNA sequences were PCR amplified and analyzed on agarose gel to determine the size of the inserts. PCR analysis showed that 36% of the phage clones had an insert size of 600bp. The sequencing results showed that this insert was 626 bp in length and contained a single open reading frame (ORF), that was predicted to encode 104 amino acids. This gene was designated C.parvum 21(CP21) and was a hypothetical protein gene of C.parvum by BLAST searches with the GenBank database. ScanProsite and InterProScan analysis showed that the gene have two N-glycosylation site, an N-terminal signal peptide and an transmembrane-regions . Localization of the CP21 protein The fragment of CP21 ORF was amplified by PCR with designed primers according to CP21. The recombinant plasmids PMD18-T-CP21 was constructed. The ligation products were transformed to competent cells of host strain DH5α. The recombinant plasmids pET-28a-CP21 was constructed and transformed into E.coli Rosetta(DE3), and the cells were cultured and induced by IPTG. The expressive levels were determined by SDS-PAGE and Western blot analysis of the cell lysates. The localization of CP21 in C. parvum was determined by immunofluorescence using antibodies specific for rCP21. The results showed that the recombinant proteins were highly expressed induced by 1mM IPTG for 4h before harvesting cells. The recombinant proteins were inclusion bodies in the cytoplasm and were specificity of C.parvum by Western blotting. Immunofluorescence staining of sporozoites and oocysts showed that CP21 protein was present on the surface of sporozoites and oocysts. It may be the vaccine candidate antigen excellently.The blocking essay of anti-rCP21 protein immune serum in vitro The blocking essay in vitro was divided into three groups, infection group, blocking group and control group. The infection group was carried out as described above. The blocking groups were carried out by adding 1%,2%,5%,10% rCP21 antiserum into standard maintence medium, respectively. The control group was HCT-8 cells. The culture medium was removed post infection 24 h, and the cells were collected and tagged with goat-anti-mice fluorescein(FITC). The fluorescence intensity of samples were analyzed by flow cytometry. So it is hypothesized that the fluorescence intensity can be able to reflect the percentage of infected cells.Our results showed that the average infection rate of HCT-8 was up to 82.0% when were infected after 24 hours. But when adding 1%,2%,5%,10% anti-rCP21 protein immune serum into standard maintence medium, the infection rate was decreased to 41.1%, 35.2%, 30.6%, 23.5%, respectively. With increasing of the concentration of antiserum specific for rCP21, the infection rate of host cell was decreased. It is concluded that anti-rCP21 protein immune serum can block the adhesion of C. parvum oocyst and sporozoite to host cell HCT-8.Immunoprotection response induced by rCP21 protein against C.parvum in ICR mice The responses of specific humoral and cell immunity were detected by indirect ELISA and flow cytometry after the mice were immunized with rCP21 protein by intraperitoneal injection. The immunoprotection of rCP21 protein against C.parvum was studied after the mice were inoculated with C.parvum oocysts. The results showed that the responses of specific humoral and cell immunity in mice were elevated by inoculating rCP21 protein. Oocysts number of immunized mice were decreased significantly compared with control groups (P< 0.05) and the shedding time shorted 4 days compared with the control groups. The oocysts reduction rate in immunized mice was 38%. The above results suggested that the rCP21 protein have Immunoprotective effect on C.parvum infection in mice.