Screening And Expression Of Adherence-Related Protein Gene Of Cryptosporidium Parvum And Its Immunoprotection

Cryptosporidium is a widespread protozoan parasite that develops in epithelialcells lining the digestive and respiratory tracts of human and other vertebrates.Cryptosporidiosis caused by the Cryptosporidium has become a serious diarrhealdisease of humans and other mammals throughout the world. Cryptosporidium hasbeen classified to 13 species according to oocysts morphous, host specificity andgene characterization. Cryptosporidium parvum is generally considered to be theparasite species responsible for serious infection in human and domestic animals.Although progress has been made, control of cryptosporidiosis remains problematicdue to the absence of approved preventive measures and lack of consistentlyeffective parasite-specific pharmaceuticals. Antibody and vaccine would be theeffective measures for treatment and prevention of cryptosporidiosis. The screeningof vaccine candidate antigen against cryptosporidiosis was critical for controllingcryptosporidiosis. The attachment to and invasion to host epithelial cells ofC.parvum sporozoites were the critical life cycle stages for causing cryptosporidiosis.Sporozoites attach to intestinal epithelial cells with surface protein and then invadethe cells. So the surface proteins of sporozoites closely related to attach to hostintestinal epithelial cells(IECs) would become the vaccine candidate antigen, and thescreening of new vaccine candidate antigen gene of C.parvum was important forprevention and cure of cryptosporidiosis. In the present study we have constructedT7 phage display library of C.parvum and identified a novel adherence-relatedprotein gene by screening this library with IECs. This gene was designatedC.parvum 12(CP12). Immunofluorescence staining of sporozoites and oocystsshowed that CP12 protein was present on the surface of sporozoites and oocysts.The responses of specific humoral and cell immunity in mice were elevated byinoculating rCP12 protein and the rCP12 protein has Immunoprotection againstC.parvum infection in mice. The results suggested that the CP12 gene may be thevaccine candidate antigen gene. This study has established foundations forprevention of cryptosporidiosis.Construction of T7 phage display library of C.parvum The total RNA wasextracted from C.parvum by Trizol reagent. The mRNA was isolated from totalRNA by PolyA Tract mRNA Isolation Kit. The ds cDNA was synthesized byOrientExpress Random Primer cDNA Synthesis Kit. The directional EcoRⅠ/HindⅢ linkers were ligated into the ends of ds cDNA. The ds cDNA was digested withEcoRⅠand HindⅢ, which resulted in ds cDNA with EcoRⅠand HindⅢ ends. Theds cDNA fragments longer than 300bp in length were fractionated by Mini Column,and then ligated into the T7 Select 10-3b vertor with EcoRⅠand HindⅢ ends.After packaging in vitro, the T7 Select 10-3b vertor was transformed into BLT5403to construct the T7 phage display library. The data showed that the library contained1.2×107 clones, and approximately 96.7% of the library were recombinant. Thetiter of the amplied library was 2.4×1010 pfu/mL. The cDNA fragments longer than300bp in length were 92% of 100 plaques by PCR identification.Primary culture of intestinal epithelial cells from newborn calves Theileum of newborn calves were cutted aseptically and the mucosa were digested by0.25% trypase. The Dispersed IECs were plated on 96-well plates in DMEMcontaining 5%fetal calf serum at 37℃ in a 5% CO2. The growth condition andmorphologic characteristics of IECs were observed and identified byimmunohistochemistry. The results showed that IECs were adherence in 24-48h,forming cell colony in 4-6d and growing the full of plates in 10-12d. The shape ofIECs were polygon and the bouncary of cells was distinct.Screening of adherence-related protein gene of C.parvum When the wellsof 96-well plates were covered with monolayer intestinal epithelial cells, the T7bacteriophage display library was added to wells. After five rounds of panning, thefinal enriched specific clones that display C.parvum adhesion protein gene productson the surface of T7 bacteriophage were plated and single pure plaques wereisolated. The phage DNA were extracted. The cDNA inserts in these plaques wereamplified by PCR using primers flanking the inserts. The phage DNA weresequenced by TaKaRa. Nucleotide sequences were analyzed by BLAST searcheswith the GenBank database. Amino acid sequence analysis were performed withScanProsite and InterProScan. About 100 clones were then randomly picked fromindividual plaques, and their DNA sequences were PCR amplified and analyzed onagarose gel to determine the size of the inserts. PCR analysis showed that 32% ofthe phage clones had an insert size of 400bp. The sequencing results showed thatthis insert was 426 bp in length and contained a single open reading frame (ORF),that was predicted to encode 104 amino acids. This gene was designated C.parvum12(CP12) and was a hypothetical protein gene of C.parvum by BLAST searcheswith the GenBank database. ScanProsite analysis showed that CP12 protein has aN-glycosylation site, a casein kinase II phosphorylation sites and twoN-myristoylation site. InterProScan analysis showed that CP12 protein has anN-terminal signal peptide and transmembrane domain.Prokaryotic expression of the CP12 gene The fragment of CP12 ORF wasamplified by PCR with designed primers according to CP12. The recombinantplasmids pMD18-T-CP12 was constructed. The ligation products were transformedto competent cells of host strain DH5α. The recombinant plasmids pET-28a-CP12was constructed and transformed into E.coli Rosetta(DE3), and the cells werecultured and induced by IPTG. The expressive levels were determined bySDS-PAGE and Western blot analysis of the cell lysates. The localization of CP12in C. parvum was determined by immunofluorescence using antibodies specific forrCP12. The results showed that the recombinant proteins were highly expressedinduced by 1mM IPTG for 4h before harvesting cells. The yields of rCP12 were upto 41% of the total bacterial protein in the cell lysates. The recombinant proteinswere inclusion bodies in the cytoplasm and were specificity of C.parvum byWestern blotting. Immunofluorescence staining of sporozoites and oocysts showedthat CP12 protein was present on the surface of sporozoites and oocysts. It may bethe vaccine candidate antigen excellently.Immunoprotection response induced by rCP12 protein against C.parvum inBALB/c mice The responses of specific humoral and cell immunity weredetected by indirect ELISA and flow cytometry after the mice were immunized withrCP12 protein by intraperitoneal injection. The immunoprotection of rCP12 proteinagainst 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 micewere elevated by inoculating rCP12 protein. Oocysts number of immunized micewere decreased significantly compared with control groups (P< 0.05) and theshedding time shorted 4 days compared with the control groups. The oocystsreduction rate in immunized mice was 43%. The above results suggested that therCP12 protein have Immunoprotective effect on C.parvum infection in mice.