Screening And Localization Of Adherence-related Protein Of Cryptosporidium Parvum Sporozoites

Parasites of Cryptosporidium genus can cause diarrhoeal disease in humans, livestock and other animals throughout the world, and can be major economic burden to the water industry. Till now, eighteen cryptosporidium species has been reported,Cryptosporidium parvum is generally considered to be the parasite species responsible for serious infection in human and domestic animals.Althrough a large number of anti-parasite 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 sould be the effective measures for treatment and prevention of Cryptosporidiosis.The screening of vaccine candidate antigen against Cryptosporidiosis was critical for controlling cryptosporidiosis. A primary event in the pathogenesis of cryptosporidiosis after parasite excystation is the attachment of sporozoites to the intestinal epithelium. The sporozoite then invaginates the host cell apical membrane, which subsequently envelops the parasite, leaving the organism in an intracellular, extracytoplasmic vacuole. Although the ultrastructural details of the attachment and invasion of the host cells have been well characterized, the molecular basis of these early events has not been elucidated.The purpose of this study is to search for the specific molecules that mediate C.parvum-host interaction and the molecular mechanisms involved in the pathogenesis.In the present study, at first , T7 phage display library of C.parvum were constructed, then 6 novel adherence-related proteins and a known CP2 proteins were identified by screening the library with Caco-2 cells and hyperimmunized sera. After that two encoded gene(CP389 and CP245) fragments were expressed in E.coil. Finally, immunofluorescence staining of sporozoite and oocysts showed that CP389 and CP245 were present on the surface of sporozoites and oocysts.Construction of T7 phage display library of C.parvum.The total RNAs was extracted from C.parvum by Trizol reagent. The mRNA was isolated from total RNA by PolyA Tract mRNA Isolation kit. The ds cDNA was synthesized by OrientExpress Random Primer cDNA Synthesis Kit. The directional EcoRâ… /Hindâ…¢linkers were ligated into the ends of ds cDNA.Next, the ds cDNA was digested with EcoRâ… and Hindâ…¢,then ligated into the T7 Select 10-3b vertor with EcoRâ… and Hindâ…¢ends. After packaging in vitro, the T7 Select 10-3b vertor was transformed into BLT5403 to construct the T7 phage display library. The titer of the initial library was 3.3×10⁷pfu/mL. There are 1.0×10⁷recombinants in this library, and approximately 94% of clones in the library have inserted fragment, 92% of insert fragments are longer than 300bp. The titer of the amplied library was 3.1×10¹⁰pfu/mL.Screening the library with Caco-2 cells. Caco-2 Cells were grown to confluence in Minimum essential medium supplemented with 5% (V/V) fetal calf serum,25 mM HEPES, penicillin (100U/mL), and streptomycin(100mg/mL) for 72 h at 37°C in 5% CO2, in 96-well tissue culture plates. When the wells of 96-well plates were covered with monolayer Caco-2 Cells,the T7 bacteriophage display library was added to wells.After seven rounds of panning,the final enriched specific clones that display C.parvum adhesion protein on the surface of T7 bacterialphage were plated and individual plaques were isolated.The Phage DNA was extracted.The inserted fragment in these plaques were amplified by PCR using T7 primers.The phage DNA were sequenced by Sangon company. Nucleotide sequences were analyzed by BLAST searches with the GeneBank database.Amino acid sequence analysis were performed with ScanProsite and InterProScan.The sequencing results showed six genes were identified, one of them encode CP2, which has been suggested to be involved in the invasion process. The others encoed proteins whose function were unknown. Bioinformatic analyses of these proteins indicated that they may participate in the host-parasite interactions.Screening of the library with hyperimmunized serum. Wells of a high binding microtiter plate was coated overnight at 4°C with 1:50 dilutions of a hyperimmunized serum. After a incubation for 1 h at 37°C, the unbound phages were discarded by washing the wells five times with PBST. The bound phages were then eluted with elution buffer and amplified by infecting Escherichia coli host BLT5403. The amplified phages were then subjected to another four rounds of selective screening as above to enrich the clones that are highly specific for anti-cryptosporidium sera. The sequencing results showed three genes were identified, one of them encode CP2, which has been suggested to be involved in the invasion process. The others encoed protein whose function were unknown.Expression of the identified CP389 and CP245 gene fragments in E.coli.The recombinant plasmid pGEX-4T-1-CP389 and pGEX-4T-1-CP245 were constructed and transformed into E.coli BL21,and the cells were cultured and induced by IPTG..The expression level were determined by SDS-PAGE and Western-blot analysis of the cell lysates.The localization of CP389 and CP245 were determined by immunofluorescence using antibodies against rCP389 and rCP245.The results showed that both recombinant protein were expressed as soluble protein with high yield.Immunofluorenscence staining of sporozoites and oocysts showed that both CP389 and CP245 were present on the surface of sporozoites and oocysts. These results further proved that these proteins might involved in the adhesion process between sporozoites and host cells.