Antigen Localization And Immunoprotection Of Rhomboid Protein For Cryptosporidium Parvum

Apicomplexan Rhomboid protein, having a potential role in microneme protein cleavage during host cell invasion, is a candidate antigen for development of a coccidiosis vaccine. In this paper, Rhomboid gene of Cryptosporidium parvum isolated from Changchun was amplified by RT-PCR. The results indicated ORF was 1395bp and compared with C. parvum Rhomboid reported in Genbank, the result indicated the homology of the nucleotide and the amino acid sequences were 99.9% and 99.6%.Prokaryotic expression of the C.parvum Rhomboid gene The Rhombiod gene was inserted into pQE-30 vector directly , transformed into E.coli Rosetta (M15) and induced by IPTG. The expressive levels were determined by SDS-PAGE and Western blotting. The results indicated that the recombinant proteins were inclusion bodies in the cytoplasm and were specificity of C.parvum by Western blotting.The expression product have immunogenicity and it may be the vaccine candidate antigen excellently.Immunoprotection response induced by C.parvum Rhomboid recombinant protein against C.parvum in BALB/c mice Prepared succeedly recombinant proteantigen of C.parvum with extraction, purification and renaturation of the cytoryctes. The responses of specific humoral and cell immunity were detected by indirect ELISA and flow cytometry after the mice were immunized with pQE-CP-Rhomboid protein. The immunoprotection of Rhomboid protein against C.parvum was studied after the BALB/c mice were inoculated with C.parvum oocysts(1×106). The results indicated that the responses of specific antibodies were elevated with the increase of immunization times. The immunity index between experiment and control groups was significant (P<0.01). The response of cell immunity showed that the level of CD4+ CD8+ lymphocyte of immunized mices was not increased significantly ,compared with control groups,. Oocysts number of immunized mice were decreased significantly compared with control groups and the time shedding by experiment shorted 2 days. The oocysts rate of the immunity was 65.48%. The above results indicated the Rhomboid protein have immunoprotective effect on C.parvum infection in mice.Localizing Rhomboid protein on C.parvum Immunofluorescence staining of sporozites showed that Rhomboid protein was present on sporozoites. Constrution and expression of eukaryotic expression vector of C.parvum Rhomboid gene The recombiant plasmid pVAX1-CP-Rhomboid was constructed by Rhomboid gene was inserted into eukaryotic expression vector pVAX1 and transfected into Hela cell.The results showed that the specific recombiant plasmid was detected in Hela cell and has immunogenicity by indirect immunofluorescence assay and Western blotting.Immunoprotection response induced by pVAX1-CP-Rhomboid plasmid against C.parvum in BALB/c mice The experiment BALB/c mice were immunized with pVAX1-CP-Rhomboid plasmid. The responses of pecific humoral and cell immunity showed that the experiment group immunized with pVAX1-CP-Rhomboid plasmid compared with control groups: the responses of specific antibodies was significant (P<0.01), the level of CD4+ CD8+ lymphocyte was not increased significantly.The experiment that the BALB/c mice were inoculated with C.parvum oocysts showed the nucleic acid vaccine have immuno-protective effect on C.parvum infection in mice, the time of oocysts shedding by experiment shorted 2 days, the decreased oocysts rate of the immunity was 63.1%.The study laid a good foundation of the further research about prevention and cure of cryptosporidiosis.