Screening Of Adherence-Related Protein Gene Of Cryptosporidium Parvum And Its Immune Characteristics

Cryptosporidium is a protozoa that can infect people and Animal, which is primarily parasitic in mucosal epithelial cells of gastrointestinal tract and respiratory tract of humans and other vertebrates. The disease caused by cryptosporidium is named cryptosporidiosis. The obvious feature of cryptosporidiosis is severe watery diarrhea in clinic. According to Cryptosporidium oocyst morphology, host specificity and genetic characteristics, that the cryptosporidium were divided into 13 species in classification, and in which cryptosporidium parvum (C.parvum) have greatest risk for human and animal health. Until now, there is no effective treatment and prevention for cryptosporidiosis. Antibody and vaccines will play an important role in therapy of cryptosporidiosis, and finding effective candidate antigen of vaccine against cryptosporidiosis becomes predominant way. The key of pathogenesis is sporozoites of C.parvum that adhering and invading host cells. Sporozoite first adhere host cell by surface protein, and then enter into host cell. Thus, all surface proteins that closly related to host cells can be used as vaccine candidate antigens. Screening more noval potential vaccine antigen becomes more important in rescerch of prevention and therapy of cryptosporidiosis. In this study, cDNA library of C.parvum was constructed, screened cDNA library with neonatal calf intestinal epithelial cells, and finally, identified C.parvum16(CP16) as a noval cryptosporidium parvum adhesion-related protein gene. In the prokaryotic expression experiment, we fonud that the CP16 protein located on the surface of sporozoite and oocyst wall, indicated that CP16 gene can be used as small cryptosporidiosis vaccine antigen. The CP16 gene was cloned into the eukaryotic expression vector pVAXI by recombinant DNA method and got PVAXI-CP16 plasmids and then plasmids transfected into Hela cells. After pressure screening by G418 we found the CP16 were stable expressing in the cell lines. The cells with transfected plasmids were detected the target proteins by indirect immunofluorescence method. Immunized BALB / c mice with plasmids, and checked the dynamic changes of their immune response induced by the plasmids, The results showed that IgG antibody titers of the plasmid immunized mice increased with the frequency of immunization, and have a gradually increasing trend. LgG titer were significant difference comparing with the negative control group after the third immunization (P<0.05). CD4+ T lymphocyte count and CD4+/CD8+T lymphocytes count was significant difference with the negative control group (P <0.05). The results of immune protection test showed that the plasmids of eukaryotic expression on protection have well effect to against infection that compared with the control group oocyst discharging significantly decreased, the average duration of oocyst discharging were 3 ~ 4d shortening, the best group of oocyst reduction rate was up to 58.68%. The recombinant plasmids, pVAX1- CP16, were inoculated pregnant goats by the nasal mucosa, and perform the oocysts attack to the later generations of the goats, the results showed that the DNA vaccine can induce immune response in goats, the antibody titers gradually increased with the the number of immunizating, the difference was significantly comparing with control group (p<0.01). After oocysts attack, the number of oocysts discharged significantly reduced compareing with the control goat, the duration of oocyst discharging was shortens. the goats of experimental group of oocyst reduction rate was up to 56.1%. This study can serve as a basis for the prevention of cryptosporidiosis.