| Cryptosporidiosis is a zoonotic parasitic protozoal in which Cryptosporidium is parasitized in the respiratory or digestive tract epithelial cells of humans and animals.Diarrhea and dehydration are the main clinical symptoms.Cryptosporidium can infect a variety of animals,among which young animals are the most susceptible and become one of the causes of diarrhea in newborn animals.Cryptosporidiosis has severely harmed human and animal health and brought serious economic losses to the animal husbandry.Currently,there are no effective therapeutic drugs.Vaccine immunization is a new strategy for prevention and control of the disease.Therefore,screening for highly immunogenic target antigens is of great significance.There are few researches on Cryptosporidium paridis in Baoding area.The study laid the foundation for the prevention and control of Cryptosporidium paridis in Baoding area and provided candidate antigens for the development of anti-Cryptosporidium parvum vaccinesThe purpose of this study was to understand the immunity characteristics of sporozoite surface antigen Cp23 of Cryptosporidium parvum from dairy cow in Baoding of Hebei Province.Cryptosporidium parvum is separated froming Baoding.The18SrRNA gene was obtained by PCR amplification.By application DNAStar software analyses sequencing results,the isolated species was identified as Cryptosporidium parvum.The Cp23 gene of Cryptosporidium parvum was cloned into prokaryotic expression vector pET-28a(+)by PCR,and the recombinant expression vector pET28a-Cp23 was constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)and induced by IPTG.The results of SDS-PAGE showed that the protein was 25 ku,and was inclusion protein.The results of Western blotting showed that the expressed product could be recognized by the positive serum of Cryptosporidium parvum,and had special immunogenicity.The 4 weeks old BALB/c mice that were immunized the recombinant plasmid pET28-Cp23.Then the humoral and cellular immunoresponses were detected.The results showed that the experiment group CD4+/CD8+T lymphocyte was increased,and IgG levels were also significantly increased its titer reached 1:12800.the concentration of IL-12,IL-4,IL-10 and IFN-γwere improved,there were significantly higher than those in control group.(P<0.01).The above results of humoral immunity and cellular immunity indicated that the recombinant protein CP23 immunized mice to induce a Th1/Th2 mixed immune response,which can effectively induce the body to produce a specific immune response,and enhance the immune function,with good immune characteristics.To construct the recombinant plasmid pEASY-18SrRNA,the plasmid was serially diluted 10 times.Six consecutive gradient plasmids were used to establish a standard curve of Cryptosporidium parvum quantitative PCR.To optimize the conditions of the fluorescent quantitative PCR,and the sensitivity and specificity were analyzed.30samples from Baoding district were detected by ordinary PCR and fluorescence quantitative PCR.The results showed that the copy number of the plasmid template was3.5×10~6～3.5×10~1,all of which could amplify the curve;the sensitivity could reach 35copies of the standard DNA plasmid.Specific analysis showed that only the amplification curves of Cryptosporidium parvum and Cryptosporidium andersoni DNA appeared.Repeatability analysis shows that the coefficient of variation is less than 10%.The positive rate of clinical samples was 30%(9/30),and 78 copy/μL could be detected.The established fluorescence quantitative PCR method can satisfy the needs of clinical detection of Cryptosporidium and provide a rapid detection method for epidemiological investigation. |
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