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Cryptosporidiosis (cryptosporidiosis) is Cryptosporidium (Cryptosporidium spp.) Infected animals and humans caused by a parasitic disease to self-limiting watery diarrhea as the main symptom. Cryptosporidium can infect more than 170 kinds of animals, of which at least 79 kinds of mammals. Cryptosporidium oocysts ubiquitous in the environment, humans can get infected through a variety of ways. Cryptosporidiosis but no treatment effects, and thus it is important to detect and prevent. In this study, restriction enzyme digestion from recombinant plasmid pMD18-T-P23 obtained Cryptosporidium mouse genotype (Cryptosporidium mouse genotype) P23 gene was cloned into the pGEX-4T-1 vector to construct a recombinant prokaryotic expression plasmid pGEX-P23. The purified recombinant plasmid was transformed into E. coli BL21 (DE3) and induced expression product by SDS-PAGE and Western blot analysis. The results showed that rP23 respectively Cryptosporidium infection with rabbit rabbit sera, Bayesian Cryptosporidium infection chicken sera, Cryptosporidium parvum infection in bovine sera, Cryptosporidium positive sera of mice infected with mouse genotype specific reaction, rather than with rabbits, chickens, cows, rats negative serum reaction, indicating that the recombinant protein had good immunogenicity. The purified recombinant protein rP23 as antigen, establish Cryptosporidium indirect ELISA method, its sensitivity, specificity and reproducibility were observed; With the established method, the clinical serum samples for testing. Results successfully established Cryptosporidium indirect ELISA technique, the optimum concentration of antigen was 2μg/mL, optimal serum dilution of 1:800, HRP secondary antibody optimal dilution of 1:2000, with good specificity, sensitivity and reproducibility. Using the established rP23-ELISA detection method for the 23 clinical serum samples were tested, and with Sheather's sucrose flotation method, nested PCR assay results were compared. The results showed that the establishment of rP23-ELISA method detection rate in Sheather's sucrose flotation method and nested PCR method. Show that the method has high sensitivity, specificity, reproducibility, low cost, simple operation, can batch testing, etc., can be used to detect clinical samples for the development of cryptosporidiosis ELISA kit foundation. To establish external parasites Cryptosporidium parvum infection model, and its intracellular growth process was observed, the use of human cecum carcinoma (HCT-8 cells) as the infected cells, cells inoculated with different concentrations of serum concentration and culture culture. Results in 6-well plates were inoculated 1 × 106,5 × 105,1 × 105 cells after 24 h, 48 h, 72 h after grow 70 to 80% single-cell layer that can used to inoculate 1 × 105 Cryptosporidium parvum. Containing 1% serum to culture medium Cryptosporidium parvum, cultured for 72 h by Crypt-a-Glo?, And Sporo-Glo? Antibody staining successfully observed various stages of the parasite Cryptosporidium form. To explore the green fluorescent protein (EGFP) in Cryptosporidium in transient expression were amplified itself Cryptosporidium parvum sporozoite surface antigen protein CP15, heat shock protein 70, histone H4, actin, tubulin and ATP enzyme protein promoter sequence, the foreign protein EGFP gene and its promoter in place of the shuttle 30 constructed transfected. Transfected by electroporation into Cryptosporidium parvum oocysts and cultured for 72 h, flow cytometry, fluorescence microscopy and RT-PCR detection of EGFP expression. Results pSEI and pAET two plasmid group detected by flow cytometry and fluorescence microscopy were observed EGFP fluorescence, RT-PCR detection of the amplified specific bands, indicating that the study established Cryptosporidium in vivo transient transfection system can be expressed in vivo Cryptosporidium green fluorescence for the next stable expression of heterologous proteins foundation.
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