Development Of Indirect ELISA Detection Method Employing Recombinant P23 And In Vivo Transient Transfection System Of Cryptosporidium

Cryptosporidiosis is a parasitic zoonosis which can cause self-limiting watery diarrhea. Cryptosporidium can infect humans and more than 170 kinds of animals, including at least 79 species of mammals. Cryptosporidium oocysts are existed generally in the environment and humans and animals can infect by varietal ways. Until now there is still no effects drugs to treatment the disease, so it becomes very important to detect and prevent the parasite.In order to express sporozoite surface antigen P23 of Cryptosporidium mouse genotype, the gene was digested from a recombinant plasmid pMD18-T-P23 with restriction enzyme EcoRⅠand XhoⅠand inserted into pGEX-4T-1 vector. The transformed Escherichia coli BL21(DE3)of recombinant plasmid pGEX-P23 was induced by IPTG. Recombinant protein was identified by SDS-PAGE and Western blot. The results showed that the recombinant plasmid pGEX-P23 was successfully constructed and the transformed E. coli was expressed with induction of IPTG for 6 h. The antigenicity of recombinant P23 (rP23) protein was confirmed by Western blot and rP23 could be recognized specifically by the positive sera collected from rabbit infected with Cryptosporidium cuniculus, chicken infected with Cryptosporidium baileyi, dairy cattle infected with Cryptosporidium parvum and mice infected with Cryptosporidium mouse genotype, respectively, but it cannot recognized specifically by the negative sera collected from the above 4 animals without Cryptosporidium infection.The results indicated that the rP23 had a good immunogenicity.An indirect ELISA method was established for rapid detection of specific Cryptosporidium antibodies in rabbit using purified recombinant P23 protein as antigen. The specificity, sensitivity and reproducibility of the method were evaluated. The developed method was used to detect 23 field rabbit sera samples and compared with faeces detection results of Sheather's sucrose flotation and nested PCR. The results showed that the indirect ELISA assay was successful established using rP23 protein as coated antigen. The optimization concentration of coated antigen was 2μg/mL. The optimal dilution of testing serum and HRP-labeled goat anti-rabbit IgG were 1:800 and 1:2000, respectively. The established rP23-ELISA method showed good specificity, repeatability and reproducibility. The results of detection of 23 field serum/faeces samples showed the rP23-ELISA method has the highest positive rate indicating that the developed method can be used to test clinical samples with characteristics of high specificity, high sensitivity, high reproducibility, low cost, simple operation and volume testing in one times. Further investigation was focused on the development of an indirect ELISA Kit for the detection of Cryptosporidium antibodies.In order to develop an in vitro culture system for Cryptosporidium parvum and observe its different development stage formation, human ileocecal adenocarcinoma(HCT-8)cell was selected to culture the parasite. Culture conditions including the number of inoculated cells and serum concentration was optimized. In order to investigate of various developmental stages of C. parvum in cell culture, Cryptosporidium specific antibodys Crypt-a-Glo? and Sporo-Glo? were selected to view the variety stages of the parasite. The results showed that the optimal culture conditions of HCT-8 cells were 1×10~6,5×10~5,1×10~5 cells cultured for 24 h,48 h,72 h, respectively, serum concentration was 1% FBS and the infected number of C. parvum oocysts was 1×10~5 in 6-well culture plate. After 72 h culturing,the complete life cycle stages of Cryptosporidium including sporozoites, trophozoites, merontⅠ, merontⅡ, merozoites, microgamonts, macrogamonts and oocysts was found in the culture system using the Crypt-a-Glo? and Sporo-Glo? staining.In order to develop the transient expression system of enhanced green fluorescent protein (EGFP) in Cryptosporidium parvum, 5' untranslated region (5' UTR) and 3' untranslated region (3' UTR) sequences of P-type ATPase(CppA-E1)gene, surface sporozoite protein(CP15) gene, heat shock protein 70(HSP 70)gene, histone H4 gene, actin gene, alpha tubulin gene of Cryptosporidium parvum were amplified by PCR from Cryptosporidium parvum genomic DNA. Thirty recombinant eukaryotic vector including promoter of Cryptosporidium parvum were constructed by inserting EGFP gene between up stream and down stream regulatory gene with restriction enzyme. The oocysts of Cryptosporidium parvum were electroporated transfered with each expression vector separately and then cultured in HCT-8 cells lines for 72h. EGFP within parasites was observed under flow cytometry and fluorescence microscope detection in pSEI and pAET eukaryotic vector transferred groups, respectively. EGFP mRNA was also detected by RT-PCR for total RNA of the two groups parasites. The results indicated that the transient transfer system was constructed and enhance green fluorescent protein was successfully expressed in the Cryptosporidium parvum.