Construction And Immunoscreening Of Cdna Library And Establishment Of An Indirect Elisa For Cryptosporidium Parvum Infection

Cryptosporidium parvum（C. parvum）is an intracellular coccidian parasite,mainly infects the host intestinal epithelial cells, which can cause zoonoticcryptosporidiosis, the disease is usually transmitted by the ingestion oocysts excretedin feces. Cryptosporidiosis usually causes abdominal cramps, fever, vomiting,diarrhea and other symptoms in immunocompetent persons typically resolved in7to14days. In immunocompromised individuals, especially those virus-infected patientswith AIDS and severely malnourished children, the disease can cause persistentdiarrhea, and even lead to death. It is very important for the early diagnosis of thedisease. At present, the detection of Cryptosporidium infection is primarily basedon fecal examination, but the method are time consuming, laborious and with poorsensitivity. The immunological methods have the advantages of time saving, simpleoperation and easy of standardization, whch is suitable for large-scale epidemiologicalscreening, but the key is to obtain antigens with higher specificity and sensitivity.This study, SMART technology was used to construct C. parvum cDNAlibrary. C. parvum oocysts were purified, then total RNA was extracted with TrizolReagent for constructing double cDNA. cDNA was digested with proteinase K and Sfiand ligated into a λTriplE×2vector to construct the expression library. The librarywas amplified by Escherichia coli(E. coli)strain XL1-Blue. The unamplified libraryhad a titre of4.5×107pfu/mL, and amplified library titer of8.34×109pfu/mL. Positiveclones could potentially encode for full-length genes with an average length of1.5kblibrary. The library was screened with rabbit anti-C. parvum serum which has beenincubated by E. coli lysates. Positive clones were connected pMD18-T and sequencedfor analysis. Two positive clones containing insert fragments of2517bp and534bp were identified respectively, which including parts of fragments of sporozoite mucin(Gp900) and lactate dehydrogenase (CpLDH) respectively. Then, Gp900gene dividedinto two parts (Gp900-1and Gp900-2) and in order to express as the recombinantproteins. The target gene were amplified by PCR, using specific primers. The finalfragment was inserted into pET-28a for expression in E.coli, and purified by Ni-NTAagarose resin. Recombinant proteins can be recognized by immunized rabbit sera andinfected bovine sera by Western blot. rCpLDH can be recognized withcryptosporidium positive human sera by ELISA, however, the immune responseshowed poor sensitivity, so rGp900-2was chosen as candidate antigen fordevelopment of indirect ELISA. The optimal concentration of coating antigen was2.5μg/mL, and the optimal dilution of human serum was1:100, the best dilutedconcentration of alkaline phosphatase-labelled goat anti-human IgG was1:20000.The serum samples of521outpatients from local hospitals in Changchun areawas detected by rGp900-2indirect ELISA. The recombinant antigen Cp23and nativeantigens were used for comparation. The seroprevalence of specific IgG to therGp900-2, rCP23and native antigens were22.8%(119/521),24.6%(128/521)and16.7%(87/521) respectively. Further, a paired analysis of all521serum samples weredone by plotting the mean absorbance result from rGp900-2and rCP23. The resultshowed absorbance values was a good linear association between rGp900-2andrCP23(r=0.406, P<0.01).In conclusion, the indirect ELISA was established by using the recombinantprotein rGp900-2, which could be used for epidemiological survellience ofCryptosporidium infection in human population.