Screening And Evaluation Of Fasciola Gigantic Antigens For Diagnosis Of Fascioliasis

[Objective] To screen antigen candidates for diagnosis of fascioliasis by screening the adult Fasciola gigantic cDNA library. The recombinant proteins of the screened F.gigantic-specific genes were prepared and the diagnostic potencies of the proteins were evaluated.[Methods] Total RNA of F.gigantica adults, which were collected from the market in Yunnan Dali and confirmed by morphological and molecular identification of coxl, ITS 1-2 and nadl genes, was isolated with a TRIzol reagent and purified into mRNA. The λ ZAP Ⅱ cDNA expression library of F.gigantica adults was constructed and Immunoscreened with pooled sera of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed. Recombinant proteins of the screened genes were prepared by prokaryotic expression and were purified. The diagnostic potencies of the proteins were evaluated by indirect ELISA.[Results] A total of 56 positive clones were screened from the adult F.gigantic cDNA library at first, and 19 positive clones were obtained after repeated screening. 11 genes were remained after discarding genes of the mitochondrial or highly homo logy with other species, and were divided into four major categories:CL, TPx, SAP2, and VB2. The TPx, SAP2 and VB2 genes encoded previously uncharacterized antigens of F.gigantica.The recombinant proteins of TPx, SAP2 and VB2 were expressed and purified successfully and their immunoreactivities were evaluated by indirect ELISA. TPx and SAP2 showed high immunoreactivities.The diagnostic potency of the rFgTPx_nt recombinant protein was assessed by indirect ELISA to detect specific antibodies in the sera of fascioliasis gigantica patients. The sensitivity, specificity and total coincidence rate of the ELISA were 66.7%,96.8% and 87.6%, respectively. And false positive rates for sera of schistosomiasis patients, clonorchiosis patients were 0.0%(0/15) and 6.7%(1/15) respectively. The OD values for serum samples collected from fascioliasis gigantica patients before and after treatment were 0.233±0.088 and 0.129±0.072 respectively, which showed a significant difference (P<0.05) between them, and indicated that rFgTPx_nt could be used for evaluation of therapeutic efficiency.The diagnostic value of rFgSAP2 was evaluated by indirect ELISA (SAP2-ELISA), compared with that of F.gigantic ESA (ESA-ELISA). The sensitivity, specificity and total coincidence rate of the SAP2-ELISA were 100%,94.8%and 95.8%, respectively. SAP2-ELISA showed no cross reaction in the sera of schistosomiasis, cysticercosis, clonorchiosis and opisthorchiasis, but a high cross reaction rate (46.1%,6/13) in the sera of paragonimiasis. There was no significant difference between ESA-ELISA and SAP2-ELISA, except that ESP-ELISA showed no reaction in the sera of paragonimiasis. The results indicated that rFgSAP2 antigen might be highly valuable in the serodiagnosis of human fascioliasis gigantica. In addition, the recombinant proteins rFgTPx_nt and rFgSAP2 could recognize sera of patients in the acute phase, which indicated that these antigens could be used for diagnosis in the early period of F.gigantic infection.[Conclusion] Four categories of antigens were obtained by screening the adult F.gigantic cDNA library with pooled sera of fescionasis gigantica patients, in which TPx, SAP2 and VB2 recombinant proteins were expressed. rFgTPx_nt and rFgSAP2 recombinant proteins showed high immunoreactivities in indirect ELISA, and could be used fer early diagnosis. In conclusion, rFgTPx_nt could be used for assessment of therapeutic efficacy, and rFgSAP2 might be highly valuable in the serodiagnosis of human fascioliasis gigantic.