| Fasciolosis is a zoonotic parasitic disease caused by Fasciola gigantica and Fasciola hepatica.The disease mainly infects ruminants and humans.It is a food-borne parasitic disease.The infected animals or humans are manifested as acute hepatitis,chronic liver fibrosis,cholangitis,etc.It can cause a large number of deaths of animals in severe cases,which is harmful to humans and animal husbandry.At present,the prevention and control of the disease mainly relies on drug treatment.Therefore,the diagnosis of the disease,especially early diagnosis,is particularly important.Early diagnosis and early treatment can reduce the harm of the disease to human and animal health,reduce the economic loss of animal husbandry.The diagnostic research of Fasciola has always been a research hotspot in this field.At present,the main clinical diagnosis method is still the classic fecal egg microscopy,but this method cannot be used for the detection of early infection.There are several better immunological diagnostic methods in the world,but the diagnostic antigens are mostly natural mixed antigens purified from parasites,so there are disadvantages such as high cost and certain cross-reactivity.In order to solve this important problem,this project was based on the proteomics in provioulsy study,and some candidate diagnostic targets were screened through bioinformatics methods,and the diagnostic targets were screened and evaluated through prokaryotic expression,protein purification,and Western bloting.A new cathepsin(named CL7)was screened,an indirect ELISA method was established,and field samples were tested using this method.Next,monoclonal antibodies and polyclonal antibodies were prepared for the selected diagnostic target protein CL7,and then a double-antibody sandwich ELISA method was established to detect the circulating antigen of Fasciola,for the early diagnosis of Fasciolosis.The specific content is summarized as follows:1.Screening,expression and purification of diagnostic targets and establishment and optimization of indirect ELISA methodsThis study selected Annexin(Ann),Leucine aminopeptidase(LAP),CUB domain protein(CUB)and a new protein(we identified it as a member of the cathepsin family and named it Cathepsin L7(CL7)).Molecular cloning,vector construction,prokaryotic expression,protein purification.Western blotting showed that the four recombinant proteins can react with the serum of buffalo infected by Fasciola.The corresponding indirect enzyme linked immunoabsordent assay(iELISA)method was established with 4 proteins as coating antigens to screen the best diagnostic targets.The positive sera of the sheep infected by Fasciola and the negative sera of healthy sheep were used to evaluate its sensitivity.The results showed that the sensitivitys(SEN)of Ann and CUB were low.The sensitivitys of LAP,Ann+LAP,CL7 were higher.Using positive sera from sheep infected with Fasciola(n=16)and positive sera from other common parasites(n=16)for cross-reaction,the results showed that CL7 specificity(SPE)was the highest,followed by the other two.CL7 has the most potential.2.CL7 bioinformatics analysis,preparation of monoclonal antibodies and establishment of sandwich ELISA methodThe CL7 gene was studied for the first time in this experiment.Bioinformatics analysis prove it is a member of the CL family(Cathepsin L),so it was named(CL7),and its gene sequence was uploaded to GenBank,and the gene number was MN150457.At the same time,this study also conducts structural domain analysis and modeling and reliability 3D modeling for CL7.In order to establish a sandwich ELISA(sELISA)suitable for early diagnosis,mouse and New Zealand rabbits were immunized with CL7,and 11 strains of CL7 monoclonal antibodies(mAb)and rabbit polyclonal antibodies were successfully prepared.Four high-titer monoclonal antibodies:2B5,3B10,5A2,and 9H9 were selected for the preparation,purification,titer determination,and phenotypic epitope identification of mouse ascites.The results showed that:2B5,3B10,5A2,and 9H9 belong to:IgG1,IgG2b,IgG1,IgG3.Superimposed ELISA results show that 2B5 and 5A2 recognize similar or identical epitopes,and 3B10 and 9H9 recognize similar or identical epitopes.Since 5A2 and 9H9 had lower titers after purification,2B5 and 3B10 were combined with rabbit polyclonal antibodies to establish a variety of sELISA methods.The result indicated that CL7 monoclonal antibody 2B5 combined with rabbit polyclonal sELISA had the best effect.And a standard curve for the CL7 sELISA had been established,which can be used for the quantitative analysis of circulating antigen CL7.ROC statistical analysis showed that:the detective limit of CL7 sELISA for Fasciola infested sheep serum(n=12)was 1.087 ng/mL(Cut-off:0.4115),and the above positive serum was used for the evaluation of SEN and SPE.The results showed that both of them reached 100%.When the CL7 sELISA was used for buffalo’s positive(3-7WPI)and negative sera,the detective limit was 2.009 ng/mL(Cut-off:0.4845),SPE reached 100%,and SEN reached 93.75%.Therefore,CL7 sELISA can be applied to the early diagnosis of Fasciolosis in ruminants.In summary,this experiment successfully expressed 4 kinds of Fasciola recombinant proteins,found a good diagnostic target protein CL7,and established a sELISA method that can be used for early diagnosis,which was important for the diagnosis of Fasciolosis. |
