Establishment Of An Indirect ELISA And Colloidal Gold Test Strip Diagnosis Method For Sheep Fascioliasis Based On Two Recombinant Proteins Of TPx And CatB4

Fascioliasis hepatica is a serious parasitic disease caused by Fasciola hepatica,parasitizing ruminants such as cattle and sheep.It can cause death in severe cases.The disease often causes huge economic losses to the animal husbandry industry.Human infections have also been reported.In recent years,the disease has a high incidence trend.Therefore,early diagnosis and early treatment are the keys to prevention and treatment of the disease.This study is based on the proteomics analysis,and selected the thioredoxin peroxidase（TPx）and cathepsin B4（Cat B4）proteins that have the potential for early diagnosis of antigens as the research objects.According to the published information in Gen Bank The TPx and Cat B4 sequences of TPx and Cat B4 are reference design two pairs of specific primers to amplify the gene fragments of the two proteins.After the target fragments are double-enzyme digested,TPx is connected to the expression vector p Cold I,and Cat B4 is connected to the expression vector p ET-32a（+）,the ligation product was transformed into BL21 competent cells,and the expression was induced by IPTG and then identified by SDS-PAGE.Western blot analysis showed that the two recombinant proteins reacted with the positive serum of Fasciola hepatica,indicating that they have good reactogenicity.Optimize the purification method and conditions of the protein,collect the purified protein and measure the concentration.The purified TPx,Cat B4 and TPx+Cat B4 recombinant proteins were used to immunize New Zealand white rabbits to prepare polyclonal antibodies.After the rabbits were immunized for three times,when the antibody titer reached 10⁵or more,blood was collected from the heart to collect serum.Detect the changes of Ig G(Ig G₁,Ig G_2a)antibody levels in serum;take spleen cells and co-culture with recombinant protein to detect the changes of Th1/Th2 cytokines.The results of antibody detection in the serum of each immune group showed that the levels of Ig G,Ig G₁,and Ig G_2aantibodies increased,which induced the body to produce Th1/Th2 immune response,and the antibody level of Ig G₁（Th2）increased most significantly.ELISA detection of cytokine secretion in the culture medium showed that the contents of IFN-γ,IL-12,IL-4 and IL-10 all increased significantly.IFN-γand IL-12 increased significantly in the culture supernatant,and IL-4 and IL-10 increased significantly in the culture supernatant.The secretion of each cytokine in the TPx+Cat B4 group was higher than that in the other experimental groups.Using purified recombinant proteins TPx,Cat B4 and TPx+Cat B4 as coating antigens,three indirect ELISA methods were established.The checkerboard method was used to optimize the optimal serum dilution and antigen coating amount for the reaction,and other reaction conditions of the indirect ELISA method were screened at the same time.The results showed that the best coating concentrations of the three groups of recombinant antigens were 0.375 ng/μL,0.75 ng/μL,and 0.75ng/μL;the best serum dilutions were 1:400,1:100,and 1:200;the best The incubation time was all coated overnight at 4°C;the best blocking solution was 1%BSA;the best secondary antibody incubation time was 30 min,60 min,45 min;the best color development time was 15 min;three indirect ELISA methods were used The positive sera for different days of artificially infected sheep with Fasciola hepatica were detected.The earliest days of infection were 14 d,21 d,and 14 d;and these three ELISA methods did not cross-react with the positive sera of Clonorchis sinensis,Schistosoma japonicum and Haemonchus contortus.261 clinical samples collected from 18 counties（cities）in Heilongjiang Province were tested.The positive detection rate of TPx was 14.94%（39/261）,Cat B4 was 16.09%（42/261）,and TPx+Cat B4 was 18.77%（49/261）.Recombinant proteins TPx,Cat B4 and TPx+Cat B4 were used as antigen-labeled colloidal gold to prepare test strips.The results showed that the optimal p H of the three recombinant antigens were 8,9,and 9,respectively;the optimal antigen marker concentrations were 18.75μg/m L,37.5μg/m L,37.5μg/m L,respectively;the best antigen coating concentration for the detection line was 1 mg/m L;the best rabbit polyclonal antibody labeling concentration for the quality control line was 1 mg/m L,0.5 mg/m L,0.5 mg/m L,respectively;Three types of colloidal gold test strips were used to detect the positive sera of different days of artificially infected sheep with Fasciola hepatica,and the earliest days of infection were detected at 14 d,21 d,and 14 d;and these three colloidal gold test strips have no cross-reactivity with Clonorchis sinensis,Haemonchus contortus and Schistosoma japonicum.261clinical samples collected from 18 counties（cities）in Heilongjiang Province were tested.The results showed that the positive detection rate of TPx was 12.64%（33/261）,the coincidence rate with ELISA was 84.62%,and the positive detection rate of Cat B4 was 14.56%（38/261）,the coincidence rate with ELISA was 90.48%,the positive detection rate of TPx+Cat B4 was 16.86%（44/261）,and the coincidence rate with ELISA was 89.8%.The results show that the prepared three colloidal gold test strips can be used for preliminary detection of clinical samples and can be used for early diagnosis of Fasciola hepatica.