| Fasciola hepatica is a kind of parasitic disease caused by Fasciola of Fasciolidae and Fasciola in liver and bile duct of human and cattle and sheep,which causes hepatitis,cholangitis and systemic nutrition disorder.The World Health Organization（WHO）has listed it as one of the important zoonoses.According to statistics,more than 300 million cattle and sheep around the world are infected with liver fluke,which greatly reduces the output of livestock products,which not only seriously affects the development of animal husbandry,but also brings huge economic losses to animal husbandry.The annual economic losses caused by liver fluke disease exceed$3.2billion.Therefore,the early diagnosis and prevention of liver fasciolomiasis are particularly important.Therefore,on the basis of our previous proteomic studies,we selected glyceraldehyde triphosphate dehydrogenase（GAPDH）and cathepsin L1（Catl-1）,which have the potential to be used as early diagnostic antigens,as the research objects.Two pairs of specific primers were designed according to the sequences of Fh GAPDH（THD20231.1）and Fh Catl-1（AY573569.1）published in Gen Bank.The Fh GAPDH and Fh Catl-1 genes were amplified by PCR after the total RNA of the adult liver fluke was extracted.The target genes were cloned into p MD18-T vector.After the identification by double enzyme digestion,PCR and plasmid sequencing,Fh GAPDH and Fh Catl-1-1 genes were successfully cloned.Sequence analysis showed that the length of nucleotide from the start codon to the stop codon was 1014 bp,encoding 337 amino acids.The length of the nucleotide from the start codon to the stop codon is 981 bp,encoding 326 amino acids.Bioinformatics analysis results showed that:The isoelectric point of Fh GAPDH protein is 7.65,which is a basic amino acid.The average hydrophilic coefficient of Fh GAPDH protein is less than zero.The B cell epitope analysis shows that Fh GAPDH protein has good antigenicity and belongs to hydrophilic protein,which mainly hasαhelical and random coil as secondary structure,and does not have signal peptide and transmembrane region.The results of spatial structure prediction showed that the consistency between the template and the sequence was 71.99%,the GMQE reliability was good,and the QMEAN4 matching degree was highly consistent.The isoelectric point of Fh Catl-1 protein is 6.15,which is an acidic amino acid,and the average hydrophilic coefficient is less than zero.B cell epitope analysis shows that Fh Catl-1 protein has good antigenicity and is a hydrophilic protein.At 1-15 amino acids,the protein has signal peptide,but does not have transmembrane region.The reliability of GMQE is excellent,and the matching degree of QMEAN4 is highly consistent.The correctly identified target gene fragment was cut off from the clone vector and linked to the expression vectors p Cold I and p ET-28a,respectively.The correct identification by double enzyme digestion indicated that the prokaryotic expression vectors p Cold I-Fh GAPDH and p ET-28a-Fh Catl-1 were successfully constructed.The recombinant proteins of Fh GAPDH and Fh Catl-1 were expressed,and the protein sizes were 36.47 k Da and36.60 k Da,respectively.The results of Western bloting showed that Fasciola hepatica antibody can specifically recognize the two recombinant proteins,indicating that the recombinant proteins have good reactivity.In order to prepare polyclonal antibodies and evaluate the immune effect of rabbits immunized with recombinant protein,rabbits were immunized with Fh GAPDH,Fh Catl-1 and Fh GAPDH+Fh Catl-1 fused with adjuvant respectively.The rabbits were immunized once every14 days.Blood samples were collected before immunization and 7 days after each immunization through ear vein.The changes of specific antibody level in serum were detected.After triple immunization,when the antibody titer reached 105or above,cardiac blood collection was performed,and the collected serum was polyclonal antibody.Subsequently,splenic lymphocytes were co-cultured with recombinant proteins to detect the changes of Th1/Th2 cytokines.Serum specific antibody detection results showed that rabbits immunized with recombinant protein could produce mixed Th1/Th2 immune response,and the Ig G,Ig G1and Ig G2aantibody levels were all increased,and the increase of Ig G1（Th2）was more significant,and the Fh GAPDH+Fh Catl-1group induced the best immune effect.The cytokines produced by splenic lymphocytes were detected by ELISA,and the results showed that the levels of IL-4,IL-10,IL-12 and IFN-γwere all increased,among which the levels of IFN-γand IL-12 were significantly increased and the levels of IL-4 and IL-10 were extremely significantly increased.The secretion of IL-4,IL-10,IL-12 and IFN-γwas the highest in Fh GAPDH+Fh Catl-1 group.The purified recombinant proteins of Fh GAPDH,Fh Catl-1,and Fh GAPDH+Fh Catl-1 were used as coated antigens to establish three indirect ELISA methods for detecting serum antibodies of the liver tablet flucifer.The optimal serum dilution and antigen coating concentration of the reaction were optimized by chessboard method,and other reaction conditions were optimized by indirect ELISA method.The results showed as follows:（1）The concentration of Fh GAPDH was0.375 ng/μL per well,and the serum dilution was 1:400;The best encapsulation time was 37℃2 h,and the best encapsulation solution was 1%BSA.The optimal incubation time of the secondary antibody was 60 min,the maximum P/N value was 5.561,and the optimal substrate reaction time was 15 min.（2）The encapsulation concentration of Fh Catl-1 was 1.5 ng/μL per well,and the serum dilution was 1:200;The best coating time of antigen was 37℃for 2 h,the best blocking solution was 1%BSA,the best incubation time of secondary antibody was 45 min,the maximum P/N value was 5.037,and the best substrate action time was 15 min.（3）The concentration of Fh GAPDH+Fh Catl-1 was 0.75 ng/μL,the serum dilution was 1:200,the best encapsulation time was 4℃overnight,the best blocking solution was 1%BSA,the best secondary antibody incubation time was 45 min,the maximum P/N value was 6.283,and the best substrate action time was 20 min.When the serum OD450nm value was greater than 0.788,0.652 and 0.595 and P/N>2.1,it was considered positive.The maximum coefficients of variation of the three indirect ELISA methods were all less than 10%in the intra-and inter-batch repeated tests.Three indirect ELISA methods were used to detect the positive sera of artificially infected sheep.The earliest detection days of infection were 14 days for Fh GAPDH,21 days for FHCATL-1 and 14 days for Fh GAPDH+Fh Catl-1.There was no cross-reaction between the three methods and the positive sera of Clonorchis sinensis,Schistosoma japonicum and Nematode twirlum.Results The positive rates of Fh GAPDH,Fh Catl-1 and Fh GAPDH+Fh Catl-1 were 15.33%（40/261）,16.86%（44/261）and18.39%（48/261）.Colloidal gold particles with a size of 20 nm were prepared by the trisodium citrate reduction method to label the purified recombinant protein.Results:（1）The optimal p H value of Fh GAPDH was added with 14μL 0.2 mol/L K2CO3,the optimal protein labeling concentration was 37.5μg/m L,and the optimal streaking concentration of the detection line（T line）was 1 mg/m L.The control line（line C）is the optimal concentration of 0.5 mg/m L.（2）The optimal p H value of Fh Catl-1 is added with 10μL 0.2 mol/L K2CO3,the optimal protein labeling concentration is 75μg/m L,the optimal streaking concentration of the detection line（T line）is 0.5 mg/m L,quality control The line（line C）is the optimal concentration of 1.0 mg/m L for the line.（3）The optimum p H value of Fh GAPDH+Fh Catl-1 is 12μL 0.2 mol/L K2CO3,the optimum protein labeling concentration is 37.5μg/m L,and the optimum streaking concentration of the detection line（T line）is 1.0 mg/m L.The best marking concentration of the quality control line（line C）is 0.5 mg/m L.Three kinds of colloidal gold test strips were used to detect the positive sera of sheep artificially infected with Fasciola hepatica in different days.The earliest days of infection were 14 days for Fh GAPDH,21 days for Fh Catl-1,and 21 days for Fh GAPDH+Fh Catl-1.And these three colloidal gold test strips did not cross-react with the serum of Clonorchis sinensis,Schistosoma japonicum and Haemonchus contortus.There were 261 clinical test samples,the positive rate of Fh GAPDH colloidal gold test strip was 14.18%（37/261）,the coincidence rate with ELISA method was 92.5%,and the positive rate of Fh Catl-1 colloidal gold test strip was 16.09%（42/261）.),the coincidence rate with ELISA method was 95.45%,the positive rate of Fh GAPDH+Fh Catl-1 colloidal gold test strip was 17.62%（46/261）,and the coincidence rate with ELISA method was 95.83%.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. |
