Proteomic Comparative Analysis Of Fasciola Hepatica In Different Developmental Stages And Development Of Colloidal Gold Strip

Fasciola hepatica is mainly parasitic in the liver and hepatobiliary ducts of ruminants such as cattle and sheep.A kind of clonorchiasis is called liver clonorchiasis.He patic clonorchiasis is a zoonotic parasitic disease.The disease is widely prevalent in the world,and the economic loss caused by clonorchiasis infection clonorchiasis infection^[1].so early diagnosis becomes particularly important.In this study,we analyzed the proteomics of four main developmental stages of metacercaria,juvenile,larva and adult of Fasciola hepatica.The total number of peptides and proteome identified in the stage of Cysticercus was significantly less than that in the other three stages.Using the label free algorithm of Max quant software to calc ulate the proteomic data,and analyzing the significant difference between the quantitative res ults,we found that the number of differential proteins between the cysticercus and the other three stages was significantly lower than that between the other three groups,and the number of differential proteins between the ad ults and the children was the most.The analysis results of Wayne map and volcano map are consistent with the results of label free algorithm.It can be seen from the cluster analysis that most of the proteins expressed up-regulated in Cysticercus are down regulated in juvenile,larva and adult,while most of the proteins expressed down regulated in Cysticercus are up regulated in juvenile,larva and adult.Go and KEGG analysis showed that the proteins involved in protein folding were the most in the biological process,followed by those involved in purine ribonucleoside tri p Hosp Hate metabolism and purine nucleoside trip Hosp Hate metabolism;in the cell components,the proteins enriched in cells were the most,followed by those enriched in cells;in the molec ular function,the proteins involved in protein folding were the most The protein of binding function was the most,while the protein of ATP binding function was the least,which was significantly lower than other groups.KEGG results showed that the differential proteins were mainly involved in metabolism,genetic information processing,environmental information processing,cell process,tissue system and other six aspects.It is mainly involved in carbon metabolism,glycolysis/gluconeogenesis,Rap1 signaling pathway,actin cytoskeleton regulation,tight junction,antigen processing and expression,neurotrop Hic signaling pathway,synaptic vesicle cycle and antibiotic biosynthesis.After the screening and analysis of the data of differential proteins,the differential proteins were divided into 50 groups with obvious trend,of which 42,49,48,12,45 and 47 groups had bette r trend.Profile 42 group was mainly composed of calcium binding protein,thioredoxin glutathione reductase,thioredoxin peroxidase,glutathione transferase and glycerin-3-p Hosp Hate dehydrogenase?GAPDH?.In profile 49 group,heat shock protein 90,heat sh ock protein 70 and actin were the main proteins.In profile 48 Group,Ca2+was not sensitive to EF hand and aldehyde dehydrogenase family proteins.In profile 12 group,RAS family protein,calmod ulin like protein 1,actin and calmodulin like protein 3 were identified.Profile 45 is secreted cathepsin L1,fatty acid binding protein III,protein disulfide isomerase,calreticulin family protein and myosin tail.In profile 47 group,myosin,protomyosin and myosin reg ulated light chain were identified.I selected glutathione transferase from profile 42 and fatty acid binding protein III from profile 45as early diagnosis antigens,among which glutathione transferase was developed and preserved in our laboratory.The prokaryotic expression vector pet-30a-FhFAB? was successfully constructed by Uni Prot.The fragment size was 399bp and the protein size was 16 k Da,Western blotting showed that the recombinant protein could be specifically recognized by the antibody of Fasciola hepatica.The recombinant Fh GST and FhFAB? proteins were used as diagnostic antigens to establish indirect ELISA The results showed that the concentration of FhFAB? was 9.275ng per well,the dilution of serum was 1:12800,the best incubation time of FhFAB? was 4?overnight,5%skimmed milk was selected as the closing solution,the second antibody was 60 min,and the best substrate was 15 min.The concentration of Fh GST recombinant antigen coating was 37.5ng per pore;the dilution of serum was 1:12800;the optimal incubation time of Fh GST recombinant antigen was 37?for 2h;10%skimmed milk was selected from the closed solution,the second antibody incubation time was 30min,and the optimal substrate action time was 15min.In sensitivity test,the earliest detection time of FhFAB? and Fh GST was In the specific test,FhFAB? and Fh GST of Fasciola hepatica had no cross reaction with Schistosoma japonicum,Clonorchis sinensis and Haemonchus contortus.The results showed that the coefficient of variation of the three methods was less than 10%.The positive rate of FhFAB? and Fh GST was16.84%?16/95?and 12.63%?74/240?respectively.New Zealand white rabbits?1mg/kg?were immunized with purified FhFAB? and Fh GST recombinant protein once every two weeks.When the antibody titer re ached over 10⁵,the heart blood could be collected and polyclonal antibodies were purified by protrina affinity chromatograp Hy.The colloidal gold particles of 20nm were prepared by trisodium citrate reduction method.The polyclonal gold was labeled with p urified polyclonal antibody.The results of the determination of the optimal polyclonal amount and p H value of FhFAB? were 5.07?g/m L and 7.5,respectively.The results of the determination of the optimal polyclonal amount and p H value of Fh GST were 10.15?g/m L and 8.0,respectively.The purified antibody was diluted by1:1,and the purified antibody was diluted by 1:4.The purified antibody was soaked in glass fiber.FhFAB? and Fh GST antigens were used to draw the detection line?T-line?on the nitrocellulose membrane,and the optimal concentration was 0.8mg/m L.The sensitivity,repeatability and specificity of the strip were tested.The results showed that the earliest infection time of the two test strips was 21 days,the lowest Dilution Times of fhfabii and Fh GST were 1:60 and 1:80respectively,and there was no significant difference in the res ults of repeated tests of different batches of test strips.The two test strips did not react with Clonorchis sinensis,Haemonchus contortus and Schistosoma japonicum,indicating that the prepared test strips had certain sensitivity Sex,repeatability and specificity.95 clinical samples were detected by two test strips,11 positive sera were detected by Fh GST test strips,the positive rate was 11.58%?11/95?,the coincidence rate with ELISA method was 91.67%,14 positive sera were detected by fhfabii test strips,the positive rate was 14.71%?14/95?,and the coincidence rate with ELISA met hod was87.5%.The results showed that the prepared immunochromatograp Hic strip based on FhFAB? and Fh GST protein polyclonal antibody could be used for the preliminary detection of clinical samples and the diagnosis of Fasciola hepatica.