| The aim of this study was to select the early diagnostic antigen from gel filtration chromatography components of Fasciola gigantica Excreto-ry-Secretory Products(FgESP)and their identified protein components Which suitable for detect Fasciolsis,and establish a diagnostic method.It lays a foundation for the diagnosis and treatment and the development of a ELISA kit for early diagnosis of Fasciolsis.In this study,the FgESP of adult were prepared.By gel filtration chromatography,the first wave peak and the nearby chromatographic components were collected.The collection chromatographic components was named F1、F2、F3...Fn et al.Using different chromatographic components as antigens,the optimal chromatographic components were selected.At the same time,chromatographic components with good diagnostic effect were selected for Mass Spectrometry,and the three proteins that could be used as the diagnostic candidate antigen were selected for prokaryotic expression.The method of indirect enzyme-linked immunosorbent assay was established and optimized by using the the better chromatographic components as the diagnostic antigens to react with standard positive and standard negative buffalo serum.The indirect ELISA method was optimized by using three recombinant proteins and their mixed proteins as diagnostic antigens to react with standard positive and standard negative buffalo serum and mice serum infected with Fasciola gigantica for 4 weeks.And compared with the diagnostic effect of the FgESP,the better chromatographic component,the recombinant protein and the mixed protein.In an optimized method,the IgG antibody level of the buffalo serum of 0～14 weeks and the serum of 700 cows in the Guangxi part region was detected.The results of FgESP and better chromatographic components as diagnostic antigens were compared.In this study,the effective chromatography components were F21、F22 and F23.They were selected for Mass Spectrometry.The results showed that Thioredoxin Peroxidase-1(TPx-1)、Cathepsin B3(Cat B3)、Cathepsin L1(Cat L1)、Heat shock protein 70(HSP70)and Tubulin proteins were potential candidate antigen for diagnosis.Screening,TPx-1、Cat B3 and Cat L1 as a diagnostic candidate antigen for prokaryotic expression.The expression of FgTPx-1 was soluble and inclusion bodies,and the expression of FgCat B3 and FgCat L1 was the inclusion body.The molecular weights of the recombinant protein of FgTPx-1、FgCat B3 and Cat L1 were 46 kDa,40 kDa and 39 kDa,respectively.The optimal conditions for using F22 as diagnostic antigen was that the coating concentration was 0.157 μg/mL,the dilution multiple of the buffalo serum was 1:400,the second anti-dilution multiple was 1:40000,the positive and negative critical value was 0.391.The optimum concentration of F22 chromatographic component was 1/16 of the commonly used dosage of FgESP antigen(2.5 μg/mL).The results of indirect ELISA showed that the P/N ratio of single protein of FgTPx-1 and FgCat B3 to buffalo positive and negative was low.The concentration of FgTPx-1 and FgCat B3 was 1.25 μg/mL,and the dilution multiple of rat serum was 1:100,and the P/N value was larger.When optimizing with the serum of buffalo and the serum of mice,the concentration of the FgCat L1 antigen was 0.157 μg/mL and the serum ratio was 1:100,and the P/N value was great.The concentration of the mixed protein was 0.313 μg/mL,the serum ratio of the buffalo was 1:100.the P/N value was larger,the concentration of the mixed protein was 1.25μg/mL,the serum ratio of the mice was 1:100,and the P/N value was larger.When the mixed protein and the FgCat L1 were detected in the 0～14 weeks buffalo serum,the OD450nmvalue of whole test group was on the rise,and the difference between the positive and negative values of the mixed protein was slightly lower than that the FgCat L1 at the time of 1～8 weeks.FgESP、F22、FgCat L1 and mixed protein were compared with indirect ELISA.When the serum of buffaloes in the 4th weeks of infection Fasciola gigantica was used to optimize,the P/N value of F22 was the largest at the serum ratio of 1:400,followed by FgESP.When the standard sera of buffalo was used to optimize,there vwere significant positive and negative differences in the four diagnostic antigens.When the serum of mice in the 4th weeks of infection Fasciola gigantica was used to optimize,the P/N value of the F22 chromatography was largest atthe serum ratio of 1:400,positive and negative difference between the four diagnostic antigens were large.FgESP with mice serum were slightly weaker than other proteins.Among them,FgCat L1 and the mixed protein were used as the diagnostic antigen,there were the differe-nce of positive and negative,but the P/N value was close to each other.Chromatographic component of F22 was used as a diagnostic antigen to detect the level of serum antibody in the 0-14 weeks of the buffalo from the second week was obviously increased,and the trend of the gradual increase from the fourth week.The result of the FgESP test was that the level of in the 0～14 weeks of the buffalo was significantly higher from the fourth week.The results of F22 test showed that the antibody level of buffalo increased obviously from the 2nd week,and increased slowly after the rapid rise to 4 weeks.The results of FgESP test showed that the antibody level of buffalo increased significantly from the 2nd week,and the antibody level of the buffalo increased gradually from the 2nd week.The level of anti-F22-IgG was significantly higher than that of anti-FgESP-IgG.The accuracy of detection of buffalo serum in the 0-14 weeks by FgESP and F22 chromatography was 100%.The positive rate of 700 serum in some parts of Guangxi was 50.43%and the FgESP was 45.29%.In this study,it was found that the effective chromatographic components were mainly concentrated on the first peak,and the detection effect of F22 was better than that of chromatographic component F22,and an indirect ELISA method with F22 as diagnostic antigen was established.Three recombinant proteins,FgTPx-1,FgCat L1 and FgCat B 3,expressed in prokaryotic cells,can lay a foundation for further functional study.In this study,the diagnostic result for fasciolasis of FgESP,F22 and FgCat L1 and mixed proteins showed that F22 and FgCat Ll have good diagnostic effect and no significant advantage of-mixed protein,and F22 can be used for the early diagnosis of buffalofasciolasis. |
