Screening Of The Diagnostic Identification Proteins About The Infection Of Fasciola Hepatica And Establishment Of The Indirect ELISA Detection Method Based On LC-MS/MS

Fascioliasis is an important zoonotic disease caused by Fasciola hepatica which parasitic on the liver and bile ducts of ruminants such as cattle and sheep.The most important measure for the prevention and control of the disease is accurate diagnosis.Thereby,it is particularly important to establish a sensitive,specific and effective method in the whole period of F.hepatica infection.In this experiment,adults of F.hepatica in artificially sheep liver were collected for vitro culture to prepare the excretion and secretory products(ESPs).The ESPs reacted with serum of 3 days post infection(dpi),7 dpi,21 dpi,63 dpi and 112 dpi and Fasciola gigantica serum of buffalo and negative serum of sheep by CoImmunoprecipitation(Co-IP)combined with LC-MS/MS to screen out interaction proteins.Specific proteins were screened out by protein expression analysis,cloned,prokaryotic and purified.The Western Blot was used to analyze the immunogenicity of the protein,the screened protein was used as the antigen for indirect ELISA.The protein with the highest P/N value was selected as the coating antigen to establish the ELISA and optimize the conditions.Then the cross reaction,sensitivity and repeatability were tested.Finally,the clinical samples of cattle and sheep were detected.In the present study,ESPs were prepared successfully.257,265,266,264 and264 proteins were screened from the F.hepatica serum at 3,7,21,63 and 112 dpi respectively.A total of 11 specific proteins were screened which are common in the five stages of F.hepatica that did not react with the F.gigantica serum.Through protein analysis,four proteins including Cystinosin,ADP-ribosylation factor,Hepatocyte nuclear factor 3-alpha and an uncharacterized protein(A0A4E0RCUO)were cloned and expressed.Cystinosin was expressed as inclusion body,and the rest were expressed as supernatant.The results of Western Blot showed that 4 proteins have good reactivity with F.hepatica serum.ADP-ribosylation factor had the highest P/N value which had the potential as a diagnostic antigen.The optimal conditions of indirect ELISA using the ADP-ribosylation factor as antigen are as follows: dilute antigen to 1 μg/m L with phosphoric acid buffer solution(PBS)as antigen to establish indirect ELISA method,coat it overnight at 4℃,block it with elisa plate stabilizer Ⅰ at37 ℃ for 1 h,dilute the primary antibody with PBST in the ratio of 1:50 and incubate at 37 ℃ for 45 min,and the secondary antibody was diluted with HRP coupling diluent Ⅰ in the ratio of 1:20 000 and incubated at 37 ℃ for 1.5 h.After adding chromogenic solution TMB,put it into 37 ℃ for 20 min.The critical value of positive and negative is 0.435,the serum sample greater than 0.435 is positive,otherwise it is negative.The method established in this study has good sensitivity,repeatability and no cross reaction.Finally,483 clinical sheep serum samples from Heilongjiang,Liaoning and Inner Mongolia as well as 92 cow serum samples were detected by this method.The infection rate of sheep was 8.70% and cow serum were negative.In the present study,11 specific proteins were successfully screened by Co-IP and LC-MS/MS.It was found that ADP-ribosylation factor has the potential to be used as diagnostic antigen.The indirect ELISA method for the diagnosis of F.hepatica based on this protein with no cross reaction and has the characteristics of high sensitivity and good repeatability,which laid a foundation for the development of diagnostic kit for fascioliasis.