Screening Of Diagnostic Antigen For Fascioliasis Hepatica And Establishment Of Indirect ELISA

Fascioliasis hepatica is a kind of fluke disease caused by the Fasciola hepatica parasitizing liver and bile duct of ruminants such as cattle and sheep.The disease is a worldwide epidemic,according to statistics,the global annual more than 250 million to 300 million cattle,sheep infected with Fasciola,as a result of Fasciola infection caused economic losses of more than $3 billion,at the same time the disease can also infect people,is a serious zoonoses pathogens.The infection of F.hepatica in cattle and sheep will decrease the production performance and bring great economic loss to animal husbandry production and great threat to human health.At present,the diagnosis of fascioliasis hepatica in China is mainly based on the method of egg examination and dissection examination.But the method of egg examination can only detect the ruminants in the late stage of F.hepatica infection,and the damage of F.hepatica to the production performance of animals in the early stage of infection is relatively serious.Autopsy to the suspected infection of the animal autopsy,not in vivo diagnosis.Other diagnostic methods most are immunological diagnostic methods,such as indirect hemagglutination assay,Allergic reaction,bidirectional electrophoresis,etc.And this among them,indirect ELISA has good specificity and sensitivity in the detection of fascioliasis.Foreign fascioliasis hepatica diagnostic kits are expensive.Therefore,we urgently need to establish a diagnostic method and lay a foundation for the marketization of indirect ELISA kit for fascioliasis hepatica.In the preliminary work of the laboratory,the positive serum of sheep fascioliasis hepatica was immunoprecipitated with the excreted and secreted antigens of F.hepatica,and 5 kinds of proteins existed in the growth and development stages were screened out by mass spectrometry.The 5 kinds of proteins are Cathepsin L1(Cat L1),Cathepsin L2(Cat L2),Heat shock protein 70(HSP70),Leucine aminopeptidase(LAP)and Legumain(LEG),and the study of LEG in F.hepatica protein has not been reported.c DNA of the target proteins were obtained by RT-PCR,and specific primers were designed to amplify gene fragments of 5 kinds of proteins.After double enzyme cut,the target fragments were connected to the expression vector p ET-28a(+),and the linked products were transformed into BL21 comp ETent cells.After IPTG induced expression,SDS-page and Western blot analysis were performed to analyze whether the proteins had reactivity.After determining the correct expression of the protein,the purification method and conditions of the protein were optimized,and the purified protein was collected for concentration determination.After protein purification,5 kinds of proteins were detected by checkerboard ELISA,and the results of checkerboard ELISA were analyzed.According to the P/N value,L1 was selected as the optimal detection protein,and CL1 as the main protein for subsequent optimization experiments.Indirect ELISA reaction conditions were optimized,including the selection of coating buffer,the concentration of coating protein,the selection of sealing fluids and the exploration of sealing time,the concentration of primary and secondary antibodies,the removal of non-specific factors in serum,the specificity experiment and the determination of negative and positive cut off values.The results showed that the best coating buffer was carbonate buffer(CBS),coating concentration of antigens was 7.5 g/m L,sealing liquid was 5% skim milk,sealing conditions were 37 ? for 2 h.The primary antibody was diluted by 1:200(37 ?,2 h incubation),the secondary antibody was diluted at 1:10 000.The established indirect ELISA diagnosis method has good specificity.No cross reactions occur with others positive serum in this study.specificity of 97.3%,the sensitivity of 94%,it had the repeatability.coefficient of variation between batch and batch were less than 10%,tested the Heilongjiang and Jilin province 300,serum positive rate was 4%.In this study,5 kinds of proteins were successfully expressed,and cathepsin L1,a protein with good reactivity and suitable for the diagnosis of fascioliasis hepatica,was screened out.An indirect ELISA method for the detection of Fasciola hepatica antibody was initially established by using this protein as the coating antigen.This method has the advantages of rapid,sensitive,specific and low price.It lays a foundation for the research on the diagnostic kit of fascioliasis hepatica and provides strong technical support for the development of animal husbandry in China.