| Alveolar echinococcosis(AE) is an important zoonosis caused by the larvae of Echinococcus multilocularis,which is also called parasitic cancer with high fatality.AE aroused great public concerns because it has a great impact on the health of human population,animal husbandry production,and social and economic development.Proteomics is an emerging discipline developed independently of genomics,which can study the characteristics of proteins with high throughput,including the expression level of proteins,post-translational modifications,and the interaction between proteins,from which the overall and comprehensive understanding of life activities at the protein level could be obtained.In this study,dogs were infected experimentally with protoscoleces of E.multilocularis,and feces were collected at different time points after infection.The total protein of canine feces was extracted at30,60 and 90 days post-infection after inactivation,and the total protein was separated by SDS-PAGE,and the highly abundant bands were collected and analyzed by mass spectrometry,and then the mass spectrometry data were analyzed using bioinformatics tools.The specific proteins and candidate diagnostic antigens of Echinocuccus were screened and identified preliminarily,and one of the candidate diagnostic antigen was selected for further research.Its protein-coding gene was cloned and expressed using a protein expression system.The polyclonal antibody against the protein was prepared using the purified recombinant protein.A sandwich ELISA method for fecal antigen test of Echinocuccus was preliminarily established,and the value of the protein for detection of Echinocuccus infection was confirmed using clinical samples.Finally,a monoclonal antibody against the recombinant protein was prepared,which laid the foundation for the development of detection kit based on fecal Echinocuccus antigen.The results showed that 66 Echinocuccus specific proteins were screened from the infected dog feces.In brief,28,44 and 21 proteins were identified from the feces collected at 30,60 and 90 days post-infection,respectively.Among them,8 proteins were common to all feces,13 were specific to day30,27 to day 60 while 7 proteins were specific to day 90.There were 5 common proteins for the feces of 30 and 60 days post-infection,2 for 30 and 90 days post-infection,and 4 for 60 and 90 days post-infection.After further screening,six candidate diagnostic proteins were selected.The encoding gene for one of the proteins(Em Snpt)was cloned,and was expressed as a fused protein followed by purification.New Zealand rabbits and BALB/c mice were immunized to prepare polyclonal antibodies against the purified Em Snpt.A sandwich ELISA for the detection of Echinococcus fecal antigen was preliminarily established using the prepared polyclonal antibody and evaluated by clinical negative and positive samples,the results showed that the specificity and sensitivity of the method established based on this protein were satisfactory and it was an ideal diagnostic target antigen.In this study,the parasite-associated antigens in dog feces after E.multilocularis infection were identified by proteomics for the first time,and one of the proteins,Em Snpt was evaluated for the application value in the detection of the infection.The study provides a basis for the subsequent development of E.multilocularis fecal antigen detection kit. |
