| Cystic echinococcosis,which is caused by Echinococcus granulosus.One of the most widespread zoonotic helminth diseases that affect humans and livestock.Echinococcus unilocularis(metacestode or larvae of Echinococcus granulosus tapeworm)may parasitize at the liver or lung of animals and humans,it makes a serious harm to human health and animal husbandry development.It is not only a special medical and veterinary problem,but also a serious social and economic problem.Proteome was first proposed at an academic conference by Marc Wilkins as early as in 1994,which refers to all the proteins expressed by a genome.It can be revealed mainly using two-Dimensional Electrophoresis,2 – DE technology.Proteomics is a subject or tool,which were used to study the characteristics of a full set of proteins among the organisms,tissues or cells by using the high resolution separation technology,mass spectrum identification technology and bioinformatics technology.In this study,dogs were infected with E.granulosus larva artificially,and their stool samples were collected at different time post infection and kept till use.Then,proteins were extracted from the samples and analyzed with mass spectrometry,combined with proteomics,bioinformatics and molecular biology.Of the identified 3 242 proteins in the supernatant of dog feces,3 107 were from the definitive host,and 135 were from the tapeworm.A set of potential candidate proteins for diagnosis were prodicted.Among them,specific proteins EMR,GAPDH and TMEM41 B were selected to be the candidates.Their encoding genes were cloned and expressed in E.coli cells,respectively and were valuated based on the stool antigen ELISA detection method,a duable antibody sandiwich ELISA assay.20 supernatants of dog stool samples,which were confirmed to be positive for Echinococcus granulosus infection,were used to evaluate these proteins' diagnostic performance.The results showed that when the animal sera against recombinant protein pET30a-EMR were used as the first coating antibody,the detection positive rate was 35%;for the packaged sera against recombinant protein pET30a-GAPDH,the positive rate was 45%;for the packaged sera against recombinant protein pET30a-TMEM41 B,the positive rate was 25%.To evaluate the specificity of the recombinant protein,the dog poop of Echinococcus multilocularis,Taenia multiceps,Taenia hydatigena infected naturally were tested.For the sera against the recombinant protein pET30a-EMR,the facal samples from dogs infected with Echinococcus multilocularis tested positive,while those from Taenia multiceps and Taenia hydatigena were negative;for the sera against the recombinant protein pET30 aAPDH,the feces from the dogs infected with Echinococcus multilocularis,Taenia multiceps and Taenia hydatigena were all copro-positive;for the sera against the recombinant protein pET30aTMEM41 B,the feces from the dogs with Echinococcus multilocularis and Taenia multiceps infection tested positive,whereas those from Taenia hydatigena were copro-negative.The aim of the present study is to establish a copro-antigen ELISA method for the detection of dogs with Echinococcus granulosus infection,which will provide technical support for detecting Echinococcus granulosus infection in final host timely and accuratly,and for developing the comprehensive prevention and control measures against echinococcosis.The method and results will offer an alternative and valuable references for further screening and identification of ideal potential antigens,whick can be used for the development of copro-antigen ELISA kits for diagnosis of dogs with Echinococcus granulosus infection. |
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