| Cystic Echinococcosis (CE), caused by larva of the Echinococcus granulosus, is a zoonosis. The disease is highly endemic in western China and is a major public health problem, it has caused considerable losses not only in human health but also in agricultural production. At present, diagnosis of the disease is greatly based on physical imaging methods. However, these methods can not provide early diagnosis, which is important for significant improvements in the quality of the management and treatment of the disease. Immunological methods are both sensitive and specific, and could be used for early diagnosis of the disease. Currently, immunodiagnosis of CE is mainly based on detecting the specific antibody in patients'sera, thus the antigen applied is pivotal to diagnosis efficiency. While up to now the antigens being researched are limited in sensitivity or specificity, therefore, finding new antigen with higher diagnostic value is the key to improve the diagnostic efficiency of CE. In this study, the protoscolex of Echinococcus granulosus was collected from the liver cyst of infected sheep in Emin County, Xinjiang. The mRNA was extracted and purified from the protoscolex and the cDNA library was constructed. Positive clones were immunoscreened using the mixed CE patients' sera, and 5 positive clones (L1,H3,H4,E1-1 and K1) with high response intensity were selected to be subcloned into pGEX-3X or pGEX-4T-1 vectors and expressed. The diagnostic value of these 5 expressed recombinant proteins were evaluated by ELISA using sera from patients with CE and other parasitic diseases and that from healthy persons. The homolog of the encoded protein from each selected gene were searched in GenBank, and the structure and function of the expressed proteins were analyzed. Result shows that the titer of the cDNA library is 1.8×105pfu/ml, the insert length of the library is between 1.0kb and 4.0kb with the average length of 2.64 kb and the insert efficiency of 93.75%. All of the predicted amino acid sequences from these 5 genes show good hydrophilicity, accessibility and flexibility, and they all contain many B cell epitopes. The Blastp search shows that the homologs of the 5 predictive proteins L1, H3, H4, El-1 and K1 are Echinococcus granulosus glutathione S-transferase, Schistosoma mansoni hypothetical protein, Echinococcus granulosus calreticulin, Echinococcus granulosus malate dehydrogenase and Dugesia japonica heat shock protein 90, respectively, with identities of 100%,28%,100%,99% and 77% respectively. Among them, H3 and Kl are found existing in Echinococcus granulosus for the first time. The sensitivity of the recombinant antigens L1, H3, H4, E1-1 and K1 were 83.95%, 64.20%,88.89%,54.32%,41.98% and 53.09% respectively, and H3 has the highest sensitivity(88.89%),higher than that of the AgB8/2(83.95%).The specificity of the 5 antigens were 79.34%,76.09%,88.43%,89.26% and 89.26% respectively and have no significance difference with one another.2 new Echinococcus granulosus gene (H3, K1) were found in this study. Among them the recombinant protein of H3 has a higher sensitivity than AgB8/2, which is considered the most promising antigen for diagnosis of CE by far, and its specificity is equivalent to AgB8/2. H3 gene has a length of about 4kb, and contains about 58 antigen epitopes, thus it could become the most promising antigen for detection of CE by reconstructing it to increase its sensitivity and specificity.Also, based on screening the cDNA library of Echinococcus granulosus constructed in this study more valuable antigens could be found in the future. |
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