[Gene cloning, expression and serological evaluation of diagnostic antigen Em18 for alveolar echinococcosis].

OBJECTIVE: To clone, express and serologically evaluate the Em18 antigen gene of Echinococcus multilocularis for diagnostic purpose. METHODS: Polymerase chain reaction (PCR) was employed for amplification of the target gene fragments which was then ligated with pET28a+ vector. The constructed plasmid was transferred into E. coli BL21 (DE3) for expression. The recombinant proteins were purified with Ni-NTA agarose by affinity chromatography. 237 sera were used for evaluating diagnostic value of the recombinant Em18 antigen. RESULTS: Two high-level expression clones (designated as ReEm18-1 and ReEm18-2) were obtained. ReEm18-1 showed the expected sequence, ReEm18-2 showed the same sequence but with 27 nucleotides deletion. The molecular weight of the two expression proteins was Mr 28,000 and 26,000, respectively. Serological evaluation by ELISA was carried out using sera from 101 patients with alveolar echinococcosis (AE), 47 with cystic echinococcosis (CE), 30 with cysticercosis (CC), 10 with hepatic cancer (HC), 9 with schistosomiasis (Sj) and 40 from healthy persons (NH) from both endemic and non-endemic areas. The results showed an overall sensitivity of 86.1% and 90.1% with ReEm18-1 and ReEm18-2 for AE sera, specificity 93.4% and 94.1%, positive predictive value 90.6% and 91.9%, negative predictive value 90.1% and 92.8% and efficiency 90.3% and 92.4%, respectively. The correlation analysis between the size of AE lesions and the serum absorbance reacted with recombinant Em18 antigens showed that there was a positive correlation between antibody level and the course of the disease. CONCLUSION: ReEm18 antigens are specific for AE diagnosis, and the serum antibody level displays a good correlation with the course of the disease at early stage. Similar results achieved by both ReEm18-1 and ReEm18-2 antigens.

[Western blotting analysis of specific antigens from different components of Echinococcus metacestodes].

OBJECTIVE: To analyze antigens for searching specific antigenic components for immunodiagnosis of echinococcosis. METHODS: Fourteen crude antigens from different tissues (cyst fluid, protoscoleces, laminated layer and germinal layer) of Echinococcus granulosus and E. multilocularis metacestodes and other 4 species of cestodes were analyzed by Western blotting. The differences of protein bands were compared for the 14 crude antigens by reacting with pooled sera from cystic echinococcosis (CE) and alveolar echinococcosis (AE) patients. RESULTS: Eleven protein bands from the antigens reacted nonspecifically with sera from both CE and AE patients were Mr 130000, 100000, 94000, 80000, 75000, 66000, 62000, 52000, 38000, 32000, 24000. The highly specific protein bands recognized by AE sera were Mr 120000, 109000, 86000, 59000, 43000, 28000, 20000, 18000, and by CE sera were Mr 41000, 40000, 22000, 16000 and 12000. CONCLUSION: Different antigens shared by the two species of Echinococcus were examined and potential antigenic proteins specific for AE or CE sera were found, providing useful information for further identifying specific antigens for immunodiagnosis.