| Objective: To clone Em 18 antigen gene and express recombinant Em18 antigen. Serological evaluate ReEm 18 antigen for diagnostic differentiation between alveolar echinococcosis (AE) and cystic echinococcosis (CE). Methods: The primers of Em18 were designed by DNAman biosoftware .The Em 18 antigen gene was amplified by PT-PCR from total RNA of Echinococcus multilocularis, and by TD-PCR from Em cDNA library for construction of plasmid pMD18/T-Em18. The Em18 antigen gene was identified by sequencing. The prokaryotic expression plasmid pET41a-Em18 was constructed and identified by sequencing. rEm18-GST fusion protein and GST were epressed by induction with IPTG and were detected by SDS-PAGE Western-Blot. rEm18-GST fusion protein was affinity purified against the GST tag. A total of 523 human serum samples were evaluated for the diagnostic sensitivity and specificity with the fusion protein by immunoblotting (IB) and enzyme-linked immunosorbent assay (ELISA), respectively (59 serum samples were collected from patients with AE, 240 serum samplesfrom patients with CE) . Results: DNA sequence analysis of Eml8 gene fragment indicated that the length of Em 18 was 486bp, code 161aa and was accepeted by Genbank as a new sequence (AY513691). The pET4la-Em 18 positive clone was the exact recombinant plasmid and rEml8-GST recombinant protein by induction with IPTG was espressed as a band of 50KDa by SDS-PAGE detection. The result of Western-Blot showed that rEml8-GST antigen has better antigenicity. 523 sera samples were detected by rEml8-GST antigen. The overall sensitivity was 91.52%/93.22% and the overall specificity was 94.61 %/94.82 by ELISA and Western Blotting, respectively. Conclusion: Eml8 antigen gene was cloned successfully. The combined levels of sensitivity and specificity achieved rather high with the rEml8-GST fusion protein for AE diagnosis, rEml8 has considerable advantage for serodiagnosis between AE and CE and possible development of a diagnosis kit. |
PDF Full Text Request