Prokaryotic Expression Of 20.9kD Tegumental Protein Gene From Echinococcus Multilocularis,Immunocalization And Establishment Of ELISA Diagnostic

Multilocular hydatid disease,which is also named alveolar echinococcosis,is an important human parasitic zoonosis.Echinococcus multilocularis metacestodes usually parasitize in liver or lung of their intermediate host,and can seriously threaten public health,which is confined to the northwest pastoral areas in China.At present the main preventive measure is enfoced domestic dogs with dosage of chemical drugs such as albendazole regularly.The long incubation periods always leads to missing the best treatment.Therefore,it is important to develop an early and specific dignostic technique for alveolar echinococcosis diagnosis.Em20.9 is a tegumental protein of E.multilocularis,which may play an important role in host interaction.Em20.9 could be a potential candidate for the diagnosis and vaccine production.In this study,we investigate the tegumental protein gene in E.multilocularis.using techniques such as clone,prokaryotic expression and diagnostic evaluations,We have achieved the following results.The Em20.9 gene CDS sequence was obtained through RT-PCR amplification using c DNA of Echinococcus multilocularis as template.The gene structure and phylogenetic evolution were analyzed with bioinformatic softwares.According to the results,the length for open reading frame was 543 bp in length,which encoded 180 amino acid,and have multiple antigenic epitopes.Then,the eukaryotic expression vector p ET28a-Em20.9 was constructed.According to the results of SDS-PAGE,the molecular mass of r Em20.9 was around 26 ku and expressed as insoluble inclusion bodies.Western Blotting analysis showed that r Em20.9 could be recognized by the positive serum of mice infected with alveolar echinococcosis.After immunized rabbits with the purified r Em20.9,a high titer of specific Ig G antibodies was produced.Morphological observation of E.multilocularis.by HE staining,results showed that one E.multilocularis cyst have various morphologies protoscoleces.Results of immunohistochemistry showed that Em20.9 was mainly expressed in the tegument of E.multilocularis metacestodes.It's confirmed that Em20.9 might be related to the interaction mechanism with host.Using purified r Em20.9 as the diagnosis antigen,we built up an indirect ELISA antibody detection strategy.The best reaction conditions were determind using Matrix method,the amount of antigen was 1 ?g per well;the dilution ratio of the serum was 1:100,and the antibody conjugated with HRP was 1:10 000;the best coating buffer was carbonate buffer(p H9.6),and the blocking buffer was 2.5% BSA.The cut-off value was 0.407,samples with OD450 that are equal to or above 0.473(cut-off+s)could be treated as positive results,while the negative was equal to or under 0.377(cut-off-s).Results showed that positive serum OD450 is still above 0.437 when diluted alveolar echinococcosis positive and negative serum to 1:51 200,revealing that the ELISA method has high specificity and sensitivity.Meanwhile,we tested 50 negative mice serumsamples and 30 positve serum samples of alveolar echinococcosis,and the results showed that the specificity and sensitivity of this ELISA method were 96.1% and 90.9%,respectively.