Prokaryotic Expression,Identification and Histolocalization of the Taenia asiatica Enolase Gene

Objective To express enolase gene of Taenia asiatica,investigate the immunoreactivity of the recombinant TaENO pretein,and immuno-histolocalize the presence of the recombinant TaENO in adults of T.asiatica.Methods The gene encoding enolase of T.asiatica(TaENO) was cloned by high throughput sequencing from the cDNA library of adult T.asiatica.The coding region of TaENO was amplified by PCR,and cloned into a prokaryotic expression vector pET-30 a(+ ).The recombinant plasmid was transformed into E.coli BL-21/DE3 and followed by expression of the protein induced by IPTG.The protein was purified by Ni-IDA affinity chromatography,and tested by SDS-PAGE.Its immunoreactivity was examined by Western blotting.The mice were immunized subcutaneously with purified TaENO formulated in Freund's adjuvant.Serum samples were collected and analyzed for specific antibodies by ELISA.The localization of TaENO in adult worms was demonstrated by immunofluorescent technique.Results The recombinant expression plasmid was identified by PCR,double endonuclease digestion and sequencing.The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml.It was recognized by antisera of SD rats immunized with TaENO,sera of taeniasis patients and sera of infected swine.The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T.asiatica adult.Conclusion The TaENO gene has been expressed with immunoreactivity,and the recombinant TaENO is immunolocalized in the tegument of T.asiatica adult.