Prokaryotic expression and identification of enolase 2 gene of Toxoplasma gondii

To explore biological function of ENO2of Toxoplasma gondii,an eno2gene was amplified by RT-PCR fromT.gondii RH strain and cloned into pET-28a(+)vector.Then the recombinant plasmid pET-28a(+)-eno2was transformed into Escherichia coli BL21(DE3)competent cells for expression via induction with IPTG.In result,the ORF of eno2gene was 1 353bp in length,encoding 444amino acids. BLAST analysis revealed that nucleotide and amino acid similarity of T.gondii RH strain eno2to homologs from other T.gondii(GenBank No.AF123457)were 99.8%,respectively.The recombinant protein ENO2 was about 55ku in size,and the optimal induction conditions were pH 7.5,28℃ and 0.1mmol/L IPTG. Western-blot analysis indicated that recombinant protein ENO2reacted strongly with the convalescent phase serum from the dog infected with T.gondii RH strain.Moreover,the indirect immunofluorescence assay using polyclonal antibodies against recombinant protein ENO2revealed that the antiserum could recognize ENO2localized in the cytoplasm and nucleus of T.gondii.The results laid the foundation for further studies on the biological functions of T.gondii ENO2.