high-level expression and one-step purification of chimeric antigen containing htlv-i-ii diagnostic epitopes in escherichia coli

Purification and preparation of three diagnostic antigens used for the detection of human t-lymphotropic virus (htlv)-i/-ii infection in e.coli are different parts of a multi-step method. in this study, our aim was to design a chimeric protein for the simultaneous detection of htlv-i and htlv-ii antibodies. immunodominant b cell linear epitopes of envelope and capsid proteins of htlv-i/-ii were selected and linked together; using a suitable amino acid linker and a chimeric antigen (ca). the codon-optimized synthetic dna encoding the ca was subcloned into the pgs21aexpression vector and ca expressed as his-gst fused protein in e. coli bl21 (de3) cells. then the recombinant ca was purified, using the ni-nta (nickle nitrilotriacetic acid) affinity chromatography under native conditions. the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and densitometric scanning results showed that ca accounted for 15% of the total cellular proteins and approximately 50% of the expressed histidine-glutathione s-transferase-chimeric antigen (his-gst-ca) proteins were soluble. the ca was successfully purified in one step with a purity of greater than 90%, which is suitable for antigenicity evaluations. enzymelinked immunosorbent assay (elisa) results showed that the gst fused ca reacted in a concentration-dependent manner with htlv-i/-ii infected sera and was able to distinguish normal serum from htlv-i/-ii infected one with a proper sensitivity. with further validation, ca, as described in the present study could be introduced as a novel reliable, cost-effective and easy alternative for the three separate htlv-i/-ii diagnostic peptide antigens, which is prepared as a fusion with gst.