High-level Expression and One-step Purification of Chimeric Antigen Containing HTLV-I-II Diagnostic Epitopes in Escherichia coli

  • Mahnaz Bezhgi Mail Department of Biochemistry, Payame Noor University, Tehran, Iran
  • Mohammad Fazilati Department of Biochemistry, Payame Noor University, Tehran, Iran
Keywords:
Antigenicity, Chimeric antigen, Enzyme-linked immunosorbent assay (ELISA), Human T-lymphotropic virus (HTLV), Protein purification

Abstract

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. Enzyme-linked 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. 

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Published
2020-04-16
How to Cite
1.
Bezhgi M, Fazilati M. High-level Expression and One-step Purification of Chimeric Antigen Containing HTLV-I-II Diagnostic Epitopes in Escherichia coli. Iran J Allergy Asthma Immunol. 19(2):149-158.
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Original Article(s)