Cloning, soluble expression and immunoreactivity of HIV-1 CRF35_AD p24 protein infusion with HP-thioredoxin from Iranian clinical isolates

Abstract

The purpose of this study was to isolate the coding sequence of the p24 protein (encoded by the gag gene) from sera collected from Iranian patients, to clone and express this sequence in Escherichia coli (E coli), and to assess the immunoreactivity of the expressed recombinant protein. The complete coding sequence for the Gag protein was isolated by nested real-time polymerase chain reaction (RT-PCR) from the serum of an infected individual and then cloned into a PTZ57R (T) vector. The resulting vector, PTZ-gag53-IR, was sequenced. Subsequently, the coding sequence of p24 protein was isolated by PCR from the PTZ-gag53-IR vector and inserted into a pET102/D-TOPO vector using TOPO directional cloning and then expressed in E coli in fusion with His-Patch (HP)-thioredoxin. The immunoreactivity of the expressed protein was tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA). RT-PCR revealed a sequence belonging to the CRF35_AD subtype of the HIV-1 virus, which is highly prevalent in Iran and Afghanistan. Recombinant p24 protein from this isolate showed 100% sensitivity and 100% specificity in an antibody assay. © 2012 by The American Society for Clinical Pathology.