Subcloning of the Single Chain Variable Fragment of Anti Human CD4 Receptor Fused to Green Fluorescent Protein in pET26b Plasmid

Antibodies are powerful tools to study the function، localization، and interaction of proteins. These molecules are also widely used for diagnostic and therapeutic purposes. Antibodies are usually conjugated to fluorescent molecules for research and diagnostic purposes. If these fluorescent molecules are conjugated to a recombinant fluorescent protein، the combination is known as the fluobody. In this study، an anti-human CD4 single-chain variable fragment antibody fused to a green fluorescent protein was extracted from pAB1 plasmid with two restriction enzyme EcoRI and NcoI. Then، the fragment was cloned into pET26b plasmid in order to increase its expression، and the recombinant plasmid was then transformed into Escherichia coli. Insertion of this fragment into pET26b plasmid was confirmed by colony PCR and double digestion. Also، the expression of fluobody was confirmed by dot blotting. The expressed recombinant floubody is a bifunctional antibody which retains both antigen binding activity and its fluorescence simultaneously. Selection of an appropriate plasmid such as pET26b for high production of fluobody seems to be essential in order to obtain high levels of the protein.