Application of antibodies in most therapeutic area is limited to extracellular or membranous targets because of their impermeability of membrane. For the purpose of biotechnological and therapeutic application, developing intracellular localizing antibody is the invaluable research field. A new recombinant single-chain variable fragment of an anti-dsDNA monoclonal antibody G2-6, IgG of which has been previously shown to have a cell-penetrating activity, was engineered and produced for the use as a delivery vehicle of biomolecule(s). The penetrating capacity of single-chain variable fragment in three mammalian cell lines, L929, NIH/3T3, and COS-7 was analyzed using flow cytometry and confocal microscopy. The results demonstrated that the single-chain variable fragment can effectively internalize all three cell lines, although the internalization level varied. It was also shown that the internalization was time- and concentration-dependent. Moreover, the single-chain variable fragment was located in nuclei as well as cytoplasm of L929 cells. Overall, the G2-6 single-chain variable fragment might be a candidate vehicle which could be used to deliver specific genes or biomolecules for therapy or diagnosis into the cytoplasm or cell nucleus.
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