1068 pathology of human infections. Old World Hantaviruses, such as Hantaan, Seoul, Dobrava, and Puumala viruses (Schmaljohn and Hjelle 1997; Sjölander et al. 2002), cause hemorrhagic fever with renal syndrome (HFRS), whereas, New World Hantaviruses, such as Sin Nombre and Andes viruses (Schmaljohn and Hjelle 1997; Meyer and Schmaljohn 2000), are the causative pathogen of Hantavirus pulmonary syndrome (HPS). HFRS is characterized by fever, renal failure, thrombocytopenia, and in severe cases hemorrhagic manifestations with a mortality rate of 2–10% (Peters et al. 1999), while HPS is featured by severe acute respiratory dysfunction and a mortality rate of approximately 50% (Nichol et al. 1993; López et al. 1996). More than 100,000 cases of HFRS occur annually worldwide and most are documented in China (Song 1999). Hantaan virus (HTNV), the prototype of the Hantavirus genus, is responsible for numerous cases of HFRS. The antiviral drug ribavirin, which is mainly effective in the early phase of HFRS, is currently under clinical investigation but has not been proven to be sufficient to prevent Hantavirus propagation. Instead, the treatment is restricted to supportive procedures to keep life-threatening symptoms under control (Linderholm and Elgh 2001). Suppression of pathogenic genes via nucleic acid-based reagents holds great promises as novel therapeutic approach against a wide variety of diseases, including infectious diseases, cancer, and genetic disorders. In this regard, antisense oligonucleotides and more recently, small inter- fering RNAs have also been used (Corey 2007; Dorsett and Tuschl 2004). However, a major challenge to the development of therapeutic nucleic acid drugs is specific and efficient in vivo delivery to target cells. To enhance the therapeutic efficiency, delivery of these innovative therapeutic agents into the cytosol of target cells is required. Recent studies suggest that specific gene silencing in vivo can be achieved by combining these nucleic acid-based reagents with cell type- specific internalizing antibodies. The antibody-directed thera- peutic complex enters target cells through receptor endocytosis and is subsequently released into the cytosol to specifically silence target gene expression. Antibody fragments fused with a small nucleic acid-binding protein and antibody fragment- directed nanoimmunoliposomes are two examples of effective delivery vehicles in vivo (Liu 2007). To achieve targeted and intracellular delivery of thera- peutic genes, antibodies with well-defined cell type-specific binding and internalizing capacity are required. Recombinant antibody technology now allows researchers to engineer low- cost antibodies with specificity and high binding affinity. Single-chain Fv antibody fragments (scFv) are polypeptides in which the variable domains of immunoglobulin heavy (VH) and light (VL) chain can be connected via a flexible polypeptide linker (Bird et al. 1988). As a delivery vehicle of therapeutic agents, scFv antibody offers several advan- tages over monoclonal antibody (MAb), e.g., efficient tissue Appl Microbiol Biotechnol (2010) 86:1067–1075 penetration due to their reduced size (∼30 kDa). Small recombinant antibodies can be expressed in Escherichia coli, providing an economic and simple method for large-scale production. Binding affinity and internalization of recombi- nant antibodies can be improved by mutagenesis in complementarity-determining regions (CDRs) and selection via phage display (Jackson et al. 1995; Yang et al. 1995; Schier et al. 1996) and fusion to protein transduction domain (Joliot and Prochiantz 2004; Lindgren et al. 2000). Therefore, scFvs provide useful tool for targeted and intracellular delivery of therapeutically effective molecules. Hantavirus envelop GPs are transmembrane proteins, which are normally located on the surface of virus-infected cells (Ogino et al. 2004). Thus, they become a suitable target for antibody-mediated delivery of therapeutics. In this study, the generation of a soluble recombinant HTNV GP antibody, scFv3G1, derived from hybridoma cells and the purification of bacterially expressed scFv3G1 were described, and the cell type-specific binding and internalizing properties of this single-chain antibody were evaluated. Materials and methods Cells The African green monkey kidney epithelial cell-derived Vero E6 cell line (Vero C1008, ATCC CRL 1586) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibro-BRL), supplemented with 10% heated inactivated fetal bovine serum (FBS) in a 5% CO2 humidified incubator. Hybridoma cell line (clone 3G1) and hybridomas producing anti-HTNV GP monoclonal antibody (MAb3G1) were provided by Dr. ZK Xu (Luo et al. 2003; Hu et al. 2005). The mouse hybridoma cell line 3G1 was propagated for scFv construction. Single-chain Fv antibody fragment construction Total RNA was isolated from about 107 freshly subcloned hybridoma 3G1 cells using Trizol Reagent (Gibco). The first-strand cDNA was synthesized from total RNA using SuperScript™ II Reverse Transcriptase and oligo (dT) primer (Invitrogen) according to the manufacturer’s protocol. cDNAs encoding the antibody variable domains (VH and VL) were amplified by separate polymerase chain reaction (PCR) under standard conditions, performed for 30 cycles, using Taq DNA polymerase (Invitrogen) in a thermocycler (Perkin Elmer, PE2400). The VH coding regions were amplified with primers VHRev and VHFor, while the VL fragments were obtained by PCR using primers VLRev and VLFor. Primers sequences are listed in Table 1. The amplified DNAs were cloned into the pGEM-T vector (Promega). The constructs