Appl Microbiol Biotechnol (2010) 86:1067–1075 Table 1 List of primers used for the generation of synthetic genes encoding scFv3G1 1069 VHRev VHFor VLRev VLFor VHRevEcoRI VHLinkFor VLLinkRev VLForSalI (GC)AGGC(GC)CAGCTGCAG(GC)AGTC(AT)GG GGTCCCTTGGCCCC GACATT(GC)(AT)G(AT)TGAC(GC)CAGTCTCC ACGTTTGATCTCGAGCTTGGTCCC TTTGAATTCGAGGCGCAGCTGCAGGAGTC TCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGGTCCC GGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGTGATGACGCAGTCT TTTGTCGACACGTTTGATCTCGAGCT Restriction sites EcoRI and SalI used for cloning the scFv are underlined were then sequenced, and blast of the resulting cDNA sequences and deduced amine acid sequences was performed using the GenBank database and Kabat database (http://www. antibodyresource.com/antibody-database), respectively. A synthetic gene encoding scFv3G1 was amplified by “splice-overlapped extension” (Horton et al. 1989). The genes encoding the variable domains were independently modified in an initial PCR amplification using primers VHRevEcoRI and VHLinkFor for the VH fragment and VLLinkRev and VLForSalI for the VL domain (Table 1). VHLinkFor and VLLinkRev carry overlapping sequences encoding the link peptide (Gly4Ser)3, while VHRevEcoRI and VLForSalI introduce EcoRI and SalI restriction sites at the upstream and downstream ends of the PCR product, respectively. This allows cloning of the amplified fragment into the corresponding sites of pET32a vector (Novagen), a prokary- otic expression vector harboring a thioredoxin gene and two groups of polyhistidine sequences to facilitate disulfide bond formation, solubility, and purification of recombinant proteins. Recombinant scFv antibody expression and purification A single clone pET32a-scFv3G1 was used to transform fresh competent E. coli BL21 (DE3) strain. Colonies were grown in LB medium supplemented with 100 μg/mL ampicillin at 37°C until OD600 reaches approximately 0.4–0.6. Then, bacteria were induced for production of scFv3G1 with 0.2 mM IPTG, and the temperature was shifted to 30°C for 3 h. Bacteria from cultures were centrifuged, and the cytoplasm was extracted after sonication. The recombinant protein scFv3G1 was purified using the His-Bind purification kit (Novagen) accord- ing to the manufacturer’s instructions. Fractions containing the recombinant protein were pooled, concentrated by ultrafiltra- tion (Millipore Corp.), and stored at −20°C until use. The protein concentration was determined using the BCA assay kit (Pierce) with bovine serum albumin (BSA) as a standard. SDS-PAGE and Western blot analysis Proteins were separated on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels using Mini-Protein 3 system (Bio-Rad Laboratories) according to the manufacturer’s instructions. Samples were mixed with an equal volume of 2× Laemmli buffer (S3401, Sigma- Aldrich) and heated at 95°C for 5 min. The sample of 15 μL protein (or cell lysate) or 10 μL low molecular weight protein marker (Pharmacia) was loaded per lane. The gels were run at 60 mA for 45 min, and resolved protein samples were visualized by staining with Coomassie brilliant blue R-250 (Sigma-Aldrich). The crude cytoplasmic prepara- tion and the purified protein were transferred to a polyviny- lidene difluoride membrane (PVDF; Amersham Biosciences) using semidry Western blot transfer apparatus (Bio-Rad Laboratories). The membranes were blocked with 3% BSA (Sigma-Aldrich) in PBS supplemented with 0.05% [v/v] Tween-20 (PBST) for 1 h at room temperature, then washed five times with PBST, and incubated with mouse anti-His- Tag antibody as a primary antibody (Qiagen) at 1:3,000 dilution overnight at 4°C. Then the membranes were washed again and incubated with HRP-conjugated goat anti-mouse IgG as a second antibody (Abcam) at 1:3,000 dilution in PBST at room temperature for 90 min. After washing with PBST, the reaction product was visualized with enhanced chemiluminescence (Amersham) for 5–10 min at room temperature in the dark. For analysis of immunoreactivity of scFv3G1, HTNV GP or unrelated Hantavirus nucleocapsid protein (HTNV NP) was transferred to a PVDF membrane, incubated with scFv3G1 at 100 nM overnight at 4°C, and followed by incubation with either HRP-conjugated anti-His- Tag antibody (Abcam) or HRP-conjugated goat anti-mouse IgG. MAb3G1 was used as positive control while HBsAg- specific scFv15 antibody acted as negative control (Wen et al. 2007a, b). Indirect ELISA Polystyrene plates (96 wells; Nalge Nunc) were coated overnight at 4°C with HTNV GP or unrelated HTNV NP antigen at 1:10 dilution in 100 µL 0.1 M carbonate– bicarbonate buffer (pH 9.6). After three washes with PBST, the wells were blocked with 200 µL of 1% BSA in PBST for 1 h at room temperature and washed as above. The