Production and characterization of a recombinant single-chain antibody against Hantaan virus envelop glycoprotein

Abstract

Hantaan virus (HTNV) is the type of Hantavirus causing hemorrhagic fever with renal syndrome, for which no specific therapeutics are available so far. Cell type-specific internalizing antibodies can be used to deliver therapeutics intracellularly to target cell and thus, have potential application in anti-HTNV infection. To achieve intracellular delivery of therapeutics, it is necessary to obtain antibodies that demonstrate sufficient cell type-specific binding, internalizing, and desired cellular trafficking. Here, we describe the prokaryotic expression, affinity purification, and functional testing of a single-chain Fv antibody fragment (scFv) against HTNV envelop glycoprotein (GP), an HTNV-specific antigen normally located on the membranes of HTNV-infected cells. This HTNV GP-targeting antibody, scFv3G1, was produced in the cytoplasm of Escherichia coli cells as a soluble protein and was purified by immobilized metal affinity chromatography. The purified scFv possessed a high specific antigen-binding activity to HTNV GP and HTNV-infected Vero E6 cells and could be internalized into HTNV-infected cells probably through the clathrin-dependent endocytosis pathways similar to that observed with transferrin. Our results showed that the E. coli-produced scFv had potential applications in targeted and intracellular delivery of therapeutics against HTNV infections.

Fig. 1

Construction of HTNV GPs targeting scFv3G1. a PCR for the construction of scFv antibody-encoding gene. The genes encoding the variable heavy chain (V_H) and variable light chain (V_L) fragments were amplified and assembled via a short nucleotide linker in a V_H-linker-V_L format. The amplified products were resolved in 1.5% agarose gel and stained with ethidium bromide. DNA markers are given in base pairs indicated at the left. b Nucleotide and deduced amino acid sequences of scFv3G1. The linker fragment (Gly4Ser)3 is shown in italics. The complementarity-determining regions (CDRs) of the V_H (H-CDR) and V_L (L-CDR) domains are underlined, and the sequences were identical to those reported in the Kabat database. The four conserved Cys are indicated by stars

Fig. 2

Expression and purification analysis. Escherichia coli BL21(DE3) was transformed with the pET32a-scFv3G1 construct. After induction, cytoplasmic proteins were extracted following sonication. Cytoplasmic fraction was purified by immobilized metal affinity chromatography. Bacterial cytoplasmic extract and purified recombinant scFv3G1 antibody were subjected to 15% SDS-PAGE, stained with Coomassie brilliant blue (a) or transferred to PVDF membrane for Western blot analysis using anti-His-Tag antibody (b). c Western blot analysis of immunoreactivity of scFv3G1. HTNV GP or unrelated HTNV NP was transferred to PVDF membrane, incubated with scFv3G1, and followed by incubation with HRP-conjugated anti-His-Tag antibody. Molecular weight markers are given in kilodaltons indicated on the left

Fig. 3

HTNV GP antigen-specific binding characteristics of scFv3G1. a Solid-phase binding of purified scFv3G1 to HTNV GP antigen as measured by ELISA. The wells were coated with HTNV GP or HTNV NP and then incubated with purified twofold serially diluted scFv3G1 from 1,000 to 0.1 nM. b To compare the HTNV GP-specific antigen-binding activity of scFv3G1 to that of MAb3G1, serially diluted scFv3G1 and MAb3G1 were assayed. Results are plotted as percentages of HTNV GP binding. c Flow cytometric analysis to evaluate whether scFv3G1 can bind HTNV GP antigen expressed on the cell membrane. Ten-day HTNV-infected Vero E6 cells or uninfected cells were incubated with 100 nM purified scFv3G1 and followed by detection with FITC-conjugated anti-His-Tag antibody

Fig. 4

Internalization of scFv3G1. Vero E6 cells infected with HTNV for 10 days were incubated with FITC-labeled scFv3G1 or FITC-labeled MAb3G1 in the absence (a) and in the presence (b) of Texas red-labeled transferrin as an indicator of clathrin-dependent endocytosis at 37°C for 30 min to allow internalization. Nuclei were counterstained with DAPI. Immunofluorescence was observed under confocal microscopy. The colocalization of the scFv3G1 antibody with clathrin-coated endocytic vesicles are indicated by arrows. Scale bar 20 μm