Construction And Expression Of Foot-and-Mouth Disease Virus Single-Chain Variable Fragment

Single-chain variable fragment (scFv) is a small molecular antibody containing the variable regions of the heavy chains (VH) and light chains (VL) of immunoglobulins, which were connected with a short Linker peptide of10-25amino acids. Despite removal of the constant regions and the introduction of the Linker, the antibody keeps the specificity of the immunoglobulin. ScFvs were created to facilitate phage display or ribosome display technology, where it is highly convenient to express the antigen-binding domain as a peptide. As an alternative antibody, variable genes of heavy chains and light chains of scFv can be subcloned directly from a hybridoma or B lymphocyte. ScFvs can be used in many ways, e.g., as antigen-binding domains of artificial T cell receptors, being used in FMC (Flow cytometric analysis) or immunohistochemistry, and have a considerably prospect for application in the prevention and detection of biological pathogens, parasites and microbial contamination.The VH and VL used in this study were amplified by PCR, which belong to variable genes of heavy chains and light chains of immunoglobulins respectively collected from Balb/c mice, and FMDV scFv genes library with a capacity of5.74×1013were constructed by SOE-PCR using a flexible Linker-(Gly4Ser)3. Antibody light chain constant region of Balb/c mice, which was amplified by PCR and have a similarity of100%with the sequences in GenBank, is as a spacer for constructing ribosome display library. Ribosome display library with a storage capacity of9.81×1013of FMDV scFv were builded up through a series of PCR and SOE-PCR based on the conditions of constructing scFv successfully. Ribosome display elements are complete, there is diversity between the target sequences and these sequences can be amplified after analysis the library. The mRNA-ribosome-scFv complexes were obtained after transcription and translation of the ribosome display library in vitro. These complexes were demonstrated by SDS-PAGE. The mRNAs were obtained from the mRNA-ribosome-scFv complexes by solid affinity selection where the FMDV antigens or purified146S virus particles were used as ligands, and then were used to amplify by RT-PCR for a DNA library. The DNA library was used in the next ribsome display, and then scFvs were obtained after the fifth round of selection. The recombinant plasmid pET-scFv was successfully constructed and transformed into BL21(DE3) cell of prokaryotic expression. The new band was observed in the SDS-PAGE gel whose wight is approximately32KD after the transformants were induced by IPTG. Western-blot results further showed that the expression of scFv.According to the characteristics of scFv, mRNA was extracted from spleen cells separated from the Balb/c mice which were inoculated with purified146S FMDV particles in this study. FMDV scFv genes library were constructed successfully by SOE-PCR and further ribosome display library of FMDV scFv were constructed. It is first to select FMDV scFv from ribosome display library of FMDV scFv through ribosome display technology. ScFv was expressed and preliminary researched. This study can provide basic datum for more research on the prevention and treatment of FMD, also scFv can be used for the development of rapid diagnostic techniques of FMD.