Expression And Puriifcation Of Anti-Foot-and-Mouth Disease Virus Asia I Type Single-Chain Variable Fragment

scFv (single chain fragment variable) antibodies have been constructed mainly from hybridoma,spleen cells from immunized mice, and B lymphocytes from human. scFv (single chain fragmentvariable) is a noncovalent heterodimer comprised of the VH and VL domains in which can then beused in the construction of recombinant scFv (single chain fragment variable) antibody. In order toattain them, mRNA is first isolated from hybridoma (or also from the spleen, lymph cells, and bonemorrow) followed by reverse transcribed into cDNA to serve as a template for antibody genesamplification (PCR).With this method, large libraries with a diverse range of antibody VH and VLgenes could be created. An antibody in scFv (single chain fragment variable) format consists of variableregions of heavy (VH) and light(VL) chains, which are joined together by a flexible peptide linker thatcan be easily expressed in functional form in E.coli, allowing protein engineering to improve theproperties of scFv (single chain fragment variable) such as increase of affinity and alteration ofspecificity.The scFv cDNA fragments were ligated to T7Select10-3b vector. After packing in vitro, it wastransformed to BLT5403cells to construct the T7phage display library. The recombinant eukaryoticexpression plasmid named pEGFP-N1-scFv was constructed and transfected into CHO cells usingLipofectamineTM2000Transfection Reagent.The observation result through the inverted fluorescencemicroscope verified that scFv gene was expressed successfully in CHO cells, and scFv protein wasmainly localized in cytoplasm of CHO cells by confocal microscopy.The gene of anti-FMDV-scFv wasampificated by PCR, and then cloned into the plasimd pSMK. The prokaryotic expression plasmidofanti-FMDV-scFv gene has been successfully constructed. The pSMK-scFv fusion protein could beexpressed in prokaryotic expression system of E. coli. The expression of pSMK-scFv fusion protein wasinduced with IPTG at16℃, and the products were analyzed by SDS-PAGE.Purified recombinantproteins was detected by ELISA, and the result shows that the expression pruducts have affinity andspecificity with AsiaⅠFMDV146S antigen.In this study, the active and functinal scFv protein was expressed and detected by ELISA usingAsiaⅠFMDV146S antigen.The result shows that expressed scFv have efficient affinity and specificitywith AsiaⅠFMDV146S antigen, and can be used for the development of rapid diagnostic techniques ofFMD.