Plasmid Contruction And Expression Of Anti-NDV Single-Chain Fragment Variable Antibody

Newcastle disease (ND) is avian respiratory disease that caused by Newcastle disease virus (NDV). ND poses a significant economic threat to the poultry industry worldwide because it can result in high mortality in susceptible hosts T Currently no effective treatment available when the chicken are infected by NDV. Single-chain fragment variable (scFv) antibody is the smallest functional unit of immunoglobulin retaining the antigen-binding activities and could be express in vertebrate and invertebrate. It could be used in medical diagnosis and therapy by neutralizing various virus. In this study, we designed and contructed two eukaryotic expression vectors of anti-NDV scFv, named pB315B-CAG-puro-pGFP-scFv and pB315B-EF1?-puro-pGFP-scFv which were driven by CAG and EF1? promoter, respectively, using a PiggyBac transposal backbone.Two cell lines, DF-1 and 293T were transfected using LipofectamineTM2000 and a total of four stable cell lines stably expressing the above two vectors were established:DF-1-CAG-scFv?293T-CAG-scFv? DF-1-EF1?-scFv and 293T-EF1?-scFv. The transfection efficiency in 293T cell is 25.8%, higher than in DF-1 cell with 19.1%. RT-PCR results indicated that the gene of anti-NDV-scFv antibody was expressed in all of four cell lines. Then, we detected the scFv in protein level by western-blot using antibody against His-tag protein, which was fused with anti-NDV-scFv antibody.Cell lysates from all four cell lines were negative for His-tag in the western-blot, whereas supernatant collected from the culture of DF-1-EFla-scFv and were positive. Moreover, the scFv level in supernatant from DF-1-EF1?-scFv was higher than DF-1-CAG-scFv. We then challenged the cells with 6,60 and 600 times for TCID50 of NDV and evaluate their resistant to the virus. The results reveale that more significant cytopathic effect was observed in DF-1 (control) and DF-1-CAG-scFv cells than DF-1-EFla-scFv in 6x and 60x TCID50.This study suggests that EF 1 apromoter was better than CAG promoter to express anti-NDV scFv antibody in DF-1 cell line. Anti-NDV scFv expressed in DF-1 demonstrated neutralizing activity and enhance the resistant of the cells to NDV.We transfected chicken primordial germ cell (PGCs) with the anti-NDV-scFv plasmid drived EFla promoter,the transfection efficiency was 2.9% after 24 h,and had established PGCs cell line stably expressed anti-NDV-scFv through puromycin screening. Our study indicates its potential use in generation of transgenic chicken for production of anti-NDV scFv.