Expression Of Single-chain Variable Fragment(scFv) Against Influenza Virus PB2 And Detection Of Its Activity

Influenza is a highly infectious disease caused by influenza virus,which poses a threat to human health.The prevention and treatment of influenza virus has always been the focus of influenza research.Single-chain variable fragment(scFv)is paid attention for its relatively size,low immunogenicity and strong penetrability.Due to the simplicity of cultivation and low cost,E.coli expression systems is often used to product scFvs.However,scFvs expressed in reducing cytoplasm mainly exist in the form of inclusion body or the production of functional scFvs is low,which limits the application of scFvs.Therefore,we intended to obtain high expression of soluble scFvs in E.coli by fusion expression and established an efficient and low-cost method of preparing anti-influenza scFvs.Previous results have confirmed the property of sf GFP for facilitating the solubility of fusion partner in our laboratory.Therefore,we designed anti-influenza virus H1N1 PB2 scFv fused sf GFP and expressed in E.coli.After purification,the biological activity of sf GFP-scFv-His was detected in vitro and cells.The results showed that sf GFP-scFv-His was more soluble than scFv-His in E.coli.After optimizing the inducing temperature,time and concentration of IPTG,the optimal expression of sf GFP-scFv-His was induction with 0.1m M IPTG for 16 h at 18?.Approximately 20 mg sf GFP-scFv-His was produced from per liter of E.coli culture.The purified scFv-His was obtained from the cleavage of sf GFP-scFv-His with TEV protease and Ni-NTA purification.The results of western blot showed that sf GFP-scFvHis was more stable than scFv-His.These results indicated that sf GFP not only facilitated the solubility of scFvs in E.coli,but also promoted the stability of scFvs.We further detected the immunological activity of anti-PB2 scFvs.The results of western blotting and ELISA shown that anti-PB2 sf GFP-scFv-His specifically bound to PB2.The results of hemagglutination assay and real-time PCR indicated that sf GFP-scFv-His and scFv-His exhibited anti-influenza virus H1N1 activity.We prepared soluble functional anti-influenza virus H1N1 scFvs by fusing sf GFP in E.coli,which provides a theoretical foundation for further developing the anti-influenza application of scFvs.In addition,the study also provides a model of soluble expression of functional scFvs,laying a theoretical foundation for large-scale preparation of anti-influenza scFvs.