Anti-lung, Single-chain Antibody Fusion Construct Of Green Fluorescent Protein Expression And Activity Of Research

A monoclonal antibody is secreted from LC-1,which single chain Fv fragment(ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. ScFv is the smallest segment being able to interact with antigen Because of small molecular weight and lack of Fc,which compares conventional antigen with strong force of penetration and immunogenecity. Up to time the expression of fusion protein on GFP and ScFv has not reported.On the theory,this fusion protein should be with green fluorescent and antibody activity, the expression of protein fusions has been used for tumor monitoring, high-throughput screening drugs or researching the function of the acceptor of certain cell factor.The total RNA was solated from high secretory volume and activity antibody LC-1 monocolony cells, which was transferred cDNA. The variable regions of heavy chain and light chain were cloned through PCR according to ScFv gene, respectively . ScFv' gene was formed after the SOE ligation of VH and modified VL. The ScFv gene amplified by PCR is cloned into pET-22b(+) plasmid between the Nco I/Sal I. and introduced into E.co//BL21 the constructed plasmid gave a high level of 40% expression after IPTG induction. SDS-PAGE analyzed the purity of protein through the purification of Ni-NTA resin. ELISA indicated that ScFv has activity similar to natural antibody.The large preparation of ScFv by gene engineering is against finiteness of ascetic fluid and reduces the cost of production.At the same time The GFP gene is cloned into pET-22b(+) plasmid between the Sal I. /Xho I sites, and subsequently I, the ScFv gene is inserted into the N-terminal of GFP gene between Nco I/Sal I sites, then transforming the recombinant vector into E.coliBL21. The constructed plasmid gave a high level of expression similar to the anticipated protein after IPTG induction. The host cells fluoresce bright green under 485nm wavelenth, ELISA indicated the target protein has the biological function needs further research.