Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag
LU Min , GONG Xing-guo YU Hong LI Jian-yong (Institute of Biologic Macromolecule and Enzyme Engineering, School of Life Science, Zhejiang University, Hangzhou 310027, China)
Total RNA was isolated from the hybridoma cell line (LC-1), which secretes anti-lung adenocarcinoma monoclonalantibody, and was transferred into cDNA. Based on the FR1 (framework region 1) and FR4 conserved regions of LC-1 gene, thevariable regions of heavy chain (Vh) and light chain (Vl) were amplified, and the Vh and modified Vl were connected to singlechain Fv (ScFv) by SOE-PCR (splice overlap extension PCR). The modified ScFv was fused with green fluorescent protein (GFP)and introduced into E. coli JM109. The fusion protein induced by IPTG (Isopropylthiogalactoside) was about 57000 on a 10%SDS-PAGE gel (10% Sds Polyacrylamide Gel Electrophoresis), and primarily manifested as inclusion bodies. The renaturedprotein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-1 lung adenocarcinoma. In addition, theinduced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activitywas expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detectimmunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.