Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag
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Total RNA was isolated from the hybridoma cell line (LC-1), which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FR1 (framework region 1) and FR4 conserved regions of LC-1 gene, the variable regions of heavy chian (Vh) and light chain (VI) were amplified, and the Vh and modified VI were connected to single chain Fv (ScFv) by SOE-PCR (splice overlap extension PCR). The modified ScFv was fused with green fluorescent protein (GFP) and introduced intoE. 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 renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-1 lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.
Key wordsScFv (single chain variable fragment) GFP (green fluorescent protein) tag Protein fusion Purification
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- Cheng, H., Liu, Y.F., Zhang, H.Z., Shen, W.A., Zhang, S.Z., 2001. Constrution and expression of eukaryotic vector with recombinant secratory anti-liver cancer double fuction antibody and GFP fusion gene.World Chinese Journal of Digestion,9(6): 640–644 (in Chinese).Google Scholar
- Gao, Y.D., Xiong, D.S., Peng, H., 2003. Expression and characterization of recombinant ScFv antibody detecting p-glycoprotein.Chinese Journal of Immunology,20(4): 267–271 (in Chinese).Google Scholar
- Hu, X., Lu, P., Wang, L.F., 1999. Expression and analysis of recombinant human IL-18 and human IL-18-GFP fusion protein.Chinese Journal of Microbiology and Immunology,19(4): 306–310 (in Chinese).Google Scholar
- Joseph, S., David, W.R., 2001. Molecular Cloning, Cold Spring Harbor Press, New York, American, p. 96–99.Google Scholar
- Shi, H.D., Su, W.W., 2000. Display of fluorescent protein on Escherichia coli cell surface.Enzyme and Microbial Technology,28(2001): 25–34.Google Scholar