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Abstract: 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 chain (Vh) and light chain (Vl) were amplified, and the Vh and modified Vl 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 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 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.

[1] Casey, J.L., Coley, A.M., Tilley, L.M., Foley, M., 2000. Green fluorescent antibodies: Novel in vitro tools. Protein Engineering, 13:445-452.

[2] 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).

[3] David, G.M., Kaija, A., Kirsi, O., 2000. Baculoviral display of functional ScFv and synthetic IgG-binding domains. Biochemical and Biophysical Research Communications, 275(1):84-90.

[4] Gao, Y.D., Xiong, D.S., Peng, H., 2003. Expression and characterization of recombinant ScFv antibody detecting p-glycoprotein. Chinese Journal of Immunolog, 20(4):267-271 (in Chinese).

[5] George, N.P., 1997. Structrue and dynamics of green fluorescent protein. Current Opinion in Structure Biology, 7:821-827.

[6] Gerard, W., Andreas, P., 1999. The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli. Protein Engineering, 12:605-611.

[7] Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling. Electrophoresis, 18:2714-2723.

[8] Haraguchi, T., Ding, D.Q., Yamamoto, A., Kaneda, T., Koujin, T., Hiraoka, Y., 1999. Multiple-color fluorescene imaging of chromosomes and microtubules in living cells. Cell Struct. Funct., 24:291-298.

[9] 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).

[10] Joseph, S., David, W.R., 2001. Molecular Cloning. Cold Spring Harbor Press, New York, American, p.96-99.

[11] Jung, S., Honegger, A., Plückthun, A., 1999. Selection for improved protein stability by phage display. J. Mol. Biol., 294(1):163-180.

[12] Mark, A., Remko, A.G., Jan, W.B., 2000. Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein. J. Biol. Chem., 275:17556-17560.

[13] Mccall, A.M., Adams, G.P., Amoroso, A.R., 1999. Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific ScFv that triggers CD16-dependent tumor cytolysis. Molecular Immunology, 36(7):433-446.

[14] Mclean, A.J., Bevan, N., Rees, S., Milligan, G., 1999. Visualizing differences in ligand regulation of wild-type and constitutively active mutant β₂-adrenoceptor-green fluorescent protein fusion proteins. Mol. Pharmacol., 56:1182-1191.

[15] Myers, K., Ryan, M.G., Stern, P.L., Shaw, D.M., Embleton, M.J., Kingsman, S.M., Carroll, M.W., 2002. Targeting immune effector molecules to human tumor cells through genetic delivery of 5T4-specific ScFv fusion proteins. Cancer Gene, 9(11):884-896.

[16] Ogawa, H., Inouye, S., Tsuji, F.I., Yasuda, K., Umesono, K., 1995. Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells. Proc. Natl. Acad. Sci. USA, 92(25):11899-11903.

[17] Orlandi, R., Gussow, D.H., Johns, P.T., 1989. Cloning immunoglobulin variable domain for expression by the polymerase chain reaction. Proceedings of the National Academy of Sciences, 86(10):3833-3837.

[18] Otsuki, M., Fukami, K., Kohno, T., Yokota, J., Takenawa, T., 1999. Identification and characterization of a new phospholipase C-like protein, PLC-L₂. Biochem. Biophys. Res. Commun., 266:97-103.

[19] Peitsch, M.C., 1995. Protein modelling. Bio/Technology, 13:658-660.

[20] Prasher, D.C., Echenrode, V.K., Ward, W.W., 1992. Primary structure of the Aequorea victorial green fluorescent protein. Gene, 111:229-233.

[21] Schwede, T., KOPP, J., Guex, N., Peitsch, M.C., 2003. MC SWISS-MODEL: An automated protein homology-modelling server. Nucleic Acids Research, 31:3381-3385.

[22] Shi, H.D., Su, W.W., 2000. Display of fluorescent protein on Escherichia coli cell surface. Enzyme and Microbial Technology, 28(2001):25-34.

[23] Tina, K., Dirk, M.N., Tina, V., 2004. Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: A comparative analysis of bacterially expressed single-chain diabody and tandem ScFv. Journal of Gene Medicine, 6:642-651.

[24] Walker, D., Htun, H., Hager, G.L., 1999. Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells. Methods Companion Methods Enzymol, 19:386-393.

[25] Youvan, D.C., Michel, B.M., 1996. Structwe and flurescence mechanism of GFP. Nat. Biotechnol., 14(10):1219-1220.

[26] Zhu, H.Y., Yamada, H., Jiang, Y.M., Yamada, M., Nishiyama, Y., 1999. Intracellular localization of the UL31 protein of herpes simplex virus type 2. Arch. Virol., 144:1923-1935.