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Received: 2005-01-25

Revision Accepted: 2005-04-28

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Journal of Zhejiang University SCIENCE B 2005 Vol.6 No.8 P.832~837


Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag

Author(s):  LU Min, GONG Xing-guo, YU Hong, LI Jian-yong

Affiliation(s):  Institute of Biologic Macromolecule and Enzyme Engineering, School of Life Science, Zhejiang University, Hangzhou 310027, China

Corresponding email(s):   sc9910@hotmail.com

Key Words:  ScFv (single chain variable fragment), GFP (green fluorescent protein) tag, Protein fusion, Purification

LU Min, GONG Xing-guo, YU Hong, LI Jian-yong. Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag[J]. Journal of Zhejiang University Science B, 2005, 6(8): 832~837.

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author="LU Min, GONG Xing-guo, YU Hong, LI Jian-yong",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag
%A LU Min
%A GONG Xing-guo
%A YU Hong
%A LI Jian-yong
%J Journal of Zhejiang University SCIENCE B
%V 6
%N 8
%P 832~837
%@ 1673-1581
%D 2005
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.2005.B0832

T1 - Cloning, expression, purification, and characterization of LC-1 ScFv with GFP tag
A1 - LU Min
A1 - GONG Xing-guo
A1 - YU Hong
A1 - LI Jian-yong
J0 - Journal of Zhejiang University Science B
VL - 6
IS - 8
SP - 832
EP - 837
%@ 1673-1581
Y1 - 2005
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.2005.B0832

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.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


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