Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

Chemical Research in Chinese Universities ›› 2005, Vol. 21 ›› Issue (2): 191-195.

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Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

WEI Jing-yan¹, LI Shan-yu^1,2, MU Ying³, ZHU Xue-jun³, LIU Lei³, GAO Li-zeng⁴, SONG Da-qian⁵, SUN Zhi-wei⁶, YAN Gang-lin³, ZHANG Han-qi⁵, JIN Qin-han⁵, LI Wei⁴, LUO Gui-min³

1. College of Pharmaceutical Science, Jilin University, Changchun 130021, P. R. China;
2. The first Colloege of Clinical Medicine Affiliated to Jilin University, Changchun 130021, P. R. China;
3. Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, P. R. China;
4. College of Life Science, Jilin University, Changchun 130023, P. R. China;
5. College of Chemistry, Jilin University, Changchun 130023, P. R. China;
6. College of Public Health, Jilin University, Changchun 130021, P. R. China

Received:2004-11-10 Online:2005-03-24 Published:2011-07-27
Supported by:
Supported by the National Natural Science Foundation of China(No.29875010) and the Health Department of Jilin Province of China(No.98047).

Abstract

Abstract: The single chain variable fragments of antibodies(scFvs) against eTnI were screened from the phage display antibody library by using cTnI as the target antigen.After four rounds of panning, four clones(H2, G5,A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing.The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions (PCR)and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins.The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE.The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins.The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules.The association constant (K_A)values range from 1.2×10⁴ to 1.7×10⁵ L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits.These antibodies can be valuable reagents for the immunoassay of cTnI.

Key words: Cardiac troponin I, Single chain variable fragments of antibody(scFv) against cTnI, Phage display, Antibody library

WEI Jing-yan, LI Shan-yu, MU Ying, ZHU Xue-jun, LIU Lei, GAO Li-zeng, SONG Da-qian, SUN Zhi-wei, YAN Gang-lin, ZHANG Han-qi, JIN Qin-han, LI Wei, LUO Gui-min. Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta[J]. Chemical Research in Chinese Universities, 2005, 21(2): 191-195.

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Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

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