Expression of intracellular domain of epidermal growth factor receptor and generation of its monoclonal antibody.
To prepare monoclonal antibody specific to epidermal growth factor receptor (EGFR) intracellular domain, its gene was amplified from total RNA of A431 cell by RT-PCR. Then the gene was cloned into prokaryotic vector pET30a(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni2+-NTA agarose. Then the anti-EGFR monoclonal antibody (mAb) was prepared with classical hybridoma technique. Positive clones were selected using indirect enzyme-linked immunoabsorbent assay (ELISA). Totally 4 hybridoma clones were obtained and these mAbs were IgG1 (3 clones) and IgG2a (1 clone), respectively. Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line. The mAbs will be useful in the study of EGFR-mediated signal transduction.
Lin
Ying
Y
Laboratory of Molecular Immunology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Liu
Zhiduo
Z
Jiang
Jianmin
J
Jiang
Ziqing
Z
Ji
Yongyong
Y
Sun
Bing
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors