Expression of Intracellular Domain of Epidermal Growth Factor Receptor and Generation of Its Monoclonal Antibody

查看全文 作　　者：YingLin ZhiduoLiu JianminJiang ZiqingJiang Yongyongji [1]BingSun 机构地区：[1]LaboratoryofMolecularImmunology,InstituteofBiochemistryandCellBiology,ShanghaiInstitutesforBiologicalSciences,ChineseAcademyofSciences,Shanghai200031,China 出　　处：《Cellular & Molecular Immunology》索引2004年第1卷第2期,共5页 基　　金：supported by Science and Technology Commission of Shanghai Municipality(03JC14085);grant of National Natural Science Foundation#30170888;the grant from E-institutes of Shanghai Universities Immunology Division. 摘　　要：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 (nAb) was prepared with classical hybridoma technique. Positive clones were selected using indirect enzyme-linked inmunoabsorbent 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. 关 键 词：单克隆抗体 EGFR 原核表达 细胞内反应 表皮生长因子 免疫反应