Lin Ying, Liu Zhiduo, Jiang Jianmin, Jiang Ziqing, Ji Yongyong, Sun Bing
Laboratory of Molecular Immunology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Cell Mol Immunol. 2004 Apr;1(2):137-41.
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.
为制备针对表皮生长因子受体（EGFR）胞内结构域的单克隆抗体，通过逆转录聚合酶链反应（RT-PCR）从A431细胞的总RNA中扩增其基因。然后将该基因克隆到原核载体pET30a(+)中。将重组质粒转化到大肠杆菌BL21(DE3)菌株中进行蛋白表达。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导重组蛋白表达，并使用镍离子-亚氨基二乙酸（Ni2+-NTA）琼脂糖进行纯化。然后采用经典杂交瘤技术制备抗EGFR单克隆抗体（mAb）。使用间接酶联免疫吸附测定（ELISA）筛选阳性克隆。共获得4个杂交瘤克隆，这些单克隆抗体分别为IgG1（3个克隆）和IgG2a（1个克隆）。它们的轻链均为κ链。蛋白质印迹分析和共聚焦免疫荧光测定表明，这些单克隆抗体能够特异性识别A431癌细胞系上表达的EGFR。这些单克隆抗体将有助于EGFR介导的信号转导研究。
