Islamic Azad Univ;
Fac Basic Sci;
Immunol Res;
Drug Appl Res Ctr;
Tabriz;
Maragheh;
Biotechnol Res Ctr;
TB & Lung Dis Res Ctr;
Ahar Branch;
Maragheh Fac Med Sci;
Tabriz Univ Med Sci;
Dept Microbiol;
Iran;
Ahar;
关键词：
Recombinant expression;
Single domain antibody;
EGFR;
期刊名称：
Protein Expression and Purification
i s s n：
1046-5928
年卷期：
2016 年
120 卷
页 码：
59-64
页 码：
摘 要：
Epidermal growth factor receptor (EGFR) plays an important role in cell growth, multiplication and differentiation. Over expression of EGFR is associated with carcinogenesis and seen in variety of cancers. Anti-EGFR monoclonal antibodies can block EGFR downstream signaling pathway resulting in inhibition of uncontrolled cell proliferation. Antibody fragments have a variety of advantages. In comparison to full length antibodies they have smaller size and therefor exhibit better tumor penetration ability. The aim of this study was to prepare a single domain antibody to target extracellular domain of EGFR. mRNA was extracted from C225 hybridoma cells producing anti-EGFR antibody and subjected to reverse transcription reaction (RT-PCR) to obtain cDNA molecules encoding VH domain of mAb C225. The cDNA encoded VH domain was in frame introduced into the pET-22b(+) vector and expressed in BL21 (DE3) bacterial cells. The resultant antibody was purified via Ni- NTA column and its reactivity was assessed by ELISA and western blot techniques using A431 cell lysate. Analysis by ELISA revealed that this single domain antibody was able to bind EGFR on A431cells. This result was further confirmed by western blotting. In conclusion, the results of this study indicated that single domain antibody can identify and bind to EGFR of A431 carcinoma cells. This recombinant fragment antibody would potentially be used for targeting of cancer cells with high EGFR expression. (C) 2015 Elsevier Inc. All rights reserved.
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