The Experiment Of DNA Aptamers For Epidermal Growth Factor Receptor(EGFR) Based On The SELEX

The epidermal growth factor receptor(EGFR), the product of proto-oncogene c-erb B1, is the member of Erb B/HER family which belongs to the type I transmembrane tyrosine kinase receptors and it is widely distributed on the surface of mammalian epithelial cells,such as fibroblasts cells,glial cells,skin cells,etc. In normal physiology, EGFR plays central role in the development and growth of tissues by moderating cell differentiation and morphology. Overexpression or elevated levels of EGFR activity is associated with many different types of solid cancers,such as glioma, skin, kidney, lung, prostate, pancreatic and breast cancer.The overexpression of EGFR is also associated with proliferation and angiogenesis, invasion and metastasis, and inhibition of apoptosis of malignant cells.At the same time,the overexpression of EGFR concerns with the radiation tolerance of glioblastoma,the radiosensitivity will be increased when EGFR is restrained.Thus,EGFR has become one of the important target for research. Currently,the drugs of anti-EGFR include monoclonal antibodies against EGFR(Cetuximab) and tyrosine kinase inhibitors(Gefitinib). However, there are many innate problems with the use of monoclonal antibodies as drugs, such as difficulty in chemical modifications, and significant immunogenicity because of belonging to protein.This charactetistics of the antibody have limited their use and efficacy.Aptamers, the output of the Systematic Evolution of Ligands by EXponential enrichment(SELEX), are the DNA or RNA oligonucleotide fragments which can bind the protein and other substance with specificity. In contrast to antibodies and peptides, aptamers have the charactrictics of high selectivity and affinity,no-toxicity and no-immunogenicity which become good instrument in protein research,especially in the research for finding the biomarker. Researchers can easily and quickly obtain high specificity and high affinity aptamers by SELEX technology, which show great prospect in basic research of biological medicine and the diagnosis and treatment of clinical aspects.Currently, the aptamers of vascular endothelial growth factor(VEGF) have been approved by the FDA as a drug to treat the age-related macular degeneration. The RNA aptamer of EGFR has been selected, but the use of RNA aptamer will be limited because it is easy to be degradated by nuclease and the selection process is complicated and expensive. Compared to RNA aptamers, DNA aptamers are less expensive and easier to be used and stored, have more inclusive application. In this experiment,we select the aptamer of EGFR using the single-stranded DNA oligonucleotides as the initial library.In this work,we provided the recombinant EGFR(extracellular domain) as target protein which coupled with Ni-beads. On the other hand,Ni-beads acted as the anti-selection protein. The 76 base single-stranded DNA oligonucleotides random library acted as initial library.After 11 rounds of selection, we obtained 22 DNA aptamers that binded the target protein EGFR-beads with high specificity and affinity. We chose one of the aptamers mamed Tu Tu22 as target aptamer to detect their function. The series experiments were carried out to test the binding of aptamer Tu Tu22 with U251,U87,A431 which are the EGFR-positive cells and Jurkat cells which belong to EGFR-negative cells.The results demonstrated that the aptamer Tu Tu22 can combine with the U251,U87,A431 cells with specificity.On the other side, the aptamer Tu Tu22 doesn’t bind the Jurkat cells which don’t express EGFR.Thus,the selected aptamer Tu Tu22 may have wide range of uses in biosensors and nanotechnology with the optimization of Tu Tu22 and it may become the inconceivable drug in novel targeted therapy.