Purification Of Polyclonal Antibody Against Type Ⅲ EGF Deletion-mutant Receptor And Its Specificity Investigation

Epidermal growth factor receptor (EGFR) is a membrane glycoprotein with multifunction and is widely distributed in human tissues. EGFR and its ligand is a part of cellular signal transduction pathway. The overexpression of EGFR is an important factor of malignancies proliferation. Mutations in EGFR are commonly found in many types of human tumors, the most common mutation is the type III EGF deletion-mutant receptor (EGFRvIII). Because the EGFRvIII is only expressed on the surface of many kinds of tumor cells, it is an ideal target for tumor therapy.In order to research the role of the EGFRvIII in tumorgenesis, in our studies, the coding sequence of EGFR, EGFRvIII gene, E33XI and E34XI fragment were respectively inserted into pET30a vector and were performed prokaryotic expression and purification to obtain the polyclonal antibody against EGFRvIII. The main achievements of our studies are as following:In the research, pET30a/EGFR, pET30a/EGFRvIII, pET30a/E33XI, pET30a/E34XI prokaryotic expression vectors were constructed with pET30a. After DNA Sequence Analysis, recombinant proteins with the molecular weight of 170kDa,145kDa,19.69kDa and 38.94kDa were expressed after the E.coli were induced by 1mM IPTG at OD₆₀₀ 0.5 and further incubated at 37°C for 4 hours. Then the recombinant proteins were purified through His tag to prove the correctness and performed quantitative analysis.Animals were immunized with the synthesized peptide against EGFRvIII, which was coupled with hemocyanin, and the antiserum against the EGFRvIII were raised. Then the polyclonal antibodies against EGFRvIII were extracted through column purification. Western blot analysis with the His-EGFR, His-EGFRvIII, His-E33 recombinant protein and immunohistochemistry with U251, U87, Hela, 293 cells were performed to test specificity of antibody. In addition, the optimal dilution of antibody for western blot analysis was also performed.