The Preparation And Evaluation Of Anti-EGFR Nanobody

Anti-EGFR nanobody has potential value in cancer diagnosis and therapeutics for its better solubility, stability and easiness to expression as fusion protein compared with monoclonal antibody. Nevertheless, high yield and activity of anti-EGFR nanobody is still hard to obtain currently which limit the progress in anti-EGFR study. In the present study,5 gene sequences modified anti-EGFR nanobodies were successively expressed and purified as inclusion bodies. Furthermore, the yield of soluble nanobodies was further raised through optimizing renaturation process. The overall evaluation of the nanobodies’ binding activity and recognizing ability confirmed its practicability. The content of the present study includs:Induced expression of the anti-EGFR nanobodies:in the current study,5 anti-EGFR nanobodies were expressed in E.coli BL21 expression strain using gene engineering approach. The effect of inducer IPTG concentration, induction timing and duration of the induction period on expression level was studied. The optimal expression condition was that at 37℃,0.4 mM IPTG was injected to the 4h cultured strain and then treating for 4 h. In the optimal condition, the expression level of anti-EGFR nanobodies was about 25% of total protein.The purification and renaturation of anti-EGFR nanobodies:6×His tag was cloned to the C-terminal of anti-EGFR nanobodies. The purity of anti-EGFR nanobodies was significantly raised to greater than 90% using immobilized metal affinity chromatography and gradient imidazole elution (100 mM and 300 mM imidazole). Furthermore, dialysis and dilution renaturation were studied comparatively considering the yield and activity of soluble nanobodies. Bradford assay and cell Elisa were used respectively. Then,6 factors (initial protein concentration, adding velocity, additives, concentration of Arg and GSH:GSSH) affecting the efficacy of dilution renaturation were studied. The optimal renaturation condition was that initial concentration of nanobodies was 0.3-0.4 mg/mL, pH=8.0, additive was 200 mM Arg and GSH:GSSH=5:1.The activity and recognition ability of anti-EGFR nanobodies:The binding activity of 5 anti-EGFR nanobodies was evaluated and confirmed using cell Elisa and nanobody-C had the highest binding activity. Furthermore, the recognition of anti-EGFR nanobody to EGFR receptor at the surface of A431 was confirmed using indirect immunofluorescence and MTT assay. The mechanism study showed that anti-EGFR nanobody could competitively binding EGFR receptor with EGF and significantly reduced the proliferation of A431 (the inhibition efficiency was about 75%at 5 μmol/L nanobody).As indicated above, the procedure of the present study could be used in fast, efficient and large scale preparation of anti-EGFR nanobodies. The study also laid the foundation to nanobody based recognition and drug delivery in the future and provide mechanism insight in anti-EGFR nanobodies studies and promote the application in cancer diagnosis and therapeutics.