Expression Of Xenogeneic Homologous EGFR Ectodomain In Pichia Pastoris And Its Antitumor Efficacy

Objective Epidermal growth factor receptor(EGFR) is a member of the src family receptor tyrosine kinase composed of an ectodomain juxtaposed a transmembrane domain to the cytoplasmic kinase entodomain.Evaluation of EGFR as therapeutic targets for tumors which surface express EGFR is highly warranted nowadays.To test this concept,we developed a secreted expression system of xenogeneic homologous Epidermal growth factor receptor(EGFR) ectodomain using P.pastoris.We found that immunotherapy with a vaccine based on xenogeneic homologous EGFR was effective at therapeutic anti-tumor immunity. Methods In order to construct the secreted expression vector pPICZαA-mer and pPICZαA-her, we recombined the cDNA of xenogeneic(human) EGFR ectodomain(her) or corresponding control mouse EGFR ectodomain (mer) with the empty plasmid pPICZαA,respectively. Then the recombinant plasmids were linearized by SacI and transformed into P.pastoris strain GS115 by electroporation. The positive transformants were selected on YPDS plate including Zeocin and induced to express by methanol. Consequently, the proteins in the culture supernatant were assayed with SDS-PAGE and Western-blot. So we canchoose the high-level secreted expression recombinant strain to produce protein vaccine vastly.Then we purified the aimed protein of EGFR using the methods of gene engineering. The hepal-6 hepatoma model was established in C57BL/6 mice. Mice were immunized with a certain dose of protein vaccine with the Al(0H)3 adjuvant by injection once a week for 4 weeks. Additional control mice were injected with normal saline and adjuvant. And Rabbits were immunized with a certain dose of protein vaccine with the Al(0H)3 adjuvant by injection once a week for 4 weeks. Results We got the secreted expression of mouse and human EGFR ectodomain in Pichia pastoris. The relative Molecular mass of the two aimed proteins is both about 95 X 103.The Western-blot analysis demonstrated that the expression proteins have much good antigenicity and specificity. The therapeutic efficacy of aimed protein EGFR was tested in the established hepatoma model. The mice were treated starting at day 3 after the injection of hepal-6 hepatoma cells, when the tumor was palpable. Treatment with EGFR once a week resulted in significant anti-tumor activity.And I've got lots of antibodies of Rabbits to mice,which appear to anti-tumor activity in ex vivo suppression experiments. Conclusion The recombinant plasmids pPICZaA-me/- and pPICZaA-her could be induced to express the EGFR aimed proteins by methanol in methanol-trophic yeast expression system.The present findings provide evidence of the anti-tumor activity of the recombinant protein EGFR, and may be of importance for the further exploration of the application of this protein in specific active immunotherapy of tumors.