| Epidermal growth factor receptor (EGFR) is a member of ErbB family. It's an important transmembran receptor with tyrosine kinase activity. This family also conteins other there members: HER2/erbB2,HER3/erbB3,HER4/erbB4. Epidermal growth factor receptor (EGFR) protein consist of extracellular domain, transmembrane domain and intracellular domain with tyrosine kinase activity. The extracellular domain can bind of their specific ligands , such as epidermal growth factor(EGF) , then induces receptor activation, modulation of cell proliferation and differentiation in normal tissues and tumors. Research results revel that activation of the EGFR can enhance processes responsible for tumor growth and progression, including the promotion of proliferation, angiogenesis, and metastasis, and inhibition of apotosis[1,2]. The expression of EGFR in tumors has been correlated with disease progression, poor surcival.poor response to therapy[3]. High levels of EGFR hace been observed in a variety of tumors, including prostate, breast, gastric, colorectal, and ovarian[1,2,4]. So targeting EGFR for tumors therapy attracts more and more attention. And there are two strategies which are successful in cancer therapy up to now. They are the monoclonal antibody and the EGFR- tyrosine kinase inhibitors(TKI).Among these agents, Cetuximab, panitumumab, Tarceva(Erlotinib), Iress(aGefitinib)have been approved by FDA for cancer therapy. So the success of these strategies can indicate that the EGFR-targeting therapy is feasible and it may represent a significant contribution to cancer therapy.In this study, the mice are immunized with xenogeneic EGFR ,then the ant-EGFR antibody dan be indced and inhibit the singal transduction, and then the growth of tumor can be inhibited. By searching target gene sequences in GenBank, one set of oligonucleotide primers were chosen from the EGFR ectodomain gene of chicken.The EGFR gene fragment which was 642bp in length was amplified by PCR, inserted into pGEX-4T-2 vetor,. After the sequence identification of the recombinant plasmid, it was transformed into dominant bacteria BL21(DE3). The correct dominant bacteria was chosen by PCR and enzyme restriction digestion.The expression of the fusion protein was induced by IPTG, and then purified by Ni-NTA Agarose.The purified molecular weights were detected by SDS-PAGE. Renaturation of the purified fusion protein was taking by dialyseing, and the fusion proteins'antigenicity was observed by western blot. Fourteen C57BL/6J mice were randomly divided into 2 groups: the fusion protein group and the normal saline(NS) group(7 mice/group). Each mouse of fusion protein group was injected with the 100μg fusion protein by week 0,2,3, and the mice of NS group were injected with 100μL normal saline. Then Lewis lung carcinoma model was established in C57BL/6J mice after 3 times immunization. Tumor growth and mouse's physical status were observed after the injection of tumor cells, and the serum level of specific anti-EGFR antibody were measured by enzyme-linked immunosorbent assay(ELISA).The ectodomain gene fragment of checken EGFR was cloned successfully., and it was ligated into prokaryotic expression vetor. The cEGFR-GST fusion protein was expressed in E.coli BL21(DE3) stably, then purified by Ni-NTA agarose and its purification efficiency was above 85%. .After renaturing in renaturation solution ,the protein exists in solutin form. the fusion protein's antigenieity was proved by westem-Blot analysis. Animal experiment results Animal experiment results indicates that serum anti-EGFR antibody level of the vaccine group was 1:5000 and the fusion protein vaccine showed inhibition effect against Lewis lung cancer cells.This study provide evidence of the anti-tumor activity of the recombinant protein EGFR, and it can break immune tolerance to self-antigens in animal experiment, and may be of importance for the further exploration of the application of this protein in spedific active immunotherapy of tumors.
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