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EGFR (epidermal growth factor receptor, EGFR) is a member of the ErbB family, having a tyrosine kinase activity, is an important transmembrane receptor. The family members also include HER2/erbB2, HER3 / erbB3, HER4/erbB4. Epidermal growth factor receptor extracellular domain, a transmembrane region and an intracellular kinase activity area. Ligand activation of the tyrosine kinase activity of the receptor intracellular segment, the main function of the extracellular region binding epidermal growth factor EGF the start signaling pathway, has played the role in the regulation of cell proliferation and differentiation in normal tissue and tumor tissue. EGFR an important role to play in the process of tumor progression, it can promote tumor cell proliferation, migration, inhibit apoptosis and promote tumor angiogenesis [1,2]. Clinical studies show that the deterioration of the tumor, the patient's lower survival rates as well as the low sensitivity of the drugs with tumor cells cells express EGFR molecules into positive correlation [3]. Detected in many patients with cancer, including prostate cancer, lung cancer, stomach cancer, colon cancer, ovarian cancer, etc. In recent years, with the development of cancer detection means to the abnormally high levels of expression of EGFR in [1,2,4]. Therefore, EGFR targeted cancer research hotspot. Two of the more successful examples of research is a monoclonal antibody and small molecule tyrosine inhibitors (TKI). Monoclonal antibody Cetuximab panitumumab, TKI aspect of Tarceva (Erlotinib), Iressa (Gefitinib) has been approved by the FDA for the clinical treatment of cancer. The success of these two aspects suggest that treatment strategies targeting EGFR is feasible, and will play a huge role in the field of cancer treatment. In the present study, we tried to xenogeneic EGFR protein on immune, so the mice to produce anti-EGFR antibody to block the EGFR signaling pathway, so as to achieve the anti-tumor effect. Chicken EGFR gene sequence published under GenBank, we designed a pair of specific primers in part of the the 642bp size of the the chicken source EGFR extracellular gene fragments obtained by PCR amplification, and cloned into the expression vector pGEX-4T-2 correct on sequencing, was transformed into the expression strain BL21 (DE3), positive clones were screened by PCR and restriction enzyme digestion, IPTG induction and further through the Ni-NTA Agarose affinity chromatographic purification of the target protein, purified product by SDS-PAGE. The purified product was dialyzed, Western blot the identified antigenic get the GST-cEGFR for immunization fusion protein. The 14 C57BL16J mice were randomly divided into 2 groups, GST-cEGFR seven experimental group and saline control group (NS) 7, the experimental and control mice each were injected 0,2,3 weeks 100μgGST- cEGFR fusion protein and 100μL saline. Inoculated with Lewis lung cancer cells were observed tumor growth and mouse status and detection of specific anti-EGFR antibody titers in mouse serum. Experiment successfully clone a the chicken contains 642bp source EGFR gene constructed prokaryotic expression the recombinant the plasmid pGEX4T2-cEGFR. cEGFR-GST fusion protein was highly expressed in E. coli by the Ni-NTA Agarose purified recombinant fusion protein purity of more than 85%. Refolding, the fusion protein exists in a soluble form, Western blot identification of the fusion protein antigenicity. Animal experimental results show that the the GST-cEGFR vaccine mice serum anti-EGFR antibody titer of 1:5000, and the growth of tumors in mice has some inhibitory effect. This study showed that the heterogeneous recombinant EGFR protein produced through genetic engineering methods, can be induced in mice and anti-EGFR antibody, successfully breaking of immune tolerance in mice, the anti-tumor effect. This foreign protein vaccine strategy will have good prospects for the development of active immunotherapy field in cancer. This study showed that the heterogeneous recombinant EGFR protein produced through genetic engineering methods, can be induced in mice and anti-EGFR antibody, successfully breaking of immune tolerance in mice, the anti-tumor effect. This foreign protein vaccine strategy, an effective specific active immunization method, there will be good prospects for the development of active immunotherapy field in cancer.
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