An Anti-Tumor Research Of Recombinant Phage Vaccine Expressed Xenogenic EGFR

Objective: To construct 5 recombinant T7 phage vaccines display extracellular regions of chicken epidermal growth factor receptor (cEGFR) and verify their EGFR antigenicity. To investigate the anti-tumor effect of the endogenous antibodies induced by these gene recombinant phage vaccines on a C57BL/6J-Lweis Lung Cancer animal model.Methods: RT-PCR was used to clone cEGFR extracellular region gene from chicken testis total RNA. The product was cloned into a vector (pMD18-T) and was transformed into E.coli DH5 α competent cells. Recombinant plasmid was extracted from the transformed positive DH5 α cell and the inserted gene was sequenced. Five subfragments with high-hydrophilicity and antigenicity was analyzed by DNAStar software selected, and primers were designed to amplify the 5 subfragments. Using T7 phage display system, these small peptide fragments were displayed as fused proteins on T7 phage 10B capsid protein, thus constructed 5 recombinant cEGFR-T7 phage vaccines. By SDS-PAGE and Western-blot assay, the expression of cEGFR-T7P10B fusion protein and the EGFR antigenicity was observed in these recombinant vaccines. These vaccines were used to immunize C57BL/6J mice, one time per week for 4 times. The anti-EGFR antibody was detected after the third immunization by flow-cytometry by incubating the mice sera with A431 cells, which expresses EGFR at high level before labeled by a fluorescence antibody. Lewis lung cancer model was constructed after 4 immunizations. The weight of the tumor tissue at 10 days was measured to evaluate the anti-tumor effect of each experimental group.Results: cEGFR extracellular region was cloned from chicken testis total RNA by RT-PCR, then verified with 1% agarose gel and gene sequencing. The subfragments of the gene were amplified by PCR and recovered from 1.5% agarose gel. Five subfregments were used to constructed 5 recombinant cEGFR-T7 phage vaccines which were verified them by PCR^ SDS-PAGE and Western-blot. EGFR antigenicity can be detected in Western-Blot analysis. A431 cancer cells expressing EGFR at high level reacted with sera from immunized mice for 3 weeks and could be labeled by fluorescence antibody. Using fluorescent flow cytometry to analyze the ratio of labeled cells showed that special mouse anti-EGFR antibodies had been produced in sera. Comparing all the experimental groups, the statistic differences between five test groups and the negative-control group were markedly (P<0.05) . Conclusion: A gene recombinant phage vaccine research framework has been developed primarily, and 5 cEGFR gene recombinant 77 phage vaccines had been constructed successfully. The endogenous antibody which was induced by these vaccines could restrain the proliferation of EGFR positive tumor in some degree. The immunoreaction can be induced with a xenogenic homologous peptide easier than others, and the recombination of the long xenogenic peptide can form its original three-dimensional structure better than the short ones.