| EGF(Epidermal Growth Factor, EGF) and EGFR(Epidermal Growth Factor Receptor) are overexpressed in a number of epithelial origin tumors providing necessary tumor signal for growth and metastasis for the tumor cell. EGF and EGFR have become the main targets about tumor vaccines immunotherapy research. How to increase the immunogenicity of EGF protein and break the immune tolerance of EGF protein have become the key for EGF vaccine research. In this study, we adopted the DNA shuffling technology to recombinant human, mouse and chicken EGF exon fragment to get some EGF recombinant proteins which fused multiple species. We immunized mice and induced anti-EGF antibodies in mice, blocking EGF/EGFR signaling pathways, to inhibition the growth of tumor. And we also explores the anti-tumor effect of combined immunodeficiency about recombinant EGF vaccine and recombinant VEGF protein vaccine. The main methods and results are following:(1) Amplification human, mouse and chicken EGF exon fragment by PCR, then use DNaseâ… enzyme to cut the amplified genes into small fragments about 50 bp. Small fragments were be mixed and become templates and primers for each other for PCR amplification.That is DNA shuffling recombination.Then PCR the DNA shuffling products with some primers of human, mice and chickens EGF gene which have cut site. The amplified products were fragment recovered and double enzyme cut to inserted into the vector pET-32 a, and then transformed into E.coli BL21.Then we get a library containing 400 transformants.We picked out 22 transformants by colony PCR and SDS-PAGE. Immunized mice with this the 22 transformants, we get 6 transformants who can induced high antibody titers both to mouse EGF protein(mEGF) protein and to human EGF(hEGF) in mice.(2) Six transformants recombinant proteins are purified by Ni-filter, and the proteins were mixed with MF59 to immunize mice. In this step, we found the recombinant proteins 300 and 380 have anti-tumor activity. 15 days after tumor-bearing, we stripped the tumor blocks and weighed. The average tumor blocks of PBS group were weighted 0.372 g.The average tumor blocks of PBS+ MF59 group were weighted 0.258 g. The average tumor blocks of 300+MF59 group were weighted 0.072 g.And the average tumor blocks of 380+MF59 group were weighted 0.038 g.The tumor weight of 300+MF59 group and 380+MF59 group were both significant differences with PBS group and PBS+MF59(P <0.01). We found 300 recombinant protein induced mice produce 3200 times of antibody titers anti mEGF, 380 recombinant protein induced mice produce 6400 times of antibody titers anti mEGF. Taken together, the 380 recombinant protein induces a stronger immune response, resulting in higher titer anti-EGF antibody, with better anti-tumor immune effect. Compared with the PBS control group, 380 recombinant protein-immunized group has a more significantly effect to inhibited tumor growth and prolonged the survival of tumor-bearing mice. Verified by Western Blotting, We found the antibodies in serum of 380+MF59 group could specifically binds with human EGF protein, mouse EGF protein and 380 recombinant protein.(3) Combined immunized with recombinant EGF protein vaccine and recombinant VEGF protein vaccine can induce mice produce both anti-EGF antibody and anti-VEGF antibody.The antibody titers can reached 12800 times and 6400 times. Combined immunodeficiency has a better herapeutic effect than a single vaccine of VEGF or EGF. Compared to a single vaccine, combined immunotherapy is more conducive to control the growth of mouse tumor(4T1 breast cancer) and prolong the survival time of tumor-bearing mice.In summary, DNA shuffling technology cloud recombinant tumor antigens, enhance the immunogenicity of tumor antigens and break the immune tolerance of the body to self-proteins.380 recombinant EGF protein can induced mice produce higher antibody titers to both mEGF and hEGF, blocking EGF / EGFR signaling pathways and inhibition the growth of 4T1 breast cancer cell. By combined immunization with recombinant VEGF protein vaccine, we explored the feasibility of tumor vaccine combined immunodeficiency.It may provides a new direction or ideas about tumor immunotherapy. |
