Plasmids for DNA vaccination were purified by using two rounds of passage over Endo-free columns (Qiagen, Chatsworth, CA), as reported (23). analysis and ELISA assay. The purified immunoglobulins were effective at the inhibition of VEGF-mediated endothelial cell proliferation and of other animals such as (16). For example, a comparison analysis made in the present study Otamixaban (FXV 673) by searching the Swissprot database at the National Center for Biotechnology Information indicates that this homologue Sema3b of VEGF (accession no. “type”:”entrez-protein”,”attrs”:”text”:”AAB63680″,”term_id”:”2271035″AAB63680.1) (4) is 75% and 73% identical in mouse VEGF 164 (accession no. “type”:”entrez-protein”,”attrs”:”text”:”AAB22253″,”term_id”:”249859″AAB22253.1) (3) and human VEGF 165 (accession no. “type”:”entrez-protein”,”attrs”:”text”:”BAA78418″,”term_id”:”4996351″BAA78418.1) (5), respectively, at the amino acid level. As a strategy for malignancy therapy, antiangiogenic therapy attempts to stop new vessels from forming around a tumor and to break up the existing network of abnormal capillaries that feeds the cancerous mass (6, 10, 17C22). VEGF has been known to be a potent vasculogenic and angiogenic factor (7C12). It has been reported that this abrogation of VEGF-induced angiogenesis, including the passive immunization of a neutralizing antibody against VEGF, can suppress tumor growth (12, 21), suggesting that VEGF plays an important role in angiogenesis in tumor growth. Thus, VEGF may be used as a ideal model molecule to explore the feasibility of immunogene tumor therapy with a vaccine based on a single xenogeneic gene by overcoming the immune tolerance of growth factors associated with tumor growth in a crossreaction between xenogeneic homologous and self-molecules. To test this concept, we constructed a plasmid DNA encoding homologous VEGF and that encoding the corresponding mouse VEGF 164 were isolated by PCR with the use of a cDNA library and a mouse skeletal muscle mass cDNA library (CLONTECH), respectively. The amplified products were inserted into PCR 2.1 plasmid (Invitrogen) and then subcloned into pSecTag 2A (Invitrogen), which contains a cytomegalovirus promoter. VEGF of and mouse inserted into pSecTag 2A Otamixaban (FXV 673) was named XVEGF-p and MVEGF-p, respectively. As a control, real plasmid was used as an empty vector (c-p). The full-length sequence of and mouse VEGF was confirmed by dideoxy sequence to be identical to those reported (3C5). Plasmids for DNA vaccination were purified by using two rounds of passage over Endo-free columns (Qiagen, Chatsworth, CA), as reported (23). The expression of plasmid DNA was confirmed in the transfected cells by reverse transcriptionCPCR and with the use of anti-VEGF antibodies in Western blotting analysis and Otamixaban (FXV 673) ELISA. Meth A fibrosarcoma, MA782/5S mammary malignancy, and H22 hepatoma models were established in BALB/c mice. Mice were immunized with different doses (5C150 g per mouse) of DNA vaccine in normal saline by intramuscular injection once a week for 4 weeks. Additional control animals were injected with normal saline. All studies involving mice were approved by the institute’s animal care and use committee. Western Blot Analysis. Western blot analysis was performed as explained (24). Briefly, recombinant VEGF proteins and other Otamixaban (FXV 673) proteins were separated by SDS/PAGE. Gels were electroblotted with Sartoblot onto a poly(vinylidene difluoride) membrane. The membrane blots were blocked at 4C in 5% nonfat dry milk, washed, and probed with mouse sera at 1:500. Blots were then washed and incubated with a biotinylated secondary antibody (biotinylated horse anti-mouse IgG or IgM), followed by transfer to Vectastain ABC (Vector Laboratories). Recombinant mouse VEGF, human VEGF, and basic fibroblast growth factor (bFGF) were obtained from Sigma-Aldrich. Anti-placenta growth factor (PIGF) and bFGF antibodies were obtained from Santa Cruz Biotechnology. Recombinant VEGF; mouse VEGF 120, 164, and 188 isoforms; VEGF-B and C; bFGF; and PlGF were expressed, refolded, and purified from Depletion of Immune Cell Subsets. Immune cell subsets were depleted as explained (29, 30). Mice were injected i.p. with 500 g of either the anti-CD4 (clone GK 1.5, rat IgG), anti-CD8 (clone 2.43, rat IgG), anti-natural killer (NK) (clone PK136) mAb, or isotype controls 1 day before the immunization, and then twice per week for 3 weeks..
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