| VEGF(Vascular endothelial growth factor) was first indentified from the serum-free conditioned medium of the tumor cell lines. It was initially indentified based on its ability to atimulate vascular permeability(called VPF,for vascular permeability factor),and was subsequently demonstrate to be an endothelial cell-specific mitogen and angiogenic factor,so named vascular endothelial growth factor.VEGF gene family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF(placenta growth factor).In particular, VEGF-A is also refered to as VEGF. The human VEGF gene is localized in chromosome 6p21.3,the full lenth of the gene is 28 kb,while the coding region spans approximately 14kb. The VEGF gene is organized in eight exons, separated by seven introns. Alternatively exon splicing of a single VEGF gene results in the generation of at least five different molecular species.having respectively 121, 145, 165, 189 and 206 amino acids. VEGFi65 is coded by exon 1-5, exon 7 and exon 8. VEGFies is secreted although a significant fraction remains bound to the cell surface and the extracellular matrix. VEGF165 is the predominant molecular species and is also widely used. Native VEGF is a homodimeric glycoprotein of 45 kDa. In each monomer, the N-terminal segment is a a -helix consisted of 16-24 amino acids. The central part is a four-tranded 0 -sheet formed by P 1 (residues 27-34), ?3(residues 51-58), # 5(residues 73-83) and # 6(residues 89-99),with the characteristic cystine knot motif on one end of the molecule. Monomers are stabilized by disulphide bonding between CysSl of one chain and Cys60 of the other. Under reductive circumstances, the dimomer is seprated into monomers and loses its bioactivities. The Flt-1 (also called VEGFR-1) and KDR(also called VEGFR-2) tyrosine kinase have been indentified as two major high affignity VEGF receptors. Flt-1 binds VEGF with higher affinity than KDR, however, KDR dominates the almost all bioactivities of VEGF. VEGF is now known as the endothelial cell-specific mitogen. Studies showed that the expression of VEGF was correlated with the mirovessel density. VEGF can induce proliferation and migration in vitro and also induce strong angiogenic response in vivo. It has been proved that in almost all types of tumor the expression VEGF is upregulated. The high expression of VEGF has a positive relationship with tumor malignancy, aggressivness, metastatic potential and prognosis. Now it becomes an important way for tumor therapy, using angiogenesis inhibitors targeted on theprocedure of angiogenesis to block tumor aggresivness and metastasis. Because the angiogenesis inhibitors selectively targeted on proliferating endothelial cells of the tumors, they can affect almost all tumors without the side effects caused usually by traditional drugs, such as bone marrow inhibition, vomiting, and hair loss. Furthermore, since endothelial cells are genetically stable with low mutation rate, anti-angiogenic therapy will not lead to drug resistance. Plenty of experiments showed that specific monoclonal antibodies of VEGF exerted a potent inhibitory effect on the growth of tumor in nude mice. But because of the high costs, it is impratical to manufacture large amounts of foreign antibodies. In addtion, foreign antibodies are potentially immunogenic compounds that may elicit antibody response, potentially limitting the long-term efficacy of such treatment.Based on these studies, we design a modified VEGFus protein, with a certain T-helper(Th) epitope in the COOH-terminal. We also leave the 1 to 61 amino acids of VEGF]65, according to its sites binding to the receptors. The modified VEGF165 cDNA was cloned into expression vector pRSET B , then transformed E. coli BL21 (DE3). The modified VEGFJ65 was highly expressed after IPTG induction. We immunized BALB/c mice with the modified VEGFiw protein, then established animal tumor models by inoculating S180 cells. The results indicated that the modifiedprotein can induce high-titer anti-VEGF165 autoantibodies. The autoantibodies cou...
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