Preparation Of Recombinant Human VEGF Protein, Polyclonal Anti-VEGF Antibody And Primary Study In Tumor Detection

Vascular endothelial growth factor(VEGF) is a dimeric glycoprotein of 34-42 kDa.It is the most potent endothelial cell mitogen and also a regulator of vascular permeability.It is expressed by a variety of normal cells.but is significantly over-expressed by malignant tumors,where it can be produced by the tumor cells themselves or by stromal cells.This glycoprotein plays a crucial role in many pathologic conditions and malignancies because it stimulates capillary tube formation,endothelial cell proliferation,tumor invasion n and metastasis,thereby playing an essential angiogenic role pivotal for tumor growth and aggressiveness.VEGF is emerging as a powerful new prognostic tool.The aim of the present study is to produce specific rabbit polyclonal antibodies against bacterially expressed human VEGF165 using a recombinant DNA approach.A plasmid has been constructed to direct the synthesis of recombinant human VEGF165(rhVEGF165) in Escherichia coli as a fusion protein containing a His6-tag at the N-terminus under the control of T7 promoter.The rhVEGF165 was synthesized in large amounts and accumulated in the form of inclusion bodies upon induction with IPTG.Inclusion bodies were solubilized in 8M urea and purified based on its 6xHis-tag by affinity chromatography using Nickel-Agarose Column and refolded by dialysis.Two rabbits were immunized with the purified recombinant protein to produce polyclonal anti-human VEGF antibody.The titer was checked with indirect ELISA and the final titer was 1:25600.We used the purified recombinant protein and polyclonal antibody to detect VEGF expression levels in the serum of 33 tumor patients and 25 healthy controls by an established sandwich ELISA.The mean antigen concentration in serum of patients was approximately 407 pg/ml and significantly higher than those in the healthy controls in which the circulating VEGF antigens concentration was not detectable(P <0.05).Thus,this newly ELISA could be a rapid,effective and cheap method for tumor diagnosis using serum VEGF as a biomarker.