Preparation of recombinant anti-vegf antibody and development of it's nanoconjugate

The role of vascular endothelial growth factor (VEGF) in cancer metabolism has been pointed in literature, within this work, it has been aimed to produce the blocking Anti-VEGF antibody which is bound to VEGF, consisting of two chains and found in human blood as well, as recombinant. In this respect, total RNA has been isolated from human blood, cDNA has been synthesized by that and Anti-VEGF genes have been cloned using specific primers on gene sequence of two chains of the Anti-VEGF antibody. After that, cloned genes have reformed in one chain by the same technique and the expression has been made with the relevant cloning and expression plasmid vectors and hostcells. In the last phase of the work, the purification and isolation of recombinant Anti-VEGF antibody of which the heavy and light chains are bound to each other have been achieved from the host bacterias. Purification and characterization of the protein have been realized by nickel affinity chromatography and the Western Blot, respectively. Then, in order to increase the durability of the Anti-VEGF antibody against outer environment circumstances, Nano-Anti-VEGF conjugate has been produced by ANADOLUCA metot which ensures photosensitive cross linking with ruthenium-based amino acid monomers. The characterizations of this nano-blocker have been exposed by investigating dimensional analysis, surface morphology and seconder structure by Zeta, SEM and CD spectroscopy, respectively. The conjugate of Nano-Anti-VEGF has been synthesized with different fluorescent substances such as acridine, BODIPY and FITC in order to observe how the course of treatment proceeds via molecular imaging technology to ensure it's traceability in pharmaceutical industry.