Vegf as a tumor marker: cloning and expression of vascular endothelial growth factor-a (vegf-a) in pichia pastoris and evaluation of its angiogenic effects on huvec cell line

Vascular endothelial growth factor (VEGF) is one of the most celebrated mediators in the process of angiogenesis. VEGF-A is a key regulator of angiogenesis, and differentiation of progenitor endothelialcells. There are receptor tyrosine kinases (RTKs or VEGFR) that mediate the angiogenic functions of VEGF family members. In normal and pathological circumstances, the formation of new blood vesselsoccurs during wound healing, organ regeneration, rheumatoid arthritis, diabetic retinopathy, tumor growth and types of Cancer, Breast Cancer, Colon Cancer , Ovarian Cancer and etc. The use of VEGFin many studies has proven to be a good tumor marker for the prognosis and diagnosis of various cancers. The aim of this study is Industrial Production of VEGF gene for Future approaches. At first,the A172 cell line was cultivated and total RNA was extracted. After synthesis cDNA, the VEGF encoding sequence was amplified by the specific cloning primers. After the sequencing of recombinantplasmid, the DNA was cloned into pBlueScript SK II(+) cloning vector. Recombinant insert was subcloned into pPICZα vector. The recombinant plasmids transferred into Pichia pastoris host cellswith electroporation. Then recombinant yeasts were induced with methanol and expression was studied by SDS-PAGE and Western Blotting, Finally protein was purified by affinity chromatographyand its angiogenic effect on HUVEC cell line was evaluated by tube formation assay. In each stage, the result of sequencing showed the success of cloning process. The result of SDS-PAGE and WesternBlotting proofed the expression of gene and viewing cellular morphology changes in the form of a tubular tube demonstrated the ability of the protein to stimulate angiogenesis. In future studies, the VEGF gene can be used for many purposes, including studies on tumor angiogenesis biology to detect and prognosis a variety of cancers by examining the increased expression of VEGF, nanobody production and the design of anticancer drugs for antiangiogenic therapy.

کلیدواژه ها:

Breast Cancer ، Colon Cancer ، Blood Cancer ، Skin Cancer ، Ovarian Cancer ، Cervical Cancer ، Cervical Cancer ، Stomach Cancer ، Cancer Diagnosis ، Cancer Prevention ، Gene and Cancer ، Cell andCancer ، Cancer Genetics ، Cancer Treatment and Management ، Targeted Cancer Therapy ، Drugs and Cancer