Expression, Purification And Biological Activity Analysis Of Human Vascular Endothelial Growth Factor 41 (VEGF₄₁) Fusion Protein

Objective:As a potential specific endothelial cell(EC)mitogen,VascularEndothelial Growth Factor(VEGF) promotes the division,proliferation andmigration of ECs,and also plays an important role in the maturing and remodeling ofblood vessels. VEGF₄₁ is a novel isoform of VEGF we isolated previously. This studywas to construct the recombinant expression plasmid pGEX-VEGF₄₁, to obtain highlevel expressed VEGF₄₁ protein in E. Coli,and to study its biological function.Methods : Human VEGF₄₁ was synthesized by chemical methods , andconstructed it with pGEX-4T-1 expression vector. The pGEX-VEGF₄₁ plasmid wasconfirmed by sequencing and transformed into E. coli bcl-2 for protein expression.The expressed fusion protein was purified by using Glutamine-Sepharose 4B affinitychromatography and isoelectric focusing,and then used for testing of its angiogenicactivity in human umbilical vein endothelial cells (HUVEC) and chorioallantoicmembrane in chicken embryo.Results:After 4 hours induction of 0.3 mM IPTG, hVEGF41 expression reachedpeak level by SDS-PAGE gel analysis. Western Blot analysis demonstrated anexcellent antigenicity and high specificity of the E. coli-expressed hVEGF₄₁. In thehVEGF₄₁-treated HUVEC cells group vs control group, the slope of cell growth graphwas significantly increased, the doubling time of the cells decreased, the absorption ofMTT is increased(P<0.001);the cell numbers increased (96±5 vs. 178±11), theratio of the S period cell cycle by FCM analysis was higher than the pure hypoxiagroup (P<0.001). In our vivo study in chick embryo, hVEGF41 obviously acceleratedformation of new blood vessels in chorioallantoic membrane. Conclusion:hVEGF41 could effectively promote the division,proliferation andmigration of ECs and accelerate the formation of new blood vessel。...