Expression and identification of recombinant chicken vascular endothelial growth factor in Pichia pastoris and its role in the pathogenesis of tibial dyschondroplasia

• DOI: 10.3382/ps.2013-03420
• Publication Date: 2013-11-14T20:09:53Z
• Authors: J.-P. Zhang; Y.-F. Deng; Z.-L. Zhou; J.-F. Hou
• Keywords: Vascular Endothelial Growth Factor A; 0303 health sciences; Organisms, Genetically Modified; Tibia; Chick Embryo; Thiram; Osteochondrodysplasias; Injections, Intramuscular; Polymerase Chain Reaction; Chorioallantoic Membrane; Pichia; Recombinant Proteins; 3. Good health; Avian Proteins; 03 medical and health sciences; Gene Expression Regulation; Animals; Electrophoresis, Polyacrylamide Gel; Growth Plate; Chickens; Blood Chemical Analysis; Poultry Diseases; Mutagens

Vascular endothelial growth factor (VEGF) is an essential mediator of angiogenesis and endochondral ossification. To explore the role of VEGF in avian diseases such as tibial dyschondroplasia (TD), a typical disorder of endochondral ossification, we expressed and identified recombinant chicken VEGF (chVEGF) protein in Pichia pastoris and evaluated its effects on thiram-induced TD in broiler chickens. The SDS-PAGE showed that 2 recombinant proteins, with molecular weights of ~46 and ~70 kDa, were obtained. Western blot analysis indicated that the 2 proteins were recognized by rabbit anti-chicken and goat anti-human VEGF polyclonal antibodies. Moreover, the mixture of the proteins significantly stimulated angiogenesis in the chick chorioallantoic membrane. In 21-d-old broilers that had been fed a thiram-enriched diet (100 mg/kg of thiram for 2 d at 8 d old) to induce TD, intramuscular injection of the chVEGF proteins (at a dosage of 10 or 30 μg/kg) significantly reduced the severity of TD but had no effect on TD incidence or BW; decreased serum Ca and P concentrations and tartrate-resistant acid phosphatase activity and elevated serum alkaline phosphatase activity; enhanced the total antioxidant capacity, superoxide dismutase, and glutathione peroxidase activities in the liver and kidney; upregulated the expression of type X collagen, matrix metalloproteinase (MMP)-13, and Runx2; and downregulated the Bcl-2 expression in the growth plates. In thiram-treated broilers at 15 d old, the chVEGF proteins upregulated the expression of MMP-13 and Runx2, and had different effects on type X collagen and Bcl-2 expression at different dosages. Our results indicate that exogenous chVEGF proteins promoted the recovery of TD-affected growth plates by improving the antioxidant capacity in the liver and kidney and by regulating differential expression of genes relating to endochondral ossification at different stages of TD development; VEGF deficiency in the growth plates was involved in the pathogenesis of TD.