| Vascular endothelial growth factor (VEGF) is an antiparallel dimer of glycoprotein with multiple biological functions. VEGF is not only a fundamental mediator of physiologic and pathophysiologic angiogenesis, but also plays a critical role in regulation of vacular invasion, matix degradation and remodeling, the differentiation, maturation, migration, survival and functional activity of chondrocyte, osteoblast, osteoclast by autocrine/paracrine way during endochondral and membranous ossification. This study was performed in order to obtain functional chicken VEGF protein and its antibody, meanwhile to investigate the role of VEGF in etiopathogenesis of tibial dyschondroplasia in broilers.Experiment I Prokaryotic expression, purification of chicken VEGF protein and its antibody preparationA pair of primers were designed to clone the encoding sequence of chicken VEGF gene from the total RNA of tibial growth plate by RT-PCR. After the PCR produce was cloned into pMD-18T vector and sequenced, the recombinant expression vector of pET-32a(+)-chVEGF was constructed and then transformed to Escherichia coli BL21(DE3). The result showed that the hexahistidine-tagged fusion protein (His-chVEGF) was expressed at a high level under isopropyl-D-1-thiogalactopyranoside induction. After the recombinant protein was purified by Ni2+ affinity chromatography and its concentration determined by BCA, its polyclonal antibody was prepared. The SDS-PAGE result showed that the fusion protein was about 38 KD and amounted to 60% of the whole protein in Escherichia coli. Western blotting analysis indicated that the expressed protein was chicken VEGF protein and had a satisfactory immuno-biological activity. The antibody prepared could bind to the chicken VEGF protein expressed in Pichia Yeast, and the titer of the polyclonal anti-serum reached to 1:10240 by ELISA.Experiment II Expression of chicken VEGF in the methylotrophic yeast Pichia pastoris and bioactivity identification of recombinant proteinIn order to obtain chicken VEGF (chVEGF) protein with biological functions, a pair of primers containing EcoR I and Xba I enzyme digestion sites were designed to clone chVEGF DNA from the recombinant cloning vector pMD18-T-VEGF. And then the target segment was subcloned into Pichia pastoris expression vector pPICZa-A. The constructed plasmid was transformed into the yeast host X-33 by electroporation, and the recombinant transformants were selected in YPDSZ plate containing different concentration of zeocin. The positive transformant with high copy number of target gene was induced by adding methanol every 24 h. At last, the supernatant of culture medium was collected and analyzed by SDS-PAGE and Western blotting. Results showed that the two expressed products of chVEGF in the yeast were about 46KD,70KD, respectively, and recognized by chVEGF and human VEGF polyclone antibodies. Moreover, the recombinant proteins could significantly stimulate angiogenesis in chick chorioallantoic membrane.Experiment III Thiram-induced tibial dyschondroplasia in broiler chickens and its corresponding mechanism120 eight-day old AA broilers were randomly allotted to 4 treatments with 4 replicates per treatment. Four treatments were control group, thiram group (thiram100 mg·kg-1), lower copper group (thiram100 mg·kg-1+CuSO4·5H2O100 mg·kg-1) and high copper group (thiram100 mg·kg-1+CuSO4·5H2O100 mg·kg-1). At 21 day old, all birds were killed. The samples of growth plate, blood and tissues were collected and analyzed for the incidence and severity of TD, serum parameters and copper content. The results showed that 100 mg·kg-1 of thiram induced typical TD lesion in broilers of 21 day, increased significantly the incidence and severity of TD, and decreased live weight and tibial length of birds (P<0.05); copper addition in diet decreased the incidence and index of TD. Levels of serum Ca, P, ALP and SGPT of the birds in thiram groups were up-regulated (P<0.05). However, no significant difference was observed in serum SGPT level between four treatments. Compared with the control group, the birds in thiram groups had a higher copper content in liver, heart and pancreas, and a decreased level of copper in kidney (P<0.05). Interestingly, supplementary copper decreased thiram-induced copper accumulation in liver and increased copper content in kidney. Besides, the destruction of capillary vessels and the massive death of endothelial cells and chondrocytes were existed in the growth plates with TD lesion induced by thiram; In the transition zone of growth plate, the accumulation of abnormal chondrocytes that failed to undergo full hypertrophic state formed TD lesions. It suggested that thiram was an effective TD inducer and may cause TD in broilers by damaging the hepatic and renal functions, the formation and invasion of blood vessels, and affecting the utilization of copper and other minerals in different tissues.Experiment IV Effect of chicken VEGF expressed in Pichia peastoris on tibial dyschondroplasia, antioxidant capacity of liver and kidney, blood biochemical parameters in broiler chickens240 broilers of 8 day were randomLy divided into 8 treatments. Group A and E were basic diet control and thiram diet control, respectively. TD in birds of group E, F, G and H were induced by a short period of adding thiram (100mg·kg-1) into basic diet from 8 to 14 day old. The chVEGF protein was injected into legs of those birds in test groups at the dosage of 10 μg·kg-1、30 μg·kg-1、50 μg·kg-1 at 9,13,17 day old. The trial was ended at the age of 21 day. Results showed that the recombinant protein had no difference on TD incidence and body weight of birds in basic diet groups or in thiram diet groups, but significantly increased serum Ca, P, ALP, StrACP levels of broilers in 6 experimental groups, and decreased TD severity in these birds fed with a short period of thiram diet. Levels of T-AOC and activity of SOD, GSH-Px in liver and kidney were significantly increased in the birds injected with the recombinant protein at the dosage of 10 μg·kg-1、30 μg·kg-1 when respectively compared with those in group A and group E. However, a high dosage of the recombinant protein may damage the antioxidant capacity of liver and kidney of broilers. It suggested that VEGF promotes TD lesion recovery by enhancing the antioxidant capacity of liver and kidney, indirectly regulating the metabolism of minerals and vitamins in vivo.Experiment V Effect of recombinant chicken VEGF protein on expression of genes relating to endochondral ossification during impaired and recovery of the avian growth plateAnimals were the same as in the experiment Ⅵ. On day 15 and 21,5 birds from each group were killed and samples of proximal growth plate of the right and left tibia were collected for analyzing genes relating to endochondral ossification by real-time quantitative PCR. Results showed that thiram significantly increased the expression of Bcl-2 and collagen X genes, but significantly down-regulated the expression of MMP-13 and Runx2 genes in growth plate at the age of 15 day; on day 21, the expression of MMP-13 in group E had no difference compared with the normal growth plate but levels of Bcl-2, collagen X and Runx2 mRNA were significant down-regulated in group E. Different dosage of the recombinant protein significant up-regulated the expression of MMP-13 and Runx2 mRNA in thiram experimental groups but only administration of the protein at 10μg·kg-1 up-regulated Bcl-2 and collagen X gene expression in group F at the age of 15 day; however, at the age of 21 day, the recombinant protein significant up-regulated the expression of genes encoding collage X, MMP-13 and Runx2 in thiram experimental groups, and meanwhile significantly down-regulated the expression of Bcl-2 gene in thiram diet groups at a dosage-dependent way. It suggested that MMP-13 and Runx2 play a important role in pathomechanism of TD, and VEGF promote TD lesion recovery by regulating differential expression of genes relating to endochondral ossification in avian growth plate during the development of TD, and defective expression of VEGF may be involved in the etiology of TD. |
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