Expression and identification of recombinant chicken vascular endothelial growth factor in Pichia pastoris and its role in the pathogenesis of tibial dyschondroplasia.
Expression and identification of recombinant chicken vascular endothelial growth factor in Pichia pastoris and its role in the pathogenesis of tibial dyschondroplasia.
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DOI：
10.3382/ps.2013-03420
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发表时间：
2013-12
期刊：
影响因子：
4.4
通讯作者：
J. Zhang;Y. Deng;Z. Zhou;J. Hou
中科院分区：
文献类型：
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作者：
J. Zhang;Y. Deng;Z. Zhou;J. Hou
血管内皮生长因子（VEGF）是血管生成和软骨内骨化的重要介质。为了探讨 VEGF 在禽类疾病如胫骨软骨发育不良 (TD)（一种典型的软骨内骨化疾病）中的作用，我们在毕赤酵母中表达并鉴定了重组鸡 VEGF (chVEGF) 蛋白，并评估了其对福美双诱导的肉鸡 TD 的影响。 SDS-PAGE显示获得了2种重组蛋白，分子量分别为~46和~70 kDa。 Western blot分析表明，兔抗鸡和羊抗人VEGF多克隆抗体均能识别这2种蛋白。此外，蛋白质的混合物显着刺激了鸡绒毛尿囊膜中的血管生成。在饲喂富含福美双的日粮（8日龄时连续2天福美双100mg/kg）诱导TD的21日龄肉鸡中，肌内注射chVEGF蛋白（剂量为10或30μg/kg）可显着降低TD的严重程度，但对TD发生率或体重没有影响；血清钙、磷浓度降低，抗酒石酸酸性磷酸酶活性降低，血清碱性磷酸酶活性升高；增强肝脏和肾脏的总抗氧化能力、超氧化物歧化酶和谷胱甘肽过氧化物酶活性；上调 X 型胶原、基质金属蛋白酶 (MMP)-13 和 Runx2 的表达；并下调生长板中 Bcl-2 的表达。在福美双处理的15日龄肉鸡中，chVEGF蛋白在不同剂量下上调MMP-13和Runx2的表达，并且对X型胶原和Bcl-2的表达有不同的影响。我们的研究结果表明，外源性 chVEGF 蛋白通过提高肝脏和肾脏的抗氧化能力以及调节 TD 发育不同阶段软骨内骨化相关基因的差异表达来促进 TD 影响的生长板的恢复。生长板中的 VEGF 缺陷参与了 TD 的发病机制。
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.