Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper -: A new member of the VEGF family of proteins
Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper -: A new member of the VEGF family of proteins
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DOI：
10.1074/jbc.m106531200
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发表时间：
2001-10-26
影响因子：
4.8
通讯作者：
Ho, PL
中科院分区：
文献类型：
--
作者：
Junqueira de Azevedo, ILM;Farsky, SHP;Ho, PL
在从蝰蛇科蛇 Bothrops insularis 毒腺中产生丰富的表达序列标签的过程中，我们首次鉴定了编码假定的血管内皮生长因子样（VEGF 样）蛋白的 cDNA。在对最长的蛇毒 VEGF (svVEGF) cDNA 进行完整测序后，推导的一级序列显示出与脊椎动物 VEGF 以及来自蝰蛇毒液的降血压因子的相似性。其cDNA被亚克隆，在大肠杆菌中表达，带有His(6)标签作为不溶性单体，并在8 m尿素提取后通过Ni2+-亲和层析纯化。产生抗 svVEGF 的抗血清，并在蛋白质印迹中针对蛇毒和细胞提取物中的蛋白质进行测试。成熟的 svVEGF 似乎普遍分布在蛇毒中，其他相关蝰蛇科物种的 Northern 印迹研究和来自白腹蝰蛇的 svVEGF 的 cDNA 克隆也证实了这一点。产生的重组蛋白在重折叠过程后二聚化并进行生物学表征，显示出增加血管通透性的能力。这些结果表明，svVEGF 是双蛇蛇咬伤早期阶段的一种新型且重要的活性毒素，代表了 VEGF 蛋白家族的新成员。
During the generation of abundant expressed sequence tags from the Viperidae snake Bothrops insularis venom glands, we identified for the first time a cDNA coding for a putative vascular endothelial growth factor-like (VEGF-like) protein. The deduced primary sequence, after complete sequencing of the longest snake venom VEGF (svVEGF) cDNA, displayed similarity with vertebrate VEGFs and with the hypotensive factor from Vipera aspis venom. Its cDNA was subcloned, expressed in Escherichia coli with a His(6) tag as an insoluble monomer, and purified by Ni2+-affinity chromatography after 8 m urea extraction. Antiserum against svVEGF was generated and tested in Western blot against proteins from snake venoms and cellular extracts. The mature svVEGF appears to be ubiquitously distributed throughout snake venoms and was also confirmed by Northern blot studies of other related Viperidae species and by cDNA cloning of svVEGF from Bothrops jararaca pit viper. The produced recombinant protein dimerizes after refolding processes and was biologically characterized, showing ability to increase vascular permeability. These results established that svVEGF is a novel and important active toxin during the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins.