【摘要】为了表达和纯化具有生物学活性的人血管内皮生长因子(vascular endothelial growth factor，VEGF)D，探讨VEGFD对血管增生作用，从人胎肺组织中提取总RNA，设计特异性引物并应用RTPCR法扩增VEGFD成熟肽片段;经测序证明目的片段序列正确后，构建重组表达质粒pGEX5X1/VEGFD并在大肠杆菌BL21(DE3)中进行GSTVEGFD融合蛋白的表达。结果表明，融合蛋白的分子量约为38 kD，表达量占菌体总蛋白的15%以上，抗GST和抗VEGFD的抗体均能识别融合蛋白;纯化的GSTVEGFD融合蛋白与VEGFR3/Fc具有结合活性，并能刺激红白血病细胞系HEL细胞生长;在鸡胚尿囊膜实验中GSTVEGFD具有促进鸡胚尿囊膜血管生成的作用。结论：成功表达了具有生物学活性的重组人VEGFD融合蛋白，为进一步研究VEGFD的作用机理提供了实验模型。
【关键词】 血管内皮生长因子D;血管内皮生长因子受体3;鸡胚尿囊膜;血管生成
Recombinant Human VEGFD Induces the Angiogenesis of the Chick Embryo Chorioallantoic Membrane
CHEN Hao, DING XiuYun, GAO Yuan1, LIU XiaoLan, GAO JianEn1, SUN QiHong1
Department of Immunology, Beijing Institute of Radiation Medicine, Beijing 100850，China; 1Department of Antibody Engineering, Beijing Proteome Research Center, Beijing 102206，China
Abstract Vascular endothelial growth factorD (VEGFD) and vascular endothelial growth factor receptor2,3 (VEGFR2,3) with their corresponding signaling pathway play significant roles in the development of the embryonic vascular system and pathological lymphangiogenesis. The study was aimed to express and purify the GSTVEGFD fusion protein, and to explore the angiogenesis effect of VEGFD. The total RNA was extracted from human fetal lung tissue, and the mature form of VEGFD was expanded by polymerase chain reaction (PCR), then the plasmid pGEX5X1/VEGFD was reconstructed and the GSTVEGFD fusion protein expressed in transformed E.coli BL21DE3.The results showed that the molecular mass of this fusion protein was 38 kD and compassed more than 15% of the total bacteria proteins.The fusion protein was recognized by antiGST and antiVEGFD antibodies.The soluble GSTVEGFD fusion protein could interact with VEGFR3/Fc and was able to stimulate the proliferation of human erythroleukemia cell line(HEL) cells. The data of chick embryo chorioallantoic membrane (CAM) experiments indicated that GSTVEGFD co
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