重组人血管内皮生长因子D诱导鸡胚尿囊膜血管增生  

Recombinant Human VEGF-D Induces the Angiogenesis of the Chick Embryo Chorioallantoic Membrane

作　　者：陈浩[1] 丁秀云[1] 高媛[2] 柳晓兰[1] 高建恩[2] 孙启鸿[2] 

机构地区：[1]军事医学科学院放射与辐射医学研究所免疫学研究室,北京100850 [2]北京蛋白质组研究中心抗体工程研究室,北京102206

出　　处：《中国实验血液学杂志》2007年第2期364-368,共5页Journal of Experimental Hematology

基　　金：国家高技术研究发展计划(863计划)资助;编号:2002AA214031

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VEGF-D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RT-PCR法扩增VEGF-D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pGEX-5X-1/VEGF-D并在大肠杆菌BL21(DE3)中进行GST-VEGF-D融合蛋白的表达。结果表明,融合蛋白的分子量约为38kD,表达量占菌体总蛋白的15%以上,抗GST和抗VEGF-D的抗体均能识别融合蛋白;纯化的GST-VEGF-D融合蛋白与VEGFR-3/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GST-VEGF-D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VEGF-D融合蛋白,为进一步研究VEGF-D的作用机理提供了实验模型。Vascular endothelial growth factor-D （VEGF-D） and vascular endothelial growth factor receptor-2,-3 （VEGFR-2,-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 GST-VEGF-D fusion protein, and to explore the angiogenesis effect of VEGF-D. The total RNA was extracted from human fetal lung tissue, and the mature form of VEGF-D was expanded by polymerase chain reaction （PCR）, then the plasmid pGEX-5X-1/VEGF-D was reconstructed and the GST-VEGF-D fusion protein expressed in transformed E. coli BL21- DE3. 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 anti-GST and anti-VEGF-D antibodies. The soluble GST- VEGF-D fusion protein could interact with VEGFR-3/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 GST-VEGF-D could induce the CAM angiogenesis. It is concluded that the GST-VEGF-D fusion protein with biological activity was successfully expressed, and which may provide an experimental model for the investigation of the VEGF-D- induced angiogenesis and lymphangiogenesis.

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 

分 类 号：R378.99[医药卫生—病原生物学]