重组人血管内皮细胞生长因子的表达及活性研究  

Expression, purification and activity assay of recombinated the vascular endothelial growth factor (VEGF)

作　　者：王晓华[1] 吉琼梅[1] 吴朝霞[1] 董文心[2] 

机构地区：[1]广州医学院生物化学与分子生物学教研室,广东广州510182 [2]上海医药工业研究院药理研究室,上海200437

出　　处：《暨南大学学报（自然科学与医学版）》2004年第4期426-430,共5页Journal of Jinan University(Natural Science & Medicine Edition)

摘　　要：目的 :用基因工程的方法在大肠杆菌中诱导表达人血管内皮生长因子 (VEGF) ,分离纯化并检测其生物学活性 ,以研究其在药学领域潜在的药用价值。方法 :利用PCR技术扩增VEGF基因片段 ,克隆到pQE30表达载体中 ,转化E .coliM15菌株后用IPTG进行诱导表达。经裂解细胞、变性、复性和Ni-NTAagarose金属螯合柱层析等方法纯化得到VEGF。用鸡胚绒毛尿囊膜 (CAM)血管生成实验检测VEGF的生物活性。结果 :重组表达质粒在大肠杆菌中成功地表达了相对分子质量为 2 0 6 0 0的融合蛋白 ,它以不溶性的包涵体形式存在 ,占菌体总蛋白的 30 %左右。经分离纯化融合蛋白SDS -PAGE显示为单一区带。CAM结果表明给药组血管生成数 (2 1 7± 3 1、39 3± 2 8)与对照组 (15 4± 1 9、2 9 2± 4 2 )相比有明显增加 (P <0 0 5 )。结论 :利用原核表达系统得到血管内皮生长因子具有天然VEGF生物学活性 ,为进一步的应用研究奠定了基础。The vascular endothelial growth factor (VEGF) expressed in E.coli in a novel type of fusion protein was produced by genic engineering. Its biological activity was assayed and studied on it in pharmacological fields. Methods: The VEGF gene was amplified by PCR, then inserted into plasmid pQE30 and sequenced. Expression of the fusion protein was induced by IPTG in E. coli M15. After the VEGF purified by means of cell disruption, washing and affinity chromatographies etc, the angiogenic activity was tested by chicken chorioallantoic assay. Results: The fusion protein with (20 600) was expressed by induction of IPTG in E.coli M15. Its type was inclusion body and its expression accounted for 30% of total bacteria weight. The fusion protein was purified by affinity chromatographies using Ni-NTA resin. VEGF purified was a single band on SDS-PAGE. The chicken chorioallantoic membrane (CAM) assay showed that the formation of novel blood vessel was increased obviously (P<0.05) between (experiment) (21.7±3.1,39.3±2.8) and blank control (15.4±1.9,29.2±4.2). Conclusion:The recombinant VEGF with better activity was successfully expressed in E.coli and laid the foundation for the application and study.

关 键 词：人血管内皮生长因子 鸡胚绒毛尿囊膜 基因表达 

分 类 号：R322.1[医药卫生—人体解剖和组织胚胎学] Q51[医药卫生—基础医学]