重组人VEGF165工程菌的发酵及表达产物的纯化与活性测定

目的:优化重组人VEGF165(rhVEGF165)工程菌的发酵条件,分离纯化目的蛋白并检查活性.方法:考察了甲醇诱导浓度和诱导时间对目的蛋白表达量的影响;发酵液经离心、透析后,经肝素亲和柱层析、超滤制得纯品;采用CAM实验和血管内皮细胞MTT法测定生物活性,比较了与人VEGF165标准品的免疫原性.结果:最佳表达条件为:甲醇诱导浓度为1%,诱导时间为5～6 d;采用Heparin-Sepharose CL6B亲和层析后,产物纯度可达90%以上;活性测定结果显示,目的蛋白具有促血管生成作用;与人VEGF165具有相似的免疫原性.结论:经发酵纯化后获得了有活性的重组人VEGF165纯品.

Pichia Expression of Recombined Human VEGF165 and Activity Determination

AIM: To optimize the expressi on condition of recombined human vascular endothelial growth factor(hVEGF 16 5) with Pichia pastroris expression system,then to purify and to determine its ac tivity. METHOD: The inducing concentration of methanol an d inducing period were reviewed in this paper.The supernatant of culture was purified wit h Heparin-Sepharose CL6B affinity chromatography. Biological activities of pur if ied product were tested by CAM experiment and HMVECnd proliferation. Furtherm o re,antigenicity and specificity of expressed product were compared with human VEGF 165 control by ELISA. RESULT: After being induced by 1% methanol for 5 d,recombinant protein was expressed best. The purity of product was ab ov e 90% after being purified by Heparin-Sepharose CL6B affinity chromatography. Results showed that expressed protein could stimulate vascular endothelial growth and ha d the similar antigenicity to human VEGF 165. CONCLUSION:Recombined human VEGF 165 was expressed and purified successfully.