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目的 为了获得大量重组人血管内皮细胞生长因子( Human Vascular EndothelialGrowth Factor, hVEGF),以研究其在心血管和药学领域具有的潜在药用价值。方法 用PCR方法扩增目的基因hVEGF165,连接到 pET-3a载体上,并转化到大肠杆菌BL21(DE3)中,建立可高效表达的pET-3a/hEVGF165重组质粒,经IPTG诱导表达, Western blot检测表达产物的抗原性,通过 Mono Q阴离子层析和FPLG Superose12分子筛柱层析,进行初步纯化,用 MTT法检测其生物学活性。结果 pET-3a/hEVGF表达产物经纯化后,其纯度可达到90%以上,表达的rhEVGF165能 呈剂量依赖性地促进人静脉内皮细胞(HUVEC)的增殖。结论组建了天然形式hEVGF165的大肠杆菌菌株,并摸索 出一套纯化工艺,为大规模生产重组hEVGF165奠定了基础。
Objective To obtain a large number of human vascular endothelial growth factor (hVEGF) in order to study its potential cardiovascular and pharmacological properties. Methods The target gene hVEGF165 was amplified by PCR, ligated into pET-3a vector, and transformed into E. coli BL21 (DE3). The recombinant plasmid pET-3a / hEVGF165 was constructed and expressed by IPTG. The antigenicity of the expressed product was determined by Mono Q anion chromatography and FPLG Superose 12 molecular sieve column chromatography. The biological activity of the product was tested by MTT assay. Results The purity of pET-3a / hEVGF was over 90% after purification. The expressed rhEVGF165 could promote the proliferation of human vein endothelial cells (HUVECs) in a dose-dependent manner. Conclusion The E. coli strain of natural form hEVGF165 was established and a purification process was developed, which laid the foundation for large-scale production of recombinant hEVGF165.