人血管内皮细胞生长因子在大肠杆菌中的表达及纯化

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。

Expression of Human Vascular Endothelial Growth Factor(h VEGF) in E. coli and Purification of Expressed Product

Objective To obtain large amounts of recombinant human vascular endothelial growth factor(h VEGF) and study on the potential value of it in cardiovasological and pharmacological fields.Methods The goal gene hVEGF165 was amplified by PCR and inserted into vector PET-3a,then transformed to E.coli BL21(DE3) cell. In this way,a recombinant Plasimd pET-3a/hEVGF165 was constructed and expressed under the induction of IPTG. The antigenicity of expressed product was detected by Western blot, and, after being preliminarily purified by Mono Q and FPLC Superose 12 chromographies, the biological activity of it was detected by MTT method. Results The purity of expressed product after being purified reached more than 90%. The product showed a dose-dependent promotive effect on the proliferation of huamn vascular endothelial cell(hVEC). Conclusion A strain of E. coli with the activity of natural hVEGF165 was constructed,and a purfication process was developed. The study laid a foundation of large scale production of recombinant hVEGF165.