重组VEGF-A_189发酵、纯化及鉴定

为了获得大量重组血管内皮生长因子(VEGF-A_(189))蛋白的可溶性表达,对重组VEGF-A_(189)工程菌进行发酵条件的优化并开展发酵研究.通过摇瓶培养进行可溶性发酵条件优化,在5 L发酵罐中发酵重组VEGF-A_(189)工程菌,将发酵产物经镍离子柱亲和层析纯化,并对发酵产物进行SDS-PAGE,Western Blot和ELISA等分析鉴定.结果表明,重组VEGF-A_(189)工程菌可溶性发酵条件为:37℃培养重组VEGF-A_(189)工程菌4 h,加入0.3 mmol/L的异丙基-β-D-硫代半乳糖苷(IPTG)于28℃诱导表达4 h.在5 L发酵罐中按比例放大发酵,在补料发酵3.5 h后菌体密度(A_(600nm))达到8.0,调整发酵温度到28℃,IPTG诱导4 h后的菌体密度为13.72,菌体产量为40 g/L.镍离子柱亲合层析纯化后重组VEGF-A_(189)纯度达到90%以上,回收率大于80%,Western Blot、ELISA分析结果显示,重组VEGF-A_(189)蛋白和抗VEGF抗体有良好的结合活性.结果表明经过发酵条件优化后发酵可获得有免疫结合活性的可溶性重组VEGF-A_(189)蛋白.

Fermentation, purification and identification of recombinant VEGF-A_189

To obtain a large amount of soluble recombinant VEGF-A_(189) protein, the fermentation of re-combinant VEGF-A_(189) engineering bacteria was performed in 5 L fermentor. The fermentation conditions were optimized and the recombinant VEGF-A_(189) can be high level expressed in the soluble protein forma-tion in flask. The fermentation products were purified by Ni-Seproase 6 FF and analyzed by SDS-PAGE, Western Blot and ELISA. When IPTG induction was carried out at 28℃, the soluble recombinant VEGF-A_(189) can be high-level expressed. The fermentation products were harvested after 4 hours induc-tion, in which the optical density (A_(600 nm)) was up to 13.72, and the wet-weight was 40 g/L. Western Blot and ELISA analysis showed that the fermentation products of recombinant VEGF-A_(189) were able to bind anti-human VEGF polyclonal antibodies. Soluble recombinant VEGF-A_(189) can be obtained by use of fermentation condition optimization.