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根据文献报道和NCBI数据库确定人源内皮抑制素(HE)和鼠源内皮抑制素(ME)DNA序列,以DNA合成法分别获得长度为554 bp的人源内和长度为565 bp的鼠源内皮抑素基因;分别构建携带谷胱甘肽巯基转移酶(GST)表达标签的原核表达质粒pGEX-4T1-HE和携带硫氧还蛋白A(TrxA)表达标签的原核表达质粒pET-32a-ME,在大肠杆菌BL21(DE3)中诱导表达,获得不同表达标签的HE、ME重组蛋白.最后通过鸡胚绒毛尿囊膜(CAM)试验验证重组蛋白的抗血管生成活性.结果表明,构建了人与鼠源内皮抑制素基因不同标签表达载体,在大肠杆菌中成功诱导表达.获得具有活性的人与鼠源ES重组蛋白.纯化复性后的重组蛋白能明显抑制新生血管的生长.GST和TrxA标签对增进重组ES可溶表达没有显著差异,两组标签的重组蛋白都主要存在于包涵体内.
The human endostatin (HE) and murine endostatin (ME) DNA sequences were determined according to the literature and the NCBI database, and the human endogenous gene of 564 bp in length and the mouse endogenous gene of 565 bp in length were obtained by DNA synthesis The prokaryotic expression plasmid pGEX-4T1-HE carrying the glutathione S-transferase (GST) expression tag and the prokaryotic expression plasmid pET-32a-ME carrying the thioredoxin A (TrxA) E.coli BL21 (DE3) induced expression of HE, ME recombinant protein with different expression tag.Finally, the anti-angiogenic activity of the recombinant protein was verified by chick chorioallantoic membrane (CAM) assay.The results showed that human and mouse Endostatin gene of different tag expression vector, successfully induced expression in E. coli to obtain active human and mouse ES recombinant protein purification of renaturation of recombinant protein can significantly inhibit angiogenesis .GST and TrxA tag pairs There was no significant difference in enhancing the soluble expression of recombinant ES. The recombinant proteins of both groups were mainly present in the inclusion body.