Feng Y, Cui L B, Liu C X, Ma Q J
Beijing Institute of Biotechnology, Beijing, 100850, China.
Sheng Wu Gong Cheng Xue Bao. 2001 May;17(3):278-82.
Endostatin is a newly found inhibitor of angiogenesis, which is identified as c-terminal 184 amino acid fragment of collagen XVIII NC1-domain. A 570 bp cDNA fragment of endostatin has been amplified by PCR from a commercial human fetal liver cDNA library. After subcloned into the yeast vector pPIC9 and subsequence to prove its correctness, Pichia pastoris was transformed with the recombinant pPIC9-endostatin. The expressed endostatin in P. pastoris was purified by heparin-sapherose affinity chromatography. It's purity identified by SDS-PAGE thin layer scanning analysis was up to 98.7% and its Mol. Weight measured by MS was 20.34 kD. The expression level was up to 40 mg/L. The first fifteen amino acid sequence of the N-terminal was completely identical with the inner sequence C-terminal fragment of collagen XVIII NC1 domain as has been designed. Bioassay indicated that the recombinant endostatin can inhibit angiogenesis stimulated by bFGF in CAM test and also the proliferation of both HUVEC and ECV304 in an in vitro test.
内皮抑素是一种新发现的血管生成抑制剂，它被鉴定为胶原蛋白 XVIII NC1 结构域的 C 末端 184 个氨基酸片段。通过 PCR 从商业人类胎儿肝脏 cDNA 文库中扩增出了一段 570 bp 的内皮抑素 cDNA 片段。将其亚克隆到酵母载体 pPIC9 中并随后验证其正确性后，用重组 pPIC9 - 内皮抑素转化毕赤酵母。毕赤酵母中表达的内皮抑素通过肝素 - 琼脂糖亲和层析进行纯化。经 SDS - PAGE 薄层扫描分析鉴定，其纯度高达 98.7%，通过质谱测定其分子量为 20.34 kD。表达水平高达 40 mg/L。N 末端的前十五个氨基酸序列与设计的胶原蛋白 XVIII NC1 结构域 C 末端片段的内部序列完全相同。生物测定表明，重组内皮抑素在鸡胚绒毛尿囊膜试验中可抑制 bFGF 刺激的血管生成，在体外试验中也可抑制人脐静脉内皮细胞（HUVEC）和人脐静脉内皮细胞系 ECV304 的增殖。
