人内皮抑素在毕赤酵母中的表达、纯化与生物功能研究

内皮抑素（Endostatin）是近年来新发现的一种内源性新生血管生成（Angiogenesis）抑制因子，通过抑制新血管生成而抑制肿瘤的形成和转移且不会引起耐药性，具有极高的临床应用前景。巴斯德毕赤酵母（Pichia pastoris)具有表达率高、产物可分泌、可对高等真核生物蛋白正确进行翻译后加工、遗传稳定、发酵工艺成熟等优点被用来进行重组人Endostatin的表达。本研究用PCR的方法从人胎肝cDNA文库中扩增出人Endostatin的cDNA，测序正确后转入毕赤巴斯德甲醇酵母，并获得了高效可溶型表达，用肝素亲和层析的方法进行纯化，纯化后产物经SDSPAGE薄层扫描分析纯度达987％以上，质谱测定分子量为2043kD与理论值一致，蛋白质N端序列测定结果为SPPAHTHRDFQPVLH与天然序列一致。生物活性检测证明可抑制鸡胚尿囊绒毛膜（CAM）的新生血管生成（Angiogenesis),并可抑制血管内皮细胞的增殖。因此用酵母表达系统可以得到具有生物活性的内皮抑素，经纯化后可用于进一步的生物功能和作用机理试验。

Inhibition Effect in vitro of Purified Endostatin Expressed in Pichia pastoris

Endostatin is a newly found inhibitor of angiogenesis, which is identified as c\|terminal 184 amino acid fragment of collagen XVIII NC1\|domain. A 570bp 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 subsequenced 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 20.34kD. The expression level was up to 40mg/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.