人血管抑制因子Vasostatin(120～180aa)的真核表达、纯化及活性鉴定

目的利用毕赤酵母表达系统表达人血管抑制因子Vasostatin(120～180aa),经纯化后,观察其抑制血管内皮细胞增殖的活性。方法采用PCR技术扩增出人血管抑制因子Vasostatin(120～180aa)的全长cDNA序列,将其克隆至真核表达载体pPIC9K中,转化毕赤酵母KM71,以甲醇诱导表达,并进行Western blot检测及金属螯合层析纯化。采用MTT法检测纯化的血管抑制因子对血管内皮细胞增殖的抑制作用。结果重组表达质粒pPIC9K-Vasostatin(120～180aa)经菌落PCR、酶切和测序鉴定,证明构建正确。经Western blot检测可见一条特异条带,纯化后的蛋白纯度达88.76%,浓度为300μg/ml,在体外可抑制人脐静脉血管内皮细胞ECV304的增殖。结论人血管抑制因子Vasostatin(120～180aa)可在毕赤酵母中以分泌形式表达,并具有抑制血管内皮细胞增殖的活性。

Eukaryotic Expression, Purification and Activity of Recombinant Human Vasostatin ( 120 ～ 180aa )

Objective To express recombinant human vasostatin(120～180aa) in Pichia pastoris,purify the expressed product and determine its activity in inhibiting the proliferation of vascular endothelial cells.Methods Amplify the full-length of cDNA se- quence of human vasostatin(120～180aa)by PCR and insert into eukaryotic expression vector pPIC9K.Transform the constructed re- combinant plasmid pPICgK-vasostatin(120～180aa)to Pichia pastoris for expression under induction of methanol.Identify the ex- pressed product by Western blot and purify by metal chelate chromatography.Determine the inhibiting effect of purified expressed prod- uct on the proliferation of vascular endothelial cells by MTr method.Results Recombinant plasmid pPIC9K-vasostatin(120～180aa) was correctly constructed as proved by PCR,restriction analysis and sequencing.Western blot showed a single specific hand.The puri- fied expressed product reached a purity of 88.76% and a protein concentration of 300μg/ml,and inhibited the proliferation of human umbilical vein endothelial cells ECV304 in vitro.Conclusion Recombinant human vasostation(120～180aa)was successfully ex- pressed in a secretory form in Pichia pastoris and showed activity in inhibiting the proliferation of vascular endothelial cells.