血管生长抑制因子angiostatin在昆虫细胞中的表达及活性研究

背景与目的:抑制新生血管的形成,有助于抑制肿瘤的生长,减少和预防肿瘤转移的发生。血管抑素(angiostatin)是一种重要的内源性新生血管生成抑制剂,对血管内皮细胞增殖有较强的抑制作用。为获得具有高活性的Angiostatin表达,本研究通过杆状病毒表达系统表达重组的angiostatin,分析其在昆虫细胞中表达和生物活性。方法:用带有angiostatin的杆状病毒转移载体pBlueBacHis2B和病毒DNA共同转染Sf9细胞,构建重组病毒,蚀斑实验筛选,PCR分析确定后扩增产生大量高滴度的病毒贮存液;用SDS-PAGE电泳和Westernblot对感染不同时间后分泌的重组蛋白做时间表达分析;用ProBondTM纯化系统对表达的重组angiostatin进行纯化,分光光度计确定蛋白含量,SDS-PAGE电泳确定蛋白纯度;采用MTT法测定重组蛋白angiostatin对原代培养的人脐静脉内皮细胞(HUVEC)的抑制作用而后通过鸡胚尿囊膜实验进一步证实其抗血管形成作用。结果:成功构建了滴度为2×108pfu/ml的angiostatin重组杆状病毒,并在昆虫细胞Sf9中高效表达了分子量为53ku的angiostatin重组蛋白,纯度约为90%,重组angiostatin蛋白不仅在体外显著抑制内皮细胞的生长(IC50为2.3μg/ml),而且显著抑制鸡胚尿囊膜血管的生长。结论:此系统可制备高滴度angiostatin重组杆状病毒贮存液,并在Sf9昆虫细胞中高效表达该重组蛋白,经在体和离体细胞学实验验证其对内皮细胞的增殖具有抑制作用。

Expression and biological activity of human angiogenesis inhibitor angiostatin in insect cells

BACKGROUND & OBJECTIVE: Angiogenesis is the essential process for tumor growth and metastasis. Angiostatin, a potent endogenous inhibitor of angiogenesis, could selectively inhibit proliferation of vascular endothelial cells. This study was to construct recombinant angiostatin-baculovirus, and explore the expression and biological activity of recombinant angiostatin in insect cells. METHODS: The angiostatin baculovirus transfer vector pBlueBacHis2B was co-transfected with virus DNA into insect Sf9 cells to construct recombinant baculovirus. The recombinant virus was screened by plaque assay, and confirmed and amplified by polymerase chain reaction (PCR) to produce large scale, high-titer virus stock. The expression of the recombinant protein at different time points was detected by SDS-PAGE and Western blot. The recombinant protein was purified by ProBond purification system. Inhibitory effect of recombinant angiostatin on human umbilical vein endothelial cells (HUVECs) was examined by MTT assay. Its anti-angiogenesis effect was confirmed by in vivo chorioallantoic membrane (CAM) test. RESULTS: Recombinant angiostatin baculovirus with a high virus titer (2 x 10(8) pfu/ml) was constructed successfully. Recombinant angiostatin (53 ku) was effectively expressed in Sf9 cells, and its pure degree was about 90% of insect cellular total soluble proteins. The recombinant angiostatin obviously inhibited proliferation of endothelial cells with the 50% inhibitory concentration (IC(50)) of 2.3 microg/ml, and remarkably inhibited the growth of vessels in CAM. CONCLUSIONS: The baculovirus expression system could be used to construct high-titer recombinant angiostatin-virus stock, and highly express the recombinant angiostatin in insect cells. In vitro and in vivo experiments confirmed that angiostatin could inhibit proliferation of vascular endothelial cells.