人血管抑素在丝氏酵母中的表达及其活性测定

利用RT－PCR方法从人肝细胞癌HepG2细胞株中扩增得到编码人血管抑素（angiostatin)的cDNA。将其插入毕氏酵母表达载体pPIC9K中,转化结氏酵母,获得了整合多拷贝血管抑素基因的菌株。了酵液经赖氨酸亲和柱纯化所得到的重组蛋白的产量为25mg/L。蛋白质氨基酸序列分析结果与国外报道一致。重组蛋白特异地抑制bFGF刺激的牛主动脉内皮细胞的增殖,ED50约为3mg/L。     

Canstatin-N基因在Pichia pastoris中表达及产物活性分析

目的：应用P.pastoris的pAOX1表达系统胞内表达canstatin-N蛋白。方法：通过PCR DNA合成技术以及DNA的酶切和连接技术,除去pPIC9K分泌型表达载体的信号肽,并使其载体的多克隆位点的3′端融合his6纯化标签,获得新的载体pPIC9Ki。以含canstatin N端序列的pET-CTN质粒为模版,PCR法扩增1～89个氨基酸的267bp的canstatin的N端基因片段,将其连接于pPIC9Ki的多克隆位点,获得重组表达质粒pPIC9Ki-CTN-N,用电转法将pPIC9Ki-CTN-N转化P.pastorisGS115,通过G418抗性筛选获得工程菌GS115（pPIC9Ki-CTN-N）。通过摇瓶发酵甲醇诱导表达Canstatin-N蛋白。用蜗牛酶裂解P.pastoris细胞,SDS-PAGE分析蛋白表达情况,在鸡胚中进行活性分析。结果：在pAOX1的调控下,canstatin-N基因能在P.pastoris中经甲醇诱导表达,摇瓶发酵表达量为65mg/L,纯化的目的蛋白具有显著的抑制鸡胚尿囊膜血管生成的作用。结论：实现应用P.pastoris表达Canstatin-N蛋白,为Canstatin-N蛋白的规模化生产和进一步的药用研究奠定了基础。     

重组人纤溶酶原Kringle1-5的制备及其

为了研究重组人纤溶酶原 Kringle1-5(K1-5)的抗血管生成活性及其对内皮细胞增殖的影响, 通过PCR扩增人纤溶酶原K1-5 cDNA,定向克隆于原核表达载体pET30a(+)中,构建重组表达载体pET-K1-5, 转化E.coli BL21(DE3), IPTG诱导表达,SDS-PAGE 和Western 杂交检测K1-5的表达。鸡胚尿囊膜 (CAM) 实验和MTT实验分别检测重组人纤溶酶原Kringle1-5对鸡胚新生血管生成和内皮细胞的抑制作用。结果表明,IPTG诱导原核表达载体pET-K1-5在E.coli BL21(DE3)中的表达量约占菌体总蛋白量的32%, K1-5主要以包涵体形式存在,包涵体经过洗涤、溶解、Ni-spin 亲合柱层析纯化以及蛋白质复性等步骤后,获得了纯度约为96%的重组K1-5蛋白。CAM实验表明,原核表达的重组人K1-5能有效地按剂量依赖的方式抑制鸡胚新生血管的形成。MTT实验结果显示,重组人K1-5特异地抑制内皮细胞的增殖, 而对非内皮细胞无抑制作用。     

重组人纤溶酶原Kringle1-3的生物学活性鉴定

目的：检测重组人纤溶酶原Kringle1-3（K1-3）的生物学活性。方法：用含重组人纤溶酶原K1-3基因的表达载体pET21a-Angio（K1-3）转化表达宿主菌大肠杆菌BL21（DE3）后诱导表达,表达产物经溶解、复性和纯化后,进行SDS-PAGE,计算其相对分子质量;用BCA法测蛋白浓度,用细胞抑制实验（MTT法）和鸡胚绒毛膜尿囊膜（CAM）实验鉴定纯化产物对血管内皮细胞增殖和血管生成的抑制效果。结果：表达产物的相对分子质量为38000,与预期值一致;细胞抑制实验和CAM实验结果表明表达产物具有特异抑制血管内皮细胞增殖、血管生成的功能。结论：所纯化的重组人纤溶酶原K1-3具有抑制血管内皮细胞的生物学活性,为该蛋白在病理性血管疾病等方面的应用研究提供了材料。     

人纤溶酶原K1-3基因的克隆、表达和产物的纯化及抗癌活性分析

纤溶酶原K13功能区是近年发现的血管生成抑制因子,具有抑制肿瘤生长和转移的活性。以人血管生成抑制素cDNA为模板用PCR技术扩增了K13功能区的基因,DNA序列分析后克隆至质粒pPIC9K上获得重组质粒pPIC9K13,转化毕节酵母GS115,用PCR和G418法筛选高拷贝转化子,进行摇瓶发酵。SDSPAGE和Western blot分析结果证实K13基因已在GS115分泌表达,并具有免疫活性。选用30L和80L罐进行高密度发酵,甲醇诱导48h细胞密度达到OD250～300,比摇瓶提高5～6倍,表达量150200mg/L。发酵上清液经Streamline SP离子交换及PhenylSephorose疏水层析纯化,在SDSPAGE上显示一条带,纯度96%,动物实验证明纯化产物具有抗血管生成和抗肿瘤的活性。      

OSF1在毕赤酵母菌中的分泌表达及其生物活性

为了研究人成骨细胞刺激因子(Human osteoblast-stimulating factor,OSF-1)的生物学活性,构建OSF-1高效毕赤酵母表达菌株并表达纯化具有生物活性的OSF-1。首先通过全基因人工合成的方法合成人osf-1基因,并克隆至毕赤酵母分泌表达载体pPIC9K,构建重组表达质粒pPIC9K/osf-1,线性化后电转化酵母宿主菌GS115,构建P.pastoris GS115/pPIC9K/osf-1,经MD、G418-YPD平板和PCR法筛选,获得多拷贝转化子。阳性表达菌株在25℃,经1%的甲醇诱导表达96 h,重组蛋白的表达量达到最大。经SDS-PAGE电泳分析所表达的重组蛋白约为18 kDa,与人成骨细胞刺激因子相符。表达上清经SP-Sephadex C-50离子交换层析进行纯化,得到纯度达98%以上的OSF-1。Western blotting鉴定该蛋白对人成骨细胞刺激因子抗体具有良好的抗原性。生物活性测定表明提纯的OSF-1能促进鼠成骨细胞MC3T3-E1的增殖和分化。利用重组毕赤酵母高效分泌表达了有生物活性的OSF-1,为进一步开展其抗骨质疏松活性研究及产业化开发奠定了基础。     

重组人血管内皮细胞生长因子121 cDNA的基因克隆、表达和鉴定

应用RT-PCR方法,扩增人VEGF₁₂₁ cDNA基因片段,与酵母表达载体pPIC9K重组,获得表达质粒p9KVEGF₁₂₁.该质粒转化毕赤酵母菌GS115,用G418-YPD平板筛选高拷贝转化子,PCR鉴定VEGF₁₂₁ cDNA与酵母染色体整合状态,高拷贝转化子用甲醇诱导表达.工程菌用5 L发酵罐发酵,表达产物r-hVEGF₁₂₁占培养液中总蛋白量70%以上.纯化产物促进牛毛细血管内皮(BCE)细胞增殖,并强烈促进血管通透.     

血管生成抑制因子K4K5 Cdna基因的克隆及其在毕赤酵母中的表达

应用PCR方法,扩增人纤溶酶原cDNA基因中K4K5 cDNA片段,与酵母表达载体pPIC9K重组,获得表达质凿p9kkk-18。该质粒转化毕赤酵母菌GS115,用G418－YPD筛选高拷贝表型,PCR筛选K4K5 cDNA与酵母染色体整全形成的阳性克隆,阳性克隆用甲醇诱导表达。表达产物r-K4K5分子量约21.5kD,占分泌总蛋白80％以上,产物浓度为150－250mg/L。初步纯化产物抑制牛毛细血管内皮（BCE）细胞增殖与鸡胚绒毛尿囊膜（CAM）新生血管生成。     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。     

重组人纤溶酶原K1-3蛋白的抗肿瘤活性研究

人纤溶酶原K1-3功能区是一个血管生成抑制因子。以人纤溶酶原k1-3基因在大肠杆菌中表达的重组K1-3蛋白进行鸡胚绒毛尿囊膜(chorioallantoicmembrane,CAM)血管生成抑制活性分析和小鼠B16黑色素瘤抑瘤实验,结果证实重组K1-3蛋白具有抑制毛细血管生成和抗肿瘤活性。     

人血管内皮抑制素在毕赤酵母中的表达纯化及活性测定

血管内皮抑制素是胶原ⅩⅧ C-末端的一个片段,是一个有效的血管生成抑制因子。本文克隆了人血管内皮抑制素的基因,并用毕赤酵母进行表达,表达量为50.5 mg/L。发酵液上清经SP Sepharose Fast Flow凝胶一步层析,产物纯度达到98%以上,纯化收率达到95%以上。毕赤酵母分泌表达的人血管内皮抑制素具有免疫活性;能明显抑制鸡胚尿囊膜新生血管的生长;并且能特异性地抑制bFGF刺激的人微血管内皮细的迁移,达到抑制效果为50%时所需的蛋白浓度（IC50）为0.4μg/ml。本研究为应用人血管内皮抑制素治疗肿瘤奠定了初步实验基础。     

Specific interaction of angiostatin with integrin alpha(v)beta(3) in endothelial cells

Angiostatin, the N-terminal four kringles (K1-4) of plasminogen, blocks tumor-mediated angiogenesis and has great therapeutic potential. However, angiostatin's mechanism of anti-angiogenic action is unclear. We found that bovine arterial endothelial (BAE) cells adhere to angiostatin in an integrin-dependent manner and that integrins alpha(v)beta(3), alpha(9)beta(1), and to a lesser extent alpha(4)beta(1), specifically bind to angiostatin. alpha(v)beta(3) is a predominant receptor for angiostatin on BAE cells, since a function-blocking antibody to alpha(v)beta(3) effectively blocks adhesion of BAE cells to angiostatin, but an antibody to alpha(9)beta(1) does not. epsilon-Aminocaproic acid, a Lys analogue, effectively blocks angiostatin binding to BAE cells, indicating that an unoccupied Lys-binding site of the kringles may be required for integrin binding. It is known that other plasminogen fragments containing three or five kringles (K1-3 or K1-5) have an anti-angiogenic effect, but plasminogen itself does not. We found that K1-3 and K1-5 bind to alpha(v)beta(3), but plasminogen does not. These results suggest that the anti-angiogenic action of angiostatin may be mediated via interaction with alpha(v)beta(3). Angiostatin binding to alpha(v)beta(3) does not strongly induce stress-fiber formation, suggesting that angiostatin may prevent angiogenesis by perturbing the alpha(v)beta(3)-mediated signal transduction that may be necessary for angiogenesis.     

人纤溶酶原K1-3基因的克隆、表达和产物的纯化及抗癌活性分析

纤溶酶原K1－3功能区是近年发现的血管生成抑制因子,具有抑制肿瘤生长和转移的活性。以人血管生成抑制素cDNA为模板用PCR技术扩增了K1－3功能区的基因,DNA序列分析后克隆至质粒pPIC9上获得重组质粒pPIC9K13转化毕节酵母GS115,用PCR和G418法筛选高拷贝转化子,进行摇瓶发酵。SDS－PAGE和Western blot分析结果证实K1－3基因已在GS115分泌表达,并具有免疫活性。选用30L和80L罐进行高密度发酵,甲醇诱导48h细胞密度达到OD250－300,比摇瓶提高5－6倍,表达量150－200mg/L,发酵上清液经Streamline SP离子交换及Phenyl-Sephorose疏水层析纯化,在SDS－PAGE上显示一条带,纯度96％,动物实验证明纯化产物具有抗血管生成和抗肿瘤的活性。     

Expression of recombinant kringle 1-5 domains of human plasminogen by a prokaryote expression system

Kringle1-5 (K1-5), a proteolytic fragment containing five kringle domains of human plasminogen generated by plasmin-mediated proteolysis, has been already identified by Cao et al. with relation to anti-angiogenesis and proliferation of endothelial cells. To investigate anti-angiogenesis activity of recombinant human K1-5 (rhK1-5) expressed in Escherichia coli BL21, the cDNA of human K1-5 obtained from cloning vector pUC57-K1-5 by PCR, was inserted into an expression vector pET30(+) to construct a prokaryotic expression vector pET-K1-5. Recombinant K1-5 efficiently expressed in E. coli BL21 after IPTG induction was monitored by SDS-PAGE and Western blotting with an anti-angiostatin monoclonal antibody and an anti-hexahistidine tag antibody. The expressed K1-5 accounted for approximately 32% of the total bacterial proteins as estimated by densitometry, and existed mainly as inclusion bodies. The inclusion bodies were washed, lysed, purified, and refolded to a purity of 96% as estimated by capillary electrophoresis and the final purification yield of K1-5 in E. coli system was approximately 5.8 mg/L. Purified K1-5 protein was tested on chicken embryo chorioallantoic membranes (CAMs), and a large number of newly formed blood vessels were significantly regressed. In the present study, we demonstrated that bacterial-expressed K1-5 effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner through CAM assay. In addition, the rhK1-5 potently inhibited endothelial cell proliferation but not non-endothelial cells. For the first time, these findings demonstrate that the rhK1-5 produced by a prokaryote expression system effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells. This fact derived from the present study further suggests the rhK1-5 can be used for anti-angiogenesis therapy of cancer.     

Constitutive expression of human angiostatin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by high-density cell culture

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol−0.8% PTM4 to the growing culture for 60 h at 30°C. Dissolved oxygen level was maintained at 25–30% and pH was controlled at 5 by the addition of 7 M NH₄OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins. Supported by the Natural Science Foundation of China (39670013) and “225” Science and Technology Program of Guangzhou Municipal Government of China (99-Z-004-001).     

Generation of biologically active angiostatin kringle 1-3 by activated human neutrophils

The contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1-3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1-3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule.     

人血管抑制素在酿酒酵母中的分泌表达和活性鉴定

将酵母交配因子 (MF)α1信号肽编码序列和人血管抑制素 (hAGN)cDNA融合序列插入穿梭载体pYADE4 ,构建得到分泌型重组表达质粒pYADEMA18.转化酿酒酵母JG110 7后 ,用 2 %乙醇和2 %甘油联合诱导表达 .ELISA分析表明 ,在诱导 14h～ 30h期间 ,hAGN获得了表达并分泌至细胞外 .发酵上清液经 75 %饱和度硫酸铵沉淀、CM 5 2纤维素离子交换层析和Superdex 75凝胶过滤层析纯化 .SDS PAGE分析显示 ,表达产物重组人血管抑制素 (rhAGN)相对分子质量约 5 0kD ,电泳纯度达到 94 %.生物活性分析证明 ,rhAGN在 0 0 1mg L～ 3 0mg L浓度范围内能够抑制人真皮内皮细胞株HDMEC增殖 ,抑制作用随剂量的增加而增强 .     

用GAP启动子在毕节酵母中组成型表达人血管抑制素

为探索用GAP启动子(PGAP)取代AOX1启动子(PAOX1),在毕节酵母(P．pastortis)中组成型表达外源蛋白的可能性,应用PCR方法从P．pastoris染色体中扩增了GAP启动子,以其取代诱导型表达载体pPIC9K上的PAOX1,构建了组成型表达载体pGAP9K。将人血管抑制素(AS)基因重组于pGAP9K的多克隆位点,获得含As基因的重组质粒pGAP9K-AS。转化P．pastorisGSll5,对获得的高拷贝转化子P．pastorisGSll5(pGAP9K-AS)进行组成型表达,同时以诱导型转化子P．pastoris GSll5(pPIC9K-AS)作为对照。SDS-PAGE结果显示：组成型转化子于培养4d后AS的表达水平已达到高峰,分泌量为58mg／L;而诱导型转化子诱导4d后表达的AS仅是组成型表达的70％,诱导6d后达到高峰,表达量也只是组成型表达系统表达高峰时(4d)的86％。CAM分析和抗癌实验结果显示：P．pastortis GS115(pGA．P9K-S)和P．pastoris GS115(pPIC9K-AS)表达的AS均具有抑制血管生成和C57BL／6J实验小鼠的B16黑色素瘤的生长,其平均瘤重抑制率分别达到90．61％和90．54％。以上结果表明,以GAP启动子构建的组成型表达系统具有发酵时间较短、表达水平较高、不用甲醇诱导、操作系统比较简单等优点,PGAP可以取代PAOX1在P．pastoris中表达AS及其他外源蛋白。     

Membrane-bound human 3beta-hydroxysteroid dehydrogenase: overexpression with His-tag using a baculovirus system and single-step purification

The membrane-bound human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) was overexpressed with His(6)-tag, using a baculovirus expression system, and then purified by nickel-chelated affinity chromatography. Overexpression of 3beta-HSD1 was confirmed by enzyme assay and Western blot analysis. The protein was purified to more than 95% homogeneity by a single-step Ni(2+)-chelated affinity chromatography after solubilization of the membrane-bound protein with the detergent C(12)E(8). High yield was repeatedly obtained, with 3-4 mg of homogeneous and active 3beta-HSD1 from 1 x 10(9) of infected Sf9 cells. The kinetic study showed a K(m) of 1.7 microM and a V(max) of 50 nmol/min/mg of purified protein using dehydroepiandrosterone as the substrate. The above preparation will facilitate the structure-function study of this important enzyme.